Glycoprofiling of Early Gastric Cancer Using Lectin Microarray Technology.

PubMed

Li, Taijie; Mo, Cuiju; Qin, Xue; Li, Shan; Liu, Yinkun; Liu, Zhiming

2018-01-01

Recently, studies have reported that protein glycosylation plays an important role in the occurrence and development of cancer. Gastric cancer is a common cancer with high morbidity and mortality owing to most gastric cancers are discovered only at an advanced stage. Here, we aim to discover novel specific serum glycanbased biomarkers for gastric cancer. A lectin microarray with 50 kinds of tumor-associated lectin was used to detect the glycan profiles of serum samples between early gastric cancer and healthy controls. Then lectin blot was performed to validate the differences. The result of the lectin microarray showed that the signal intensities of 13 lectins showed significant differences between the healthy controls and early gastric cancer. Compared to the healthy, the normalized fluorescent intensities of the lectins PWA, LEL, and STL were significantly increased, and it implied that their specifically recognized GlcNAc showed an especially elevated expression in early gastric cancer. Moreover, the binding affinity of the lectins EEL, RCA-II, RCA-I, VAL, DSA, PHA-L, UEA, and CAL were higher in the early gastric cancer than in healthy controls. These glycan structures containing GalNAc, terminal Galβ 1-4 GlcNAc, Tri/tetraantennary N-glycan, β-1, 6GlcNAc branching structure, α-linked fucose residues, and Tn antigen were elevated in gastric cancer. While the two lectins CFL GNL reduced their binding ability. In addition, their specifically recognized N-acetyl-D-galactosamine structure and (α-1,3) mannose residues were decreased in early gastric cancer. Furthermore, lectin blot results of LEL, STL, PHA-L, RCA-I were consistent with the results of the lectin microarray. The findings of our study clarify the specific alterations for glycosylation during the pathogenesis of gastric cancer. The specific high expression of GlcNAc structure may act as a potential early diagnostic marker for gastric cancer.

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses*

PubMed Central

Yu, Ying; Lasanajak, Yi; Song, Xuezheng; Hu, Liya; Ramani, Sasirekha; Mickum, Megan L.; Ashline, David J.; Prasad, B. V. Venkataram; Estes, Mary K.; Reinhold, Vernon N.; Cummings, Richard D.; Smith, David F.

2014-01-01

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSn analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MSn are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures. PMID:25048705

Fabrication of Carbohydrate Microarrays by Boronate Formation.

PubMed

Adak, Avijit K; Lin, Ting-Wei; Li, Ben-Yuan; Lin, Chun-Cheng

2017-01-01

The interactions between soluble carbohydrates and/or surface displayed glycans and protein receptors are essential to many biological processes and cellular recognition events. Carbohydrate microarrays provide opportunities for high-throughput quantitative analysis of carbohydrate-protein interactions. Over the past decade, various techniques have been implemented for immobilizing glycans on solid surfaces in a microarray format. Herein, we describe a detailed protocol for fabricating carbohydrate microarrays that capitalizes on the intrinsic reactivity of boronic acid toward carbohydrates to form stable boronate diesters. A large variety of unprotected carbohydrates ranging in structure from simple disaccharides and trisaccharides to considerably more complex human milk and blood group (oligo)saccharides have been covalently immobilized in a single step on glass slides, which were derivatized with high-affinity boronic acid ligands. The immobilized ligands in these microarrays maintain the receptor-binding activities including those of lectins and antibodies according to the structures of their pendant carbohydrates for rapid analysis of a number of carbohydrate-recognition events within 30 h. This method facilitates the direct construction of otherwise difficult to obtain carbohydrate microarrays from underivatized glycans.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

PubMed Central

Halder, Sujata; Cotmore, Susan; Heimburg-Molinaro, Jamie; Smith, David F.; Cummings, Richard D.; Chen, Xi; Trollope, Alana J.; North, Simon J.; Haslam, Stuart M.; Dell, Anne; Tattersall, Peter; McKenna, Robert; Agbandje-McKenna, Mavis

2014-01-01

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen. PMID:24475195

A Versatile Method for Functionalizing Surfaces with Bioactive Glycans

PubMed Central

Cheng, Fang; Shang, Jing; Ratner, Daniel M.

2011-01-01

Microarrays and biosensors owe their functionality to our ability to display surface-bound biomolecules with retained biological function. Versatile, stable, and facile methods for the immobilization of bioactive compounds on surfaces have expanded the application of high-throughput ‘omics’-scale screening of molecular interactions by non-expert laboratories. Herein, we demonstrate the potential of simplified chemistries to fabricate a glycan microarray, utilizing divinyl sulfone (DVS)-modified surfaces for the covalent immobilization of natural and chemically derived carbohydrates, as well as glycoproteins. The bioactivity of the captured glycans was quantitatively examined by surface plasmon resonance imaging (SPRi). Composition and spectroscopic evidence of carbohydrate species on the DVS-modified surface were obtained by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. The site-selective immobilization of glycans based on relative nucleophilicity (reducing sugar vs. amine- and sulfhydryl-derived saccharides) and anomeric configuration was also examined. Our results demonstrate straightforward and reproducible conjugation of a variety of functional biomolecules onto a vinyl sulfone-modified biosensor surface. The simplicity of this method will have a significant impact on glycomics research, as it expands the ability of non-synthetic laboratories to rapidly construct functional glycan microarrays and quantitative biosensors. PMID:21142056

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

NASA Astrophysics Data System (ADS)

Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten

2018-05-01

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

PubMed

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah; Dallabernadina, Pietro; Boos, Irene; Andersen, Mathias C F; Kotake, Toshihisa; Knox, J Paul; Hahn, Michael G; Clausen, Mads H; Pfrengle, Fabian

2017-11-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision. © 2017 American Society of Plant Biologists. All Rights Reserved.

Carbohydrate Microarray Technology Applied to High-Throughput Mapping of Plant Cell Wall Glycans Using Comprehensive Microarray Polymer Profiling (CoMPP).

PubMed

Kračun, Stjepan Krešimir; Fangel, Jonatan Ulrik; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Vidal-Melgosa, Silvia; Willats, William George Tycho

2017-01-01

Cell walls are an important feature of plant cells and a major component of the plant glycome. They have both structural and physiological functions and are critical for plant growth and development. The diversity and complexity of these structures demand advanced high-throughput techniques to answer questions about their structure, functions and roles in both fundamental and applied scientific fields. Microarray technology provides both the high-throughput and the feasibility aspects required to meet that demand. In this chapter, some of the most recent microarray-based techniques relating to plant cell walls are described together with an overview of related contemporary techniques applied to carbohydrate microarrays and their general potential in glycoscience. A detailed experimental procedure for high-throughput mapping of plant cell wall glycans using the comprehensive microarray polymer profiling (CoMPP) technique is included in the chapter and provides a good example of both the robust and high-throughput nature of microarrays as well as their applicability to plant glycomics.

Advances in analytical methodologies to guide bioprocess engineering for bio-therapeutics.

PubMed

Saldova, Radka; Kilcoyne, Michelle; Stöckmann, Henning; Millán Martín, Silvia; Lewis, Amanda M; Tuite, Catherine M E; Gerlach, Jared Q; Le Berre, Marie; Borys, Michael C; Li, Zheng Jian; Abu-Absi, Nicholas R; Leister, Kirk; Joshi, Lokesh; Rudd, Pauline M

2017-03-01

This study was performed to monitor the glycoform distribution of a recombinant antibody fusion protein expressed in CHO cells over the course of fed-batch bioreactor runs using high-throughput methods to accurately determine the glycosylation status of the cell culture and its product. Three different bioreactors running similar conditions were analysed at the same five time-points using the advanced methods described here. N-glycans from cell and secreted glycoproteins from CHO cells were analysed by HILIC-UPLC and MS, and the total glycosylation (both N- and O-linked glycans) secreted from the CHO cells were analysed by lectin microarrays. Cell glycoproteins contained mostly high mannose type N-linked glycans with some complex glycans; sialic acid was α-(2,3)-linked, galactose β-(1,4)-linked, with core fucose. Glycans attached to secreted glycoproteins were mostly complex with sialic acid α-(2,3)-linked, galactose β-(1,4)-linked, with mostly core fucose. There were no significant differences noted among the bioreactors in either the cell pellets or supernatants using the HILIC-UPLC method and only minor differences at the early time-points of days 1 and 3 by the lectin microarray method. In comparing different time-points, significant decreases in sialylation and branching with time were observed for glycans attached to both cell and secreted glycoproteins. Additionally, there was a significant decrease over time in high mannose type N-glycans from the cell glycoproteins. A combination of the complementary methods HILIC-UPLC and lectin microarrays could provide a powerful and rapid HTP profiling tool capable of yielding qualitative and quantitative data for a defined biopharmaceutical process, which would allow valuable near 'real-time' monitoring of the biopharmaceutical product. Copyright © 2016 Elsevier Inc. All rights reserved.

Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags.

PubMed

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-06-01

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in "Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans" (Kang et al., 2016 [1]).

Defining the interaction of human soluble lectin ZG16p and mycobacterial phosphatidylinositol mannosides

PubMed Central

Ikeda, Akemi; Kojima-Aikawa, Kyoko; Taniguchi, Naoyuki; Varón Silva, Daniel; Feizi, Ten; Seeberger, Peter H.; Yamaguchi, Yoshiki

2018-01-01

ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analyses of the interactions of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs), using glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation Transfer Difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interacts with ZG16p using the mannose residues. Binding site of PIMs is identified by chemical shift perturbation experiments using uniformly 15N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan, which would help to consider the physiological role of ZG16p. PMID:25919894

On-line capillary electrophoresis/laser-induced fluorescence/mass spectrometry analysis of glycans labeled with Teal™ fluorescent dye using an electrokinetic sheath liquid pump-based nanospray ion source.

PubMed

Khan, Shaheer; Liu, Jenkuei; Szabo, Zoltan; Kunnummal, Baburaj; Han, Xiaorui; Ouyang, Yilan; Linhardt, Robert J; Xia, Qiangwei

2018-06-15

N-linked glycan analysis of recombinant therapeutic proteins, such as monoclonal antibodies, Fc-fusion proteins, and antibody-drug conjugates, provides valuable information regarding protein therapeutics glycosylation profile. Both qualitative identification and quantitative analysis of N-linked glycans on recombinant therapeutic proteins are critical analytical tasks in the biopharma industry during the development of a biotherapeutic. Currently, such analyses are mainly carried out using capillary electrophoresis/laser-induced fluorescence (CE/LIF), liquid chromatography/fluorescence (LC/FLR), and liquid chromatography/fluorescence/mass spectrometry (LC/FLR/MS) technologies. N-linked glycans are first released from glycoproteins by enzymatic digestion, then labeled with fluorescence dyes for subsequent CE or LC separation, and LIF or MS detection. Here we present an on-line CE/LIF/MS N-glycan analysis workflow that incorporates the fluorescent Teal™ dye and an electrokinetic pump-based nanospray sheath liquid capillary electrophoresis/mass spectrometry (CE/MS) ion source. Electrophoresis running buffer systems using ammonium acetate and ammonium hydroxide were developed for the negative ion mode CE/MS analysis of fluorescence-labeled N-linked glycans. Results show that on-line CE/LIF/MS analysis can be readily achieved using this versatile CE/MS ion source on common CE/MS instrument platforms. This on-line CE/LIF/MS method using Teal™ fluorescent dye and electrokinetic pump-based nanospray sheath liquid CE/MS coupling technology holds promise for on-line quantitation and identification of N-linked glycans on recombinant therapeutic proteins. Copyright © 2018 John Wiley & Sons, Ltd.

Glycome Diagnosis of Human Induced Pluripotent Stem Cells Using Lectin Microarray*

PubMed Central

Tateno, Hiroaki; Toyota, Masashi; Saito, Shigeru; Onuma, Yasuko; Ito, Yuzuru; Hiemori, Keiko; Fukumura, Mihoko; Matsushima, Asako; Nakanishi, Mio; Ohnuma, Kiyoshi; Akutsu, Hidenori; Umezawa, Akihiro; Horimoto, Katsuhisa; Hirabayashi, Jun; Asashima, Makoto

2011-01-01

Induced pluripotent stem cells (iPSCs) can now be produced from various somatic cell (SC) lines by ectopic expression of the four transcription factors. Although the procedure has been demonstrated to induce global change in gene and microRNA expressions and even epigenetic modification, it remains largely unknown how this transcription factor-induced reprogramming affects the total glycan repertoire expressed on the cells. Here we performed a comprehensive glycan analysis using 114 types of human iPSCs generated from five different SCs and compared their glycomes with those of human embryonic stem cells (ESCs; nine cell types) using a high density lectin microarray. In unsupervised cluster analysis of the results obtained by lectin microarray, both undifferentiated iPSCs and ESCs were clustered as one large group. However, they were clearly separated from the group of differentiated SCs, whereas all of the four SCs had apparently distinct glycome profiles from one another, demonstrating that SCs with originally distinct glycan profiles have acquired those similar to ESCs upon induction of pluripotency. Thirty-eight lectins discriminating between SCs and iPSCs/ESCs were statistically selected, and characteristic features of the pluripotent state were then obtained at the level of the cellular glycome. The expression profiles of relevant glycosyltransferase genes agreed well with the results obtained by lectin microarray. Among the 38 lectins, rBC2LCN was found to detect only undifferentiated iPSCs/ESCs and not differentiated SCs. Hence, the high density lectin microarray has proved to be valid for not only comprehensive analysis of glycans but also diagnosis of stem cells under the concept of the cellular glycome. PMID:21471226

Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags

PubMed Central

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-01-01

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in “Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans” (Kang et al., 2016 [1]). PMID:27222848

A novel broad specificity fucosidase capable of core α1-6 fucose release from N-glycans labeled with urea-linked fluorescent dyes.

PubMed

Vainauskas, Saulius; Kirk, Charlotte H; Petralia, Laudine; Guthrie, Ellen P; McLeod, Elizabeth; Bielik, Alicia; Luebbers, Alex; Foster, Jeremy M; Hokke, Cornelis H; Rudd, Pauline M; Shi, Xiaofeng; Taron, Christopher H

2018-06-22

Exoglycosidases are often used for detailed characterization of glycan structures. Bovine kidney α-fucosidase is commonly used to determine the presence of core α1-6 fucose on N-glycans, an important modification of glycoproteins. Recently, several studies have reported that removal of core α1-6-linked fucose from N-glycans labeled with the reactive N-hydroxysuccinimide carbamate fluorescent labels 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) and RapiFluor-MS is severely impeded. We report here the cloning, expression and biochemical characterization of an α-fucosidase from Omnitrophica bacterium (termed fucosidase O). We show that fucosidase O can efficiently remove α1-6- and α1-3-linked core fucose from N-glycans. Additionally, we demonstrate that fucosidase O is able to efficiently hydrolyze core α1-6-linked fucose from N-glycans labeled with any of the existing NHS-carbamate activated fluorescent dyes.

Mucin-type O-glycans in Tears of Normal Subjects and Patients with Non-Sjögren’s Dry Eye

PubMed Central

Guzman-Aranguez, Ana; Mantelli, Flavio; Argüeso, Pablo

2009-01-01

Purpose O-linked carbohydrates (O-glycans) contribute to the hydrophilic character of mucins in mucosal tissues. This study aimed to identify the repertoire of O-glycans in the tear film, and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögren’s dry eye. Methods Human tear fluid was collected from the inferior conjunctival fornix. O-glycans were released by hydrazinolysis, labeled with 2-aminobenzamide, and analyzed by fluorometric, high-performance liquid chromatography (HPLC) coupled with exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium using a glycogene microarray database. Lectin-binding analyses were performed using agglutinins from Arachis hypogaea, Maackia amurensis, and Sambucus nigra. Results The O-glycan profile of human tears consisted primarily of core 1 (Galβ1-3GalNAcα1-Ser/Thr)-based structures. Mono-sialyl O-glycans represented approximately 66% of the glycan pool, being α2-6-sialyl core 1 the predominant O-glycan structure in human tears (48%). Four families of glycosyltranferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included thirteen polypeptide-GalNAc-transferases (GALNT), the core 1 β-3-galactosyltransferase (T-synthase), three α2-6-sialyltransferases (ST6GalNAc), and two α2-3-sialyltransferases (ST3Gal). No significant differences in total amount of O-glycans were detected between tears of normal subjects and dry eye patients, by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray. Conclusions This study identifies the most common mucin-type O-glycans in human tears and their expected biosynthetic pathways in ocular surface epithelia. Patients with non-Sjögren’s dry eye show no alterations in composition and amount of O-glycans in the tear fluid. PMID:19407012

Computational approaches to define a human milk metaglycome

PubMed Central

Agravat, Sanjay B.; Song, Xuezheng; Rojsajjakul, Teerapat; Cummings, Richard D.; Smith, David F.

2016-01-01

Motivation: The goal of deciphering the human glycome has been hindered by the lack of high-throughput sequencing methods for glycans. Although mass spectrometry (MS) is a key technology in glycan sequencing, MS alone provides limited information about the identification of monosaccharide constituents, their anomericity and their linkages. These features of individual, purified glycans can be partly identified using well-defined glycan-binding proteins, such as lectins and antibodies that recognize specific determinants within glycan structures. Results: We present a novel computational approach to automate the sequencing of glycans using metadata-assisted glycan sequencing, which combines MS analyses with glycan structural information from glycan microarray technology. Success in this approach was aided by the generation of a ‘virtual glycome’ to represent all potential glycan structures that might exist within a metaglycomes based on a set of biosynthetic assumptions using known structural information. We exploited this approach to deduce the structures of soluble glycans within the human milk glycome by matching predicted structures based on experimental data against the virtual glycome. This represents the first meta-glycome to be defined using this method and we provide a publically available web-based application to aid in sequencing milk glycans. Availability and implementation: http://glycomeseq.emory.edu Contact: sagravat@bidmc.harvard.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26803164

Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans

NASA Astrophysics Data System (ADS)

Zhao, Nan; Martin, Brigitte E.; Yang, Chun-Kai; Luo, Feng; Wan, Xiu-Feng

2015-10-01

Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galβ, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galβ1-4GlcNAc substructures; Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.

Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans.

PubMed

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-05-15

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags--2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)--were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses. Copyright © 2016 Elsevier Inc. All rights reserved.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

PubMed

Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom

2016-10-10

Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection.

PubMed

Mauko, Lea; Nordborg, Anna; Hutchinson, Joseph P; Lacher, Nathan A; Hilder, Emily F; Haddad, Paul R

2011-01-15

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) column coupled with electrospray ionization mass spectrometry (ESI-MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC-HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC-HILIC column and ESI-MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC-HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies. Copyright © 2010 Elsevier Inc. All rights reserved.

Carbohydrate Microarrays Identify Blood Group Precursor Cryptic Epitopes as Potential Immunological Targets of Breast Cancer

PubMed Central

Wang, Denong; Tang, Jin; Liu, Shaoyi

2015-01-01

Using carbohydrate microarrays, we explored potential natural ligands of antitumor monoclonal antibody HAE3. This antibody was raised against a murine mammary tumor antigen but was found to cross-react with a number of human epithelial tumors in tissues. Our carbohydrate microarray analysis reveals that HAE3 is specific for an O-glycan cryptic epitope that is normally hidden in the cores of blood group substances. Using HAE3 to screen tumor cell surface markers by flow cytometry, we found that the HAE3 glycoepitope, gpHAE3, was highly expressed by a number of human breast cancer cell lines, including some triple-negative cancers that lack the estrogen, progesterone, and Her2/neu receptors. Taken together, we demonstrate that HAE3 recognizes a conserved cryptic glycoepitope of blood group precursors, which is nevertheless selectively expressed and surface-exposed in certain breast tumor cells. The potential of this class of O-glycan cryptic antigens in breast cancer subtyping and targeted immunotherapy warrants further investigation. PMID:26539555

Regulation of Glycan Structures in Animal Tissues

PubMed Central

Nairn, Alison V.; York, William S.; Harris, Kyle; Hall, Erica M.; Pierce, J. Michael; Moremen, Kelley W.

2008-01-01

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes. PMID:18411279

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galβ1-4GlcNAc) and LacdiNAc (GalNAcβ1-4GlcNAc) motifs found on vertebrate and insect cells.

PubMed

Frederiksen, Rikki F; Yoshimura, Yayoi; Storgaard, Birgit G; Paspaliari, Dafni K; Petersen, Bent O; Chen, Kowa; Larsen, Tanja; Duus, Jens Ø; Ingmer, Hanne; Bovin, Nicolai V; Westerlind, Ulrika; Blixt, Ola; Palcic, Monica M; Leisner, Jørgen J

2015-02-27

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1-4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1-6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1-6 bond in LacNAcβ1-6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural analysis of N-glycans by the glycan-labeling method using 3-aminoquinoline-based liquid matrix in negative-ion MALDI-MS.

PubMed

Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-ichirou; Tanaka, Koichi

2012-11-06

Negative-ion fragmentation of underivatized N-glycans has been proven to be more informative than positive-ion fragmentation. Fluorescent labeling via reductive amination is often employed for glycan analysis, but little is known about the influence of the labeling group on negative-ion fragmentation. We previously demonstrated that the on-target glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) liquid matrix enables highly sensitive, rapid, and quantitative N-glycan profiling analysis. The current study investigates the suitability of 3AQ-labeled N-glycans for structural analysis based on negative-ion collision-induced dissociation (CID) spectra. 3AQ-labeled N-glycans exhibited simple and informative CID spectra similar to those of underivatized N-glycans, with product ions due to cross-ring cleavages of the chitobiose core and ions specific to two antennae (D and E ions). The interpretation of diagnostic fragment ions suggested for underivatized N-glycans could be directly applied to the 3AQ-labeled N-glycans. However, fluorescently labeled N-glycans by conventional reductive amination, such as 2-aminobenzamide (2AB)- and 2-pyrydilamine (2PA)-labeled N-glycans, exhibited complicated CID spectra consisting of numerous signals formed by dehydration and multiple cleavages. The complicated spectra of 2AB- and 2PA-labeled N-glycans was found to be due to their open reducing-terminal N-acetylglucosamine (GlcNAc) ring, rather than structural differences in the labeling group in the N-glycan derivative. Finally, as an example, the on-target 3AQ labeling method followed by negative-ion CID was applied to structurally analyze neutral N-glycans released from human epidermal growth factor receptor type 2 (HER2) protein. The glycan-labeling method using 3AQ-based liquid matrix should facilitate highly sensitive quantitative and qualitative analyses of glycans.

Recognition of microbial glycans by human intelectin-1

DOE PAGES

Wesener, Darryl A.; Wangkanont, Kittikhun; McBride, Ryan; ...

2015-07-06

The glycans displayed on mammalian cells can differ markedly from those on microbes. Such differences could, in principle, be 'read' by carbohydrate-binding proteins, or lectins. In this paper, we used glycan microarrays to show that human intelectin-1 (hIntL-1) does not bind known human glycan epitopes but does interact with multiple glycan epitopes found exclusively on microbes: β-linked D-galactofuranose (β-Galf), D-phosphoglycerol–modified glycans, heptoses, D-glycero-D-talo-oct-2-ulosonic acid (KO) and 3-deoxy-D-manno-oct-2-ulosonic acid (KDO). The 1.6-Å-resolution crystal structure of hIntL-1 complexed with β-Galf revealed that hIntL-1 uses a bound calcium ion to coordinate terminal exocyclic 1,2-diols. N-acetylneuraminic acid (Neu5Ac), a sialic acid widespread in humanmore » glycans, has an exocyclic 1,2-diol but does not bind hIntL-1, probably owing to unfavorable steric and electronic effects. hIntL-1 marks only Streptococcus pneumoniae serotypes that display surface glycans with terminal 1,2-diol groups. Finally, this ligand selectivity suggests that hIntL-1 functions in microbial surveillance.« less

Analysis and quality control of carbohydrates in therapeutic proteins with fluorescence HPLC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Kun; Huang, Jian; Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054

Conbercept is an Fc fusion protein with very complicated carbohydrate profiles which must be carefully monitored through manufacturing process. Here, we introduce an optimized fluorescence derivatization high-performance liquid chromatographic method for glycan mapping in conbercept. Compared with conventional glycan analysis method, this method has much better resolution and higher reproducibility making it excellent for product quality control.

Bacterial Surface Glycans: Microarray and QCM Strategies for Glycophenotyping and Exploration of Recognition by Host Receptors.

PubMed

Kalograiaki, Ioanna; Campanero-Rhodes, María A; Proverbio, Davide; Euba, Begoña; Garmendia, Junkal; Aastrup, Teodor; Solís, Dolores

2018-01-01

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria-host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment. © 2018 Elsevier Inc. All rights reserved.

Large scale preparation of high mannose and paucimannose N-glycans from soybean proteins by oxidative release of natural glycans (ORNG).

PubMed

Zhu, Yuyang; Yan, Maomao; Lasanajak, Yi; Smith, David F; Song, Xuezheng

2018-07-15

Despite the important advances in chemical and chemoenzymatic synthesis of glycans, access to large quantities of complex natural glycans remains a major impediment to progress in Glycoscience. Here we report a large-scale preparation of N-glycans from a kilogram of commercial soy proteins using oxidative release of natural glycans (ORNG). The high mannose and paucimannose N-glycans were labeled with a fluorescent tag and purified by size exclusion and multidimensional preparative HPLC. Side products are identified and potential mechanisms for the oxidative release of natural N-glycans from glycoproteins are proposed. This study demonstrates the potential for using the ORNG approach as a complementary route to synthetic approaches for the preparation of multi-milligram quantities of biomedically relevant complex glycans. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of O-glycans as 9-fluorenylmethyl derivatives and its application to the studies on glycan array.

PubMed

Yamada, Keita; Hirabayashi, Jun; Kakehi, Kazuaki

2013-03-19

A method is proposed for the analysis of O-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the O-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled O-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of O-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of O-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized O-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins.

Disposable pen-shaped capillary gel electrophoresis cartridge for fluorescence detection of bio-molecules

NASA Astrophysics Data System (ADS)

Amirkhanian, Varoujan; Tsai, Shou-Kuan

2014-03-01

We introduce a novel and cost-effective capillary gel electrophoresis (CGE) system utilizing disposable pen-shaped gelcartridges for highly efficient, high speed, high throughput fluorescence detection of bio-molecules. The CGE system has been integrated with dual excitation and emission optical-fibers with micro-ball end design for fluorescence detection of bio-molecules separated and detected in a disposable pen-shaped capillary gel electrophoresis cartridge. The high-performance capillary gel electrophoresis (CGE) analyzer has been optimized for glycoprotein analysis type applications. Using commercially available labeling agent such as ANTS (8-aminonapthalene-1,3,6- trisulfonate) as an indicator, the capillary gel electrophoresis-based glycan analyzer provides high detection sensitivity and high resolving power in 2-5 minutes of separations. The system can hold total of 96 samples, which can be automatically analyzed within 4-5 hours. This affordable fiber optic based fluorescence detection system provides fast run times (4 minutes vs. 20 minutes with other CE systems), provides improved peak resolution, good linear dynamic range and reproducible migration times, that can be used in laboratories for high speed glycan (N-glycan) profiling applications. The CGE-based glycan analyzer will significantly increase the pace at which glycoprotein research is performed in the labs, saving hours of preparation time and assuring accurate, consistent and economical results.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma.

PubMed

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong; Li, Ning

2016-12-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma

PubMed Central

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong

2016-01-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC (n = 65) and healthy control subjects (n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC. PMID:27885040

Chemoenzymatic method for glycomics: isolation, identification, and quantitation

PubMed Central

Yang, Shuang; Rubin, Abigail; Eshghi, Shadi Toghi; Zhang, Hui

2015-01-01

Over the past decade, considerable progress has been made with respect to the analytical methods for analysis of glycans from biological sources. Regardless of the specific methods that are used, glycan analysis includes isolation, identification, and quantitation. Derivatization is indispensable to increase their identification. Derivatization of glycans can be performed by permethylation or carbodiimide coupling / esterification. By introducing a fluorophore or chromophore at their reducing end, glycans can be separated by electrophoresis or chromatography. The fluorogenically labeled glycans can be quantitated using fluorescent detection. The recently developed approaches using solid-phase such as glycoprotein immobilization for glycan extraction and on-tissue glycan mass spectrometry imaging demonstrate advantages over methods performed in solution. Derivatization of sialic acids is favorably implemented on the solid support using carbodiimide coupling, and the released glycans can be further modified at the reducing end or permethylated for quantitative analysis. In this review, methods for glycan isolation, identification, and quantitation are discussed. PMID:26390280

Glycan microarray analysis of the carbohydrate-recognition specificity of native and recombinant forms of the lectin ArtinM.

PubMed

Liu, Y; Cecílio, N T; Carvalho, F C; Roque-Barreira, M C; Feizi, T

2015-12-01

This article contains data related to the researc.h article entitled "Yeast-derived ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM" by Cecílio et al. (2015) [1]. ArtinM, a D-mannose-binding lectin isolated from the seeds of Artocarpus heterophyllus, exerts immunomodulatory and regenerative activities through its Carbohydrate Recognition Domain (CRD) (Souza et al., 2013; Mariano et al., 2014 [2], [3]). The limited availability of the native lectin (n-ArtinM) led us to characterize a recombinant form of the protein, obtained by expression in Saccharomyces cerevisiae (y-ArtinM). We compared the carbohydrate-binding specificities of y-ArtinM and n-ArtinM by analyzing the binding of biotinylated preparations of the two lectin forms using a neoglycolipid (NGL)-based glycan microarray. Data showed that y-ArtinM mirrored the specificity exhibited by n-ArtinM.

Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

PubMed

Lei, Y; Yu, H; Dong, Y; Yang, J; Ye, W; Wang, Y; Chen, W; Jia, Z; Xu, Z; Li, Z; Zhang, F

2015-01-01

DENV envelope glycoprotein (E) is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD) of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

PubMed

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides.

PubMed

Struwe, Weston B; Rudd, Pauline M

2012-08-01

In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λ(ex)=355 nm and λ(em)=440 nm. We concluded that the optimal wavelengths for 3-AQ were λ(ex)=355 nm and λ(em)=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λ(ex/em) values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent properties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.

Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry.

PubMed

Yuan, Jiangbei; Wang, Chengjian; Sun, Yujiao; Huang, Linjuan; Wang, Zhongfu

2014-10-01

A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate-peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies. Copyright © 2014 Elsevier Inc. All rights reserved.

Recent Developments in Synthetic Carbohydrate-Based Diagnostics, Vaccines, and Therapeutics.

PubMed

Fernández-Tejada, Alberto; Cañada, F Javier; Jiménez-Barbero, Jesús

2015-07-20

Glycans are everywhere in biological systems, being involved in many cellular events with important implications for medical purposes. Building upon a detailed understanding of the functional roles of carbohydrates in molecular recognition processes and disease states, glycans are increasingly being considered as key players in pharmacological research. On the basis of the important progress recently made in glycochemistry, glycobiology, and glycomedicine, we provide a complete overview of successful applications and future perspectives of carbohydrates in the biopharmaceutical and medical fields. This review highlights the development of carbohydrate-based diagnostics, exemplified by glycan imaging techniques and microarray platforms, synthetic oligosaccharide vaccines against infectious diseases (e.g., HIV) and cancer, and finally carbohydrate-derived therapeutics, including glycomimetic drugs and glycoproteins. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MCAW-DB: A glycan profile database capturing the ambiguity of glycan recognition patterns.

PubMed

Hosoda, Masae; Takahashi, Yushi; Shiota, Masaaki; Shinmachi, Daisuke; Inomoto, Renji; Higashimoto, Shinichi; Aoki-Kinoshita, Kiyoko F

2018-05-11

Glycan-binding protein (GBP) interaction experiments, such as glycan microarrays, are often used to understand glycan recognition patterns. However, oftentimes the interpretation of glycan array experimental data makes it difficult to identify discrete GBP binding patterns due to their ambiguity. It is known that lectins, for example, are non-specific in their binding affinities; the same lectin can bind to different monosaccharides or even different glycan structures. In bioinformatics, several tools to mine the data generated from these sorts of experiments have been developed. These tools take a library of predefined motifs, which are commonly-found glycan patterns such as sialyl-Lewis X, and attempt to identify the motif(s) that are specific to the GBP being analyzed. In our previous work, as opposed to using predefined motifs, we developed the Multiple Carbohydrate Alignment with Weights (MCAW) tool to visualize the state of the glycans being recognized by the GBP under analysis. We previously reported on the effectiveness of our tool and algorithm by analyzing several glycan array datasets from the Consortium of Functional Glycomics (CFG). In this work, we report on our analysis of 1081 data sets which we collected from the CFG, the results of which we have made publicly and freely available as a database called MCAW-DB. We introduce this database, its usage and describe several analysis results. We show how MCAW-DB can be used to analyze glycan-binding patterns of GBPs amidst their ambiguity. For example, the visualization of glycan-binding patterns in MCAW-DB show how they correlate with the concentrations of the samples used in the array experiments. Using MCAW-DB, the patterns of glycans found to bind to various GBP-glycan binding proteins are visualized, indicating the binding "environment" of the glycans. Thus, the ambiguity of glycan recognition is numerically represented, along with the patterns of monosaccharides surrounding the binding region. The profiles in MCAW-DB could potentially be used as predictors of affinity of unknown or novel glycans to particular GBPs by comparing how well they match the existing profiles for those GBPs. Moreover, as the glycan profiles of diseased tissues become available, glycan alignments could also be used to identify glycan biomarkers unique to that tissue. Databases of these alignments may be of great use for drug discovery. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

PubMed Central

Szekrényes, Ákos; Park, SungAe Suhr; Santos, Marcia; Lew, Clarence; Jones, Aled; Haxo, Ted; Kimzey, Michael; Pourkaveh, Shiva; Szabó, Zoltán; Sosic, Zoran; Feng, Peng; Váradi, Csaba; de l'Escaille, François; Falmagne, Jean-Bernard; Sejwal, Preeti; Niedringhaus, Thomas; Michels, David; Freckleton, Gordon; Hamm, Melissa; Manuilov, Anastasiya; Schwartz, Melissa; Luo, Jiann-Kae; van Dyck, Jonathan; Leung, Pui-King; Olajos, Marcell; Gu, Yingmei; Gao, Kai; Wang, Wenbo; Wegstein, Jo; Tep, Samnang; Guttman, András

2016-01-01

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established. PMID:26466659

Glycans from avian influenza virus are recognized by chicken dendritic cells and are targets for the humoral immune response in chicken.

PubMed

de Geus, Eveline D; Tefsen, Boris; van Haarlem, Daphne A; van Eden, Willem; van Die, Irma; Vervelde, Lonneke

2013-12-01

To increase our understanding of the interaction between avian influenza virus and its chicken host, we identified receptors for putative avian influenza virus (AIV) glycan determinants on chicken dendritic cells. Chicken dendritic cells (DCs) were found to recognize glycan determinants containing terminal αGalNAc, Galα1-3Gal, GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ (chitotriose) and Galα1-2Gal. Infection of chicken dendritic cells with either low pathogenic (LP) or highly pathogenic (HP) AIV results in elevated mRNA expression of homologs of the mouse C-type lectins DEC205 and macrophage mannose receptor (MMR), whereas expression levels of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) homolog remained unchanged. Following uptake and subsequent presentation of avian influenza virus by DCs, adaptive immunity, including humoral immune responses are induced. We have investigated the antibody responses against virus glycan epitopes after avian influenza virus infection. Using glycan micro-array analysis we showed that chicken contained antibodies that predominantly recognize terminal Galα1-3Gal-R, chitotriose and Fucα1-2Galβ1-4GlcNAc-R (H-type 2). After influenza-infection, glycan array analysis showed that both levels and repertoire of glycan-recognizing antibodies decreased. However, analysis of the sera by ELISA indicated that the levels of different isotypes of anti-glycan Abs against specific glycan antigens was increased after influenza-infection, suggesting that the presentation of the glycan antigens and iso-type of the Abs are critical parameters to take into account when measuring anti-glycan Abs. This novel approach in avian influenza research may contribute to the development of a broad spectrum vaccine and improves our mechanistic understanding of innate and adaptive responses to glycans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Screening and characterization of plant cell walls using carbohydrate microarrays.

PubMed

Sørensen, Iben; Willats, William G T

2011-01-01

Plant cells are surrounded by cell walls built largely from complex carbohydrates. The primary walls of growing plant cells consist of interdependent networks of three polysaccharide classes: cellulose, cross-linking glycans (also known as hemicelluloses), and pectins. Cellulose microfibrils are tethered together by cross-linking glycans, and this assembly forms the major load-bearing component of primary walls, which is infiltrated with pectic polymers. In the secondary walls of woody tissues, pectins are much reduced and walls are reinforced with the phenolic polymer lignin. Plant cell walls are essential for plant life and also have numerous industrial applications, ranging from wood to nutraceuticals. Enhancing our knowledge of cell wall biology and the effective use of cell wall materials is dependent to a large extent on being able to analyse their fine structures. We have developed a suite of techniques based on microarrays probed with monoclonal antibodies with specificity for cell wall components, and here we present practical protocols for this type of analysis.

Glycosyltransferase gene expression identifies a poor prognostic colorectal cancer subtype associated with mismatch repair deficiency and incomplete glycan synthesis.

PubMed

Noda, Masaru; Okayama, Hirokazu; Tachibana, Kazunoshin; Sakamoto, Wataru; Saito, Katsuharu; Thar Min, Aung Kyi; Ashizawa, Mai; Nakajima, Takahiro; Aoto, Keita; Momma, Tomoyuki; Katakura, Kyoko; Ohki, Shinji; Kono, Koji

2018-05-29

We aimed to discover glycosyltransferase gene (glycogene)-derived molecular subtypes of colorectal cancer (CRC) associated with patient outcomes. Transcriptomic and epigenomic datasets of non-tumor, pre-cancerous, cancerous tissues and cell lines with somatic mutations, mismatch repair status, clinicopathological and survival information, were assembled (n=4223) and glycogene profiles were analyzed. Immunohistochemistry for a glycogene, GALNT6, was conducted in adenoma and carcinoma specimens (n=403). The functional role and cell surface glycan profiles were further investigated by in vitro loss-of-function assays and lectin microarray analysis. We initially developed and validated a 15-glycogene signature that can identify a poor-prognostic subtype, which closely related to deficient mismatch repair (dMMR) and GALNT6 downregulation. The association of decreased GALNT6 with dMMR was confirmed in multiple datasets of tumors and cell lines, and was further recapitulated by immunohistochemistry, where approximately 15% tumors exhibited loss of GALNT6 protein. GALNT6 mRNA and protein was expressed in premalignant/preinvasive lesions but was subsequently downregulated in a subset of carcinomas, possibly through epigenetic silencing. Decreased GALNT6 was independently associated with poor prognosis in the immunohistochemistry cohort and an additional microarray meta-cohort, by multivariate analyses, and its discriminative power of survival was particularly remarkable in stage III patients. GALNT6 silencing in SW480 cells promoted invasion, migration, chemoresistance and increased cell surface expression of a cancer-associated truncated O-glycan, Tn-antigen. The 15-glycogene signature and the expression levels of GALNT6 mRNA and protein each serve as a novel prognostic biomarker, highlighting the role of dysregulated glycogenes in cancer-associated glycan synthesis and poor prognosis. Copyright ©2018, American Association for Cancer Research.

Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Rui; McBride, Ryan; Nycholat, Corwin M.

2012-02-13

Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only formore » {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.« less

Hexose rearrangements upon fragmentation of N-glycopeptides and reductively aminated N-glycans.

PubMed

Wuhrer, Manfred; Koeleman, Carolien A M; Deelder, André M

2009-06-01

Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescently tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.

Microchip assays for screening monoclonal antibody product quality.

PubMed

Chen, Xiaoyu; Tang, Kaiyan; Lee, Maximilian; Flynn, Gregory C

2008-12-01

Microchip CE-SDS was evaluated as a high-throughput alternative to conventional CE-SDS for monitoring monoclonal antibody protein quality. A commercial instrument (LabChip) 90) was used to separate dodecyl sulfate coated proteins through a sieving polymer based on the proteins' sizes. Under reducing conditions, the microchip CE-SDS separation was similar to that of conventional CE-SDS, providing reasonable resolution of the non-glycosylated and the glycosylated heavy chains. The fluorescence detection on LabChip 90 using non-covalent fluorescent labeling method was about as sensitive as the 220 nm UV detection used in a conventional CE instrument. A simple glycan typing assay was developed for the reducing microchip CE-SDS format. Antibodies, either pure or in crude cell culture media are treated with Endoglycosidase H, which specifically cleaves the hybrid and high mannose type glycans. A heavy chain migration shift on reducing CE-SDS resulting from the loss of glycan is used to measure the level of high mannose/hybrid type glycans as a percentage of the total glycans. Microchip CE-SDS, under both non-reducing and reducing conditions, can be used in a variety of antibody product screening assays. The microchip analyses provide sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s versus 50 min per sample) than conventional CE separation under typical operational conditions.

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

NASA Astrophysics Data System (ADS)

Shah, Bhavana; Jiang, Xinzhao Grace; Chen, Louise; Zhang, Zhongqi

2014-06-01

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

PubMed

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans.

PubMed

Schmidt, Deborah; Schuhmacher, Frank; Geissner, Andreas; Seeberger, Peter H; Pfrengle, Fabian

2015-04-07

Monoclonal antibodies that recognize plant cell wall glycans are used for high-resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid-phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2-naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid-phase synthesis. Conjugation-ready oligosaccharides were obtained and the binding specificities of xylan-directed antibodies were determined on microarrays. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

PubMed

Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

2016-07-15

Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Direct labeling of serum proteins by fluorescent dye for antibody microarray.

PubMed

Klimushina, M V; Gumanova, N G; Metelskaya, V A

2017-05-06

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum. Copyright © 2017 Elsevier Inc. All rights reserved.

Parallel, confocal, and complete spectrum imager for fluorescent detection of high-density microarray

NASA Astrophysics Data System (ADS)

Bogdanov, Valery L.; Boyce-Jacino, Michael

1999-05-01

Confined arrays of biochemical probes deposited on a solid support surface (analytical microarray or 'chip') provide an opportunity to analysis multiple reactions simultaneously. Microarrays are increasingly used in genetics, medicine and environment scanning as research and analytical instruments. A power of microarray technology comes from its parallelism which grows with array miniaturization, minimization of reagent volume per reaction site and reaction multiplexing. An optical detector of microarray signals should combine high sensitivity, spatial and spectral resolution. Additionally, low-cost and a high processing rate are needed to transfer microarray technology into biomedical practice. We designed an imager that provides confocal and complete spectrum detection of entire fluorescently-labeled microarray in parallel. Imager uses microlens array, non-slit spectral decomposer, and high- sensitive detector (cooled CCD). Two imaging channels provide a simultaneous detection of localization, integrated and spectral intensities for each reaction site in microarray. A dimensional matching between microarray and imager's optics eliminates all in moving parts in instrumentation, enabling highly informative, fast and low-cost microarray detection. We report theory of confocal hyperspectral imaging with microlenses array and experimental data for implementation of developed imager to detect fluorescently labeled microarray with a density approximately 103 sites per cm2.

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

USDA-ARS?s Scientific Manuscript database

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal and polyclonal anti...

Live-cell MRI with xenon hyper-CEST biosensors targeted to metabolically labeled cell-surface glycans.

PubMed

Witte, Christopher; Martos, Vera; Rose, Honor May; Reinke, Stefan; Klippel, Stefan; Schröder, Leif; Hackenberger, Christian P R

2015-02-23

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper-CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper-CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live-cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell-surface glycans at nanomolar concentrations. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells.

PubMed

Sun, Qing; Zhao, Lixiang; Song, Qingqing; Wang, Zheng; Qiu, Xusheng; Zhang, Wenjun; Zhao, Mingjun; Zhao, Guo; Liu, Wenbo; Liu, Haiyan; Li, Yunsen; Liu, Xiufan

2012-03-01

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

Polydiacetylene liposomes with phenylboronic acid tags: a fluorescence turn-on sensor for sialic acid detection and cell-surface glycan imaging.

PubMed

Wang, Dong-En; Yan, Jiahang; Jiang, Jingjing; Liu, Xiang; Tian, Chang; Xu, Juan; Yuan, Mao-Sen; Han, Xiang; Wang, Jinyi

2018-03-01

Sialic acid (SA) located at the terminal end of glycans on cell membranes has been shown to play an important yet distinctive role in various biological and pathological processes. Effective methods for the facile, sensitive and in situ analysis of SA on living cell surfaces are of great significance in terms of clinical diagnostics and therapeutics. Here, a new polydiacetylene (PDA) liposome-based sensor system bearing phenylboronic acid (PBA) and 1,8-naphthalimide derived fluorophore moieties was developed as a fluorescence turn-on sensor for the detection of free SA in aqueous solution and the in situ imaging of SA-terminated glycans on living cell surfaces. In the sensor system, three diacetylene monomers, PCDA-pBA, PCDA-Nap and PCDA-EA, were designed and synthesized to construct the composite PDA liposome sensor. The monomer PCDA-pBA modified with PBA molecules was employed as a receptor for SA recognition, while the monomer PCDA-Nap containing a 1,8-naphthalimide derivative fluorophore was used for fluorescence signaling. When the composite PDA liposomes were formed, the energy transfer between the fluorophore and the conjugated backbone could directly quench the fluorescence of the fluorophore. In the presence of additional SA or SA abundant cells, the strong binding of SA with PBA moieties disturbed the pendent side chain conformation, resulting in the fluorescence restoration of the fluorophore. The proposed methods realized the fluorescence turn-on detection of free SA in aqueous solution and the in situ imaging of SA on living MCF-7 cell surfaces. This work provides a new potential tool for simple and selective analysis of SA on living cell membranes.

Comparative glycan profiling of Ceratopteris richardii ‘C-Fern’ gametophytes and sporophytes links cell-wall composition to functional specialization

PubMed Central

Eeckhout, Sharon; Leroux, Olivier; Willats, William G. T.; Popper, Zoë A.; Viane, Ronald L. L.

2014-01-01

Background and Aims Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii ‘C-Fern’, a widely used model system for ferns. Methods Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of ‘C-Fern’ sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. Key Results While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. Conclusions The differences and similarities in glycan cell-wall composition between ‘C-Fern’ gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte. PMID:24699895

Sequential glycan profiling at single cell level with the microfluidic lab-in-a-trench platform: a new era in experimental cell biology.

PubMed

O'Connell, Tríona M; King, Damien; Dixit, Chandra K; O'Connor, Brendan; Walls, Dermot; Ducrée, Jens

2014-09-21

It is now widely recognised that the earliest changes that occur on a cell when it is stressed or becoming diseased are alterations in its surface glycosylation. Current state-of-the-art technologies in glycoanalysis include mass spectrometry, protein microarray formats, techniques in cytometry and more recently, glyco-quantitative polymerase chain reaction (Glyco-qPCR). Techniques for the glycoprofiling of the surfaces of single cells are either limited to the analysis of large cell populations or are unable to handle multiple and/or sequential probing. Here, we report a novel approach of single live cell glycoprofiling enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform for performing capture and retention of cells, along with shear-free reagent loading and washing. The significant technical improvement on state-of-the-art is the demonstration of consecutive, spatio-temporally profiling of glycans on a single cell by sequential elution of the previous lectin probe using their corresponding free sugar. We have qualitatively analysed glycan density on the surface of individual cells. This has allowed us to qualitatively co-localise the observed glycans. This approach enables exhaustive glycoprofiling and glycan mapping on the surface of individual live cells with multiple lectins. The possibility of sequentially profiling glycans on cells will be a powerful new tool to add to current glycoanalytical techniques. The LiaT platform will enable cell biologists to perform many high sensitivity assays and also will also make a significant impact on biomarker research.

Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis

PubMed Central

Abbott, Karen L.; Lim, Jae-Min; Wells, Lance; Benigno, Benedict B.; McDonald, John F.; Pierce, Michael

2016-01-01

Epithelial ovarian cancer is diagnosed less than 25% of the time when the cancer is confined to the ovary, leading to 5-year survival rates of less than 30%. Therefore, there is an urgent need for early diagnostics for ovarian cancer. Our study using glycotranscriptome comparative analysis of endometrioid ovarian cancer tissue and normal ovarian tissue led to the identification of distinct differences in the transcripts of a restricted set of glycosyltransferases involved in N-linked glycosylation. Utilizing lectins that bind to glycan structures predicted to show changes, we observed differences in lectin-bound glycoproteins consistent with some of the transcript differences. In this study, we have extended our observations by the use of selected lectins to perform a targeted glycoproteomic analysis of ovarian cancer and normal ovarian tissues. Our results have identified several glycoproteins that display tumor-specific glycosylation changes. We have verified these glycosylation changes on glycoproteins from tissue using immunoprecipitation followed by lectin blot detection. The glycoproteins that were verified were then analyzed further using existing microarray data obtained from benign ovarian adenomas, borderline ovarian adenocarcinomas, and malignant ovarian adenocarcinomas. The verified glycoproteins found to be expressed above control levels in the microarray data sets were then screened for tumor-specific glycan modifications in serum from ovarian cancer patients. Results obtained from two of these glycoprotein markers, periostin and thrombospondin, have confirmed that tumor-specific glycan changes can be used to distinguish ovarian cancer patient serum from normal serum. PMID:19953551

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses.

PubMed

Okamatsu, Masatoshi; Feng, Fei; Ohyanagi, Tatsuya; Nagahori, Noriko; Someya, Kazuhiko; Sakoda, Yoshihiro; Miura, Nobuaki; Nishimura, Shin-Ichiro; Kida, Hiroshi

2013-02-01

Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in α-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with α-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus. Copyright © 2012 Elsevier B.V. All rights reserved.

Solid-phase reductive amination for glycomic analysis.

PubMed

Jiang, Kuan; Zhu, He; Xiao, Cong; Liu, Ding; Edmunds, Garrett; Wen, Liuqing; Ma, Cheng; Li, Jing; Wang, Peng George

2017-04-15

Reductive amination is an indispensable method for glycomic analysis, as it tremendously facilitates glycan characterization and quantification by coupling functional tags at the reducing ends of glycans. However, traditional in-solution derivatization based approach for the preparation of reductively aminated glycans is quite tedious and time-consuming. Here, a simpler and more efficient strategy termed solid-phase reductive amination was investigated. The general concept underlying this new approach is to streamline glycan extraction, derivatization, and purification on non-porous graphitized carbon sorbents. Neutral and sialylated standard glycans were utilized to test the feasibility of the solid-phase method. As results, almost complete labeling of those glycans with four common labels of aniline, 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA) and 2-amino-N-(2-aminoethyl)-benzamide (AEAB) was obtained, and negligible desialylation occurred during sample preparation. The labeled glycans derived from glycoproteins showed excellent reproducibility in high performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Direct comparisons based on fluorescent absorbance and relative quantification using isotopic labeling demonstrated that the solid-phase strategy enabled 20-30% increase in sample recovery. In short, the solid-phase strategy is simple, reproducible, efficient, and sensitive for glycan analysis. This method was also successfully applied for N-glycan profiling of HEK 293 cells with MALDI-TOF MS, showing its attractive application in the high-throughput analysis of mammalian glycome. Published by Elsevier B.V.

Determination of N-glycans by high performance liquid chromatography using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as the glycosylamine labeling reagent.

PubMed

Wu, Yike; Sha, Qiuyue; Du, Juan; Wang, Chang; Zhang, Liang; Liu, Bi-Feng; Lin, Yawei; Liu, Xin

2018-02-02

Robust, efficient identification and accurate quantification of N-glycans are of great significance in N-glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N-glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N-glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N-glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N-glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparative glycan profiling of Ceratopteris richardii 'C-Fern' gametophytes and sporophytes links cell-wall composition to functional specialization.

PubMed

Eeckhout, Sharon; Leroux, Olivier; Willats, William G T; Popper, Zoë A; Viane, Ronald L L

2014-10-01

Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii 'C-Fern', a widely used model system for ferns. Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of 'C-Fern' sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. The differences and similarities in glycan cell-wall composition between 'C-Fern' gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte. © The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Multiplexing N-glycan analysis by DNA analyzer.

PubMed

Feng, Hua-Tao; Li, Pingjing; Rui, Guo; Stray, James; Khan, Shaheer; Chen, Shiaw-Min; Li, Sam F Y

2017-07-01

Analysis of N-glycan structures has been gaining attentions over the years due to their critical importance to biopharma-based applications and growing roles in biological research. Glycan profiling is also critical to the development of biosimilar drugs. The detailed characterization of N-glycosylation is mandatory because it is a nontemplate driven process and that significantly influences critical properties such as bio-safety and bio-activity. The ability to comprehensively characterize highly complex mixtures of N-glycans has been analytically challenging and stimulating because of the difficulties in both the structure complexity and time-consuming sample pretreatment procedures. CE-LIF is one of the typical techniques for N-glycan analysis due to its high separation efficiency. In this paper, a 16-capillary DNA analyzer was coupled with a magnetic bead glycan purification method to accelerate the sample preparation procedure and therefore increase N-glycan assay throughput. Routinely, the labeling dye used for CE-LIF is 8-aminopyrene-1,3,6-trisulfonic acid, while the typical identification method involves matching migration times with database entries. Two new fluorescent dyes were used to either cross-validate and increase the glycan identification precision or simplify sample preparation steps. Exoglycosidase studies were carried out using neuramididase, galactosidase, and fucosidase to confirm the results of three dye cross-validation. The optimized method combines the parallel separation capacity of multiple-capillary separation with three labeling dyes, magnetic bead assisted preparation, and exoglycosidase treatment to allow rapid and accurate analysis of N-glycans. These new methods provided enough useful structural information to permit N-glycan structure elucidation with only one sample injection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of a Conserved Glycan Signature for Microvesicles

PubMed Central

Batista, Bianca S.; Eng, William S.; Pilobello, Kanoelani T.; Hendricks-Muñoz, Karen D.; Mahal, Lara K.

2011-01-01

Microvesicles (exosomes) are important mediators of intercellular communication, playing a role in immune regulation, cancer progression and the spread of infectious agents. The biological functions of these small vesicles are dependent upon their composition, which is regulated by mechanisms that are not well understood. Although numerous proteomic studies of these particles exist, little is known about their glycosylation. Carbohydrates are involved in protein trafficking and cellular recognition. Glycomic analysis may thus provide valuable insights into microvesicle biology. In this study, we analyzed glycosylation patterns of microvesicles derived from a variety of biological sources using lectin microarray technology. Comparison of the microvesicle glycomes with their parent cell membranes revealed both enrichment and depletion of specific glycan epitopes in these particles. These include enrichment in high mannose, polylactosamine, α-2,6 sialic acid, and complex N-linked glycans and exclusion of terminal blood group A and B antigens. The polylactosamine signature derives from distinct glycoprotein cohorts in microvesicles of different origins. Taken together our data point to the emergence of microvesicles from a specific membrane microdomain, implying a role for glycosylation in microvesicle protein sorting. PMID:21859146

Shrink-induced silica multiscale structures for enhanced fluorescence from DNA microarrays.

PubMed

Sharma, Himanshu; Wood, Jennifer B; Lin, Sophia; Corn, Robert M; Khine, Michelle

2014-09-23

We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30-45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays.

Shrink-Induced Silica Multiscale Structures for Enhanced Fluorescence from DNA Microarrays

PubMed Central

2015-01-01

We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30–45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays. PMID:25191785

Comparative Profiling of N-Glycans Isolated from Serum Samples of Ovarian Cancer Patients and Analyzed by Microchip Electrophoresis

PubMed Central

Mitra, Indranil; Alley, William R.; Goetz, John A.; Vasseur, Jacqueline A.; Novotny, Milos V.; Jacobson, Stephen C.

2013-01-01

Ovarian cancer is the fifth leading cause of cancer-related mortalities for women in the United States and the most lethal gynecological cancer. Aberrant glycosylation has been linked to several human diseases, including ovarian cancer, and accurate measurement of changes in glycosylation may provide relevant diagnostic and prognostic information. In this work, we used microchip electrophoresis coupled with laser-induced fluorescence detection to determine quantitative differences among the N-glycan profiles of control individuals and late-stage recurrent ovarian cancer patients prior to and after an experimental drug treatment that combined docetaxel and imatinib mesylate. N-Glycans were enzymatically released from 5-μL aliquots of serum samples, labeled with the anionic fluorescent tag, 8-aminopyrene-1,3,6-trisulfonic acid, and analyzed on microfluidic devices. A 22-cm long separation channel, operated at 1250 V/cm, generated analysis times less than 100 s, separation efficiencies up to 8 × 105 plates (3.6 × 106 plates/m), and migration time reproducibilities better than 0.1% relative standard deviation after peak alignment. Principal component analysis (PCA) and analysis of variance (ANOVA) tests showed significant differences between the control and both pre- and post-treatment cancer samples and subtle differences between the pre- and post-treatment cancer samples. Area-under-the curve (AUC) values from receiver operating characteristics (ROC) tests were used to evaluate the diagnostic merit of N-glycan peaks, and specific N-glycan peaks used in combination provided AUCs > 0.90 (highly accurate test) when the control and pre-treatment cancer samples and control and post-treatment samples were compared. PMID:23984816

Alteration of matrix metalloproteinase-3 O-glycan structure as a biomarker for disease activity of rheumatoid arthritis.

PubMed

Takeshita, Masaru; Kuno, Atsushi; Suzuki, Katsuya; Matsuda, Atsushi; Shimazaki, Hiroko; Nakagawa, Tomomi; Otomo, Yuki; Kabe, Yasuaki; Suematsu, Makoto; Narimatsu, Hisashi; Takeuchi, Tsutomu

2016-05-21

Nearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker. Serum was taken from patients with RA (n = 24) whose disease activity was scored using composite measures, and MMP-3 was immunoprecipitated and subjected to lectin microarray analysis. A disease activity index (DAI) based on lectin signal was developed and validated using another cohort (n = 60). Synovial fluid MMP-3 in patients with RA and patients with osteoarthritis (OA) was also analyzed. Intense signals were observed on a sialic acid-binding lectin (Agrocybe cylindracea galectin [ACG]) and O-glycan-binding lectins (Jacalin, Agaricus bisporus agglutinin [ABA], and Amaranthus caudatus agglutinin [ACA]) by applying subnanogram levels of serum MMP-3. ACG, ABA, and ACA revealed differences in MMP-3 quantity, and Jacalin revealed differences in MMP-3 quality. The resultant index, ACG/Jacalin, correlated well with disease activity. Further validation using another cohort confirmed that this index correlated well with several DAIs and their components, and reflected DAI changes following RA treatment, with correlations greater than those for MMP-3 and CRP. Furthermore, MMP-3, which generated a high ACG/Jacalin score, accumulated in synovial fluid of patients with RA but not in that of patients with OA. Sialidase digestion revealed that the difference in quality was derived from O-glycan α-2,6-sialylation. This is the first report of a glycoprotein biomarker using glycan change at a local lesion to assess disease activity in autoimmune diseases. Differences in the degree of serum MMP-3 α-2,6-sialylation may be a useful index for estimating disease activity.

Dual-Responsive Metabolic Precursor and Light-Up AIEgen for Cancer Cell Bio-orthogonal Labeling and Precise Ablation.

PubMed

Hu, Fang; Yuan, Youyong; Wu, Wenbo; Mao, Duo; Liu, Bin

2018-06-05

Metabolic glycoengineering of unnatural glycans with bio-orthogonal chemical groups and a subsequent click reaction with fluorescent probes have been widely used in monitoring various bioprocesses. Herein, we developed a dual-responsive metabolic precursor that could specifically generate unnatural glycans with azide groups on the membrane of targeted cancer cells with high selectivity. Moreover, a water-soluble fluorescent light-up probe with aggregation-induced emission (AIE) was synthesized, which turned its fluorescence on upon a click reaction with azide groups on the cancer cell surface, enabling special cancer cell imaging with low background signal. Furthermore, the probe can generate 1 O 2 upon light irradiation, fulfilling its dual role as an imaging and therapeutic agent for cancer cells. Therefore, the concepts of the cancer-cell-specific metabolic precursor cRGD-S-Ac 3 ManNAz and the AIE light-up probe are promising in bio-orthogonal labeling and cancer-specific imaging and therapy.

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis.

PubMed

Hilliard, Mark; Alley, William R; McManus, Ciara A; Yu, Ying Qing; Hallinan, Sinead; Gebler, John; Rudd, Pauline M

Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.

Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

PubMed

Mehta, Nickita; Porterfield, Mindy; Struwe, Weston B; Heiss, Christian; Azadi, Parastoo; Rudd, Pauline M; Tiemeyer, Michael; Aoki, Kazuhiro

2016-09-02

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

Automatic Identification and Quantification of Extra-Well Fluorescence in Microarray Images.

PubMed

Rivera, Robert; Wang, Jie; Yu, Xiaobo; Demirkan, Gokhan; Hopper, Marika; Bian, Xiaofang; Tahsin, Tasnia; Magee, D Mitchell; Qiu, Ji; LaBaer, Joshua; Wallstrom, Garrick

2017-11-03

In recent studies involving NAPPA microarrays, extra-well fluorescence is used as a key measure for identifying disease biomarkers because there is evidence to support that it is better correlated with strong antibody responses than statistical analysis involving intraspot intensity. Because this feature is not well quantified by traditional image analysis software, identification and quantification of extra-well fluorescence is performed manually, which is both time-consuming and highly susceptible to variation between raters. A system that could automate this task efficiently and effectively would greatly improve the process of data acquisition in microarray studies, thereby accelerating the discovery of disease biomarkers. In this study, we experimented with different machine learning methods, as well as novel heuristics, for identifying spots exhibiting extra-well fluorescence (rings) in microarray images and assigning each ring a grade of 1-5 based on its intensity and morphology. The sensitivity of our final system for identifying rings was found to be 72% at 99% specificity and 98% at 92% specificity. Our system performs this task significantly faster than a human, while maintaining high performance, and therefore represents a valuable tool for microarray image analysis.

Characterization of kallikrein-related peptidase 4 glycosylations

PubMed Central

Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C.; Simmer, James P.

2012-01-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1–6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. PMID:22243251

Characterization of kallikrein-related peptidase 4 glycosylations.

PubMed

Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

2011-12-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

Profiling and characterization of sialylated N-glycans by 2D-HPLC (HIAX/PGC) with online orbitrap MS/MS and offline MSn.

PubMed

Hanneman, Andrew J S; Strand, James; Huang, Chi-Ting

2014-02-01

Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor-Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization-sequential mass spectrometry (NSI-MS(n)). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (-) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(-) NSI-MS(n) investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Lectin-Array Blotting.

PubMed

Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

2017-09-01

Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Development of a Digital Microarray with Interferometric Reflectance Imaging

NASA Astrophysics Data System (ADS)

Sevenler, Derin

This dissertation describes a new type of molecular assay for nucleic acids and proteins. We call this technique a digital microarray since it is conceptually similar to conventional fluorescence microarrays, yet it performs enumerative ('digital') counting of the number captured molecules. Digital microarrays are approximately 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing (i.e., many different tests on the same sample simultaneously). Digital microarrays use gold nanorods to label the captured target molecules. Each gold nanorod on the array is individually detected based on its light scattering, with an interferometric microscopy technique called SP-IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Digital DNA microarrays may have utility in applications where sequencing is prohibitively expensive or slow. As an example, we describe a digital microarray assay for gene expression markers of bacterial drug resistance.

Microfluidic extraction and microarray detection of biomarkers from cancer tissue slides

NASA Astrophysics Data System (ADS)

Nguyen, H. T.; Dupont, L. N.; Jean, A. M.; Géhin, T.; Chevolot, Y.; Laurenceau, E.; Gijs, M. A. M.

2018-03-01

We report here a new microfluidic method allowing for the quantification of human epidermal growth factor receptor 2 (HER2) expression levels from formalin-fixed breast cancer tissues. After partial extraction of proteins from the tissue slide, the extract is routed to an antibody (Ab) microarray for HER2 titration by fluorescence. Then the HER2-expressing cell area is evaluated by immunofluorescence (IF) staining of the tissue slide and used to normalize the fluorescent HER2 signal measured from the Ab microarray. The number of HER2 gene copies measured by fluorescence in situ hybridization (FISH) on an adjacent tissue slide is concordant with the normalized HER2 expression signal. This work is the first study implementing biomarker extraction and detection from cancer tissue slides using microfluidics in combination with a microarray system, paving the way for further developments towards multiplex and precise quantification of cancer biomarkers.

Development of Fluorescent Polymerization-based Signal Amplification for Sensitive and Non-enzymatic Biodetection in Antibody Microarrays

PubMed Central

Avens, Heather J.; Bowman, Christopher N.

2009-01-01

Antibody microarrays are a critical tool for proteomics, requiring broad, highly sensitive detection of numerous low abundance biomarkers. Fluorescent polymerization-based amplification (FPBA) is presented as a novel, non-enzymatic signal amplification method that takes advantage of the chain-reaction nature of radical polymerization to achieve a highly amplified fluorescent response. A streptavidin-eosin conjugate localizes eosin photoinitiators for polymerization on the chip where biotinylated target protein is bound. The chip is contacted with acrylamide as a monomer, N-methyldiethanolamine as a coinitiator and yellow/green fluorescent nanoparticles (NPs) which, upon initiation, combine to form a macroscopically visible and highly fluorescent film. The rapid polymerization kinetics and the presence of cross-linker favor entrapment of the fluorescent NPs in the polymer, enabling highly sensitive fluorescent biodetection. This method is demonstrated as being appropriate for antibody microarrays and is compared to detection approaches which utilize streptavidin-FITC (SA-FITC) and streptavidin-labeled yellow/green NPs (SA-NPs). It is found that FPBA is able to detect 0.16 (+/− 0.01) biotin-antibody/µm2 (or 40 zeptomole surface-bound target molecules), while SA-FITC has a limit of detection of 31 (+/− 1) biotin-antibody/µm2 and SA-NPs fail to achieve any significant signal under the conditions evaluated here. Further, FPBA in conjunction with fluorescent stereomicroscopy yields equal or better sensitivity compared to fluorescent detection of SA-eosin using a much more costly microarray scanner. By facilitating highly sensitive detection, FPBA is expected to enable detection of low abundance antigens and also make possible a transition towards less expensive fluorescence detection instrumentation. PMID:19508906

Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics.

PubMed

Miura, Yuri; Hashii, Noritaka; Ohta, Yuki; Itakura, Yoko; Tsumoto, Hiroki; Suzuki, Junya; Takakura, Daisuke; Abe, Yukiko; Arai, Yasumichi; Toyoda, Masashi; Kawasaki, Nana; Hirose, Nobuyoshi; Endo, Tamao

2018-06-01

Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. Plasma proteins from Japanese semi-supercentenarians (SSCs, 106-109 years), aged controls (70-88 years), and young controls (20-38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. We found abundant glycans in SSCs, which may be associated with human longevity. Copyright © 2018 Elsevier B.V. All rights reserved.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

PubMed Central

Shewell, Lucy K.; Harvey, Richard M.; Higgins, Melanie A.; Day, Christopher J.; Hartley-Tassell, Lauren E.; Chen, Austen Y.; Gillen, Christine M.; James, David B. A.; Alonzo, Francis; Torres, Victor J.; Walker, Mark J.; Paton, Adrienne W.; Paton, James C.; Jennings, Michael P.

2014-01-01

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a Kd of 1.88 × 10−5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism. PMID:25422425

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

PubMed

Shewell, Lucy K; Harvey, Richard M; Higgins, Melanie A; Day, Christopher J; Hartley-Tassell, Lauren E; Chen, Austen Y; Gillen, Christine M; James, David B A; Alonzo, Francis; Torres, Victor J; Walker, Mark J; Paton, Adrienne W; Paton, James C; Jennings, Michael P

2014-12-09

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Analysis of nonhuman N-glycans as the minor constituents in recombinant monoclonal antibody pharmaceuticals.

PubMed

Maeda, Eiki; Kita, Soichiro; Kinoshita, Mitsuhiro; Urakami, Koji; Hayakawa, Takao; Kakehi, Kazuaki

2012-03-06

Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk-benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.

Multiple N-glycans cooperate in the subcellular targeting and functioning of Arabidopsis KORRIGAN1.

PubMed

Rips, Stephan; Bentley, Nolan; Jeong, In Sil; Welch, Justin L; von Schaewen, Antje; Koiwa, Hisashi

2014-09-01

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-β1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells. © 2014 American Society of Plant Biologists. All rights reserved.

N-glycan profiles in H9N2 avian influenza viruses from chicken eggs and human embryonic lung fibroblast cells.

PubMed

Chen, Wentian; Zhong, Yaogang; Su, Rui; Qi, Huicai; Deng, Weina; Sun, Yu; Ma, Tianran; Wang, Xilong; Yu, Hanjie; Wang, Xiurong; Li, Zheng

2017-11-01

N-glycosylation can affect the host specificity, virulence and infectivity of influenza A viruses (IAVs). In this study, the distribution and evolution of N-glycosylation sites in the hemagglutinin (HA) and neuraminidase (NA) of H9N2 virus were explored using phylogenetic analysis. Then, one strain of the H9N2 subtypes was proliferated in the embryonated chicken eggs (ECE) and human embryonic lung fibroblast cells (MRC-5) system. The proliferated viral N-glycan profiles were analyzed by a glycomic method that combined the lectin microarray and MALDI-TOF/TOF-MS. As a result, HA and NA of H9N2 viruses prossess six and five highly conserved N-glycosylation sites, respectively. Sixteen lectins (e.g., MAL-II, SNA and UEA-I) had increased expression levels of the glycan structures in the MRC-5 compared with the ECE system; however, 6 lectins (e.g., PHA-E, PSA and DSA) had contrasting results. Eleven glycans from the ECE system and 13 glycans from the MRC-5 system were identified. Our results showed that the Fucα-1,6GlcNAc(core fucose) structure was increased, and pentaantennary N-glycans were only observed in the ECE system. The SAα2-3/6Gal structures were highly expressed and Fucα1-2Galβ1-4GlcNAc structures were only observed in the MRC-5 system. We conclude that the existing SAα2-3/6Gal sialoglycans make the offspring of the H9N2 virus prefer entially attach to each other, which decreases the virulence. Alterations in the glycosylation sites for the evolution and role of IAVs have been widely described; however, little is known about the exact glycan structures for the same influenza strain from different hosts. Our findings may provide a novel way for further discussing the molecular mechanism of the viral transmission and virulence associated with viral glycosylation in avian and human hosts as well as vital information for designing a vaccine against influenza and other human viruses. Copyright © 2017. Published by Elsevier B.V.

Exploiting fluorescence for multiplex immunoassays on protein microarrays

NASA Astrophysics Data System (ADS)

Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

2014-09-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Natural antibodies to glycans.

PubMed

Bovin, N V

2013-07-01

A wide variety of so-called natural antibodies (nAbs), i.e. immunoglobulins generated by B-1 cells, are directed to glycans. nAbs to glycans can be divided in three groups: 1) conservative nAbs, i.e. practically the same in all healthy donors with respect to their epitope specificity and level in blood; 2) allo-antibodies to blood group antigens; 3) plastic antibodies related to the first or the second group but discussed separately because their level changes considerably during diseases and some temporary conditions, in particular inflammation and pregnancy. Antibodies from the third group proved to be prospective markers of a number of diseases, whereas their unusual level (below or above the norm) is not necessarily the consequence of disease/state. Modern microarrays allowed the determination of the human repertoire, which proved to be unexpectedly broad. It was observed that the content of some nAbs reaches about 0.1% of total immunoglobulins. Immunoglobulins of M class dominate for most nAbs, constituting up to 80-90%. Their affinity (to a monovalent glycan, in KD terms) were found to be within the range 10(-4)-10(-6) M. Antibodies to Galβ1-3GlcNAc (Le(C)), 4-HSO3Galβ1-4GalNAc (4'-O-SuLN), Fucα1-3GlcNAc, Fucα1-4GlcNAc, GalNAcα1-3Gal (Adi), Galα1-4Galβ1-4Glc (P(k)), Galα1-4Galβ1-4GlcNAc (P1), GlcNAcα-terminated glycans, and hyaluronic acid should be noted among the nAbs revealed and studied during the last decade. At the same time, a kind of "taboo" is observed for a number of glycans: antibodies to Le(X) and Le(Y), and almost all gangliosides have not been observed in healthy persons. Many of the revealed nAbs were directed to constrained inner (core) part of glycan, directly adjoined to lipid of cell membrane or protein. The biological function of these nAbs remains unclear; for anti-core antibodies, a role of surveillance on appearance of aberrant, especially cancer, antigens is supposed. The first data related to oncodiagnostics based on quantitation of anti-glycan nAbs are reported.

Comparative analysis of monoclonal antibody N-glycosylation using stable isotope labelling and UPLC-fluorescence-MS.

PubMed

Millán Martín, Silvia; Delporte, Cédric; Farrell, Amy; Navas Iglesias, Natalia; McLoughlin, Niaobh; Bones, Jonathan

2015-03-07

A twoplex method using (12)C6 and (13)C6 stable isotope analogues (Δmass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection.

Rational and Computational Design of Stabilized Variants of Cyanovirin-N which Retain Affinity and Specificity for Glycan Ligands

PubMed Central

Patsalo, Vadim; Raleigh, Daniel P.; Green, David F.

2011-01-01

Cyanovirin-N (CVN) is an 11-kDa pseudo-symmetric cyanobacterial lectin that has been shown to inhibit infection by the Human Immunodeficiency Virus (HIV) by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work we describe rationally-designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson–Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 m of GuaHCl against chemical denaturation, relative to a previously-characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations, and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding. PMID:22032696

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes

PubMed Central

Liao, Shih-Fen; Liang, Chi-Hui; Hsu, Tsui-Ling; Tsai, Tsung-I; Hsieh, Yves S.-Y.; Tsai, Chih-Ming; Li, Shiou-Ting; Cheng, Yang-Yu; Tsao, Shu-Ming; Lin, Tung-Yi; Lin, Zong-Yan; Yang, Wen-Bin; Ren, Chien-Tai; Lin, Kuo-I; Khoo, Kay-Hooi; Lin, Chun-Hung; Hsu, Hsien-Yeh; Wu, Chung-Yi; Wong, Chi-Huey

2013-01-01

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans. PMID:23908400

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

PubMed

Liao, Shih-Fen; Liang, Chi-Hui; Ho, Ming-Yi; Hsu, Tsui-Ling; Tsai, Tsung-I; Hsieh, Yves S-Y; Tsai, Chih-Ming; Li, Shiou-Ting; Cheng, Yang-Yu; Tsao, Shu-Ming; Lin, Tung-Yi; Lin, Zong-Yan; Yang, Wen-Bin; Ren, Chien-Tai; Lin, Kuo-I; Khoo, Kay-Hooi; Lin, Chun-Hung; Hsu, Hsien-Yeh; Wu, Chung-Yi; Wong, Chi-Huey

2013-08-20

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Protein structure controls the processing of the N-linked oligosaccharides and glycosylphosphatidylinositol glycans of variant surface glycoproteins expressed in bloodstream form Trypanosoma brucei.

PubMed

Zitzmann, N; Mehlert, A; Carrouée, S; Rudd, P M; Ferguson, M A; Carroué, S

2000-03-01

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are a family of homodimeric glycoproteins that adopt similar shapes. An individual trypanosome expresses one VSG at a time in the form of a dense protective mono-layer on the plasma membrane. VSG genes are expressed from one of several polycistronic transcription units (expression sites) that contain several expression site associated genes. We used a transformed trypanosome clone expressing two different VSGs (VSG121 and VSG221) from the same expression site (that of VSG221) to establish whether the genotype of the trypanosome clone or the VSG structure itself controls VSG N-linked oligosaccharide and GPI anchor glycan processing. In-gel release and fluorescent labeling of N-linked oligosaccharides and on-blot fluorescent labeling and release of GPI anchor glycans were employed to compare the carbohydrate structures of VSG121 and VSG221 when expressed individually in wild-type trypanosome clones and when expressed together in the transformed trypanosome clone. The data indicate that the genotype of the trypanosome clone has no effect on the N-linked oligosaccharide structures present on a given VSG variant and only a minor effect on the GPI anchor glycans. The latter is most likely an effect of changes in inter-VSG packing when two VGSs are expressed simultaneously. Thus, N-linked oligosaccharide and GPI anchor processing enzymes appear to be constitutively expressed in bloodstream form African trypanosomes and the tertiary and quaternary structures of the VSG homodimers appear to dictate the processing and glycoform microheterogeneity of surface-expressed VSGs.

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

PubMed

Mittermayr, Stefan; Albrecht, Simone; Váradi, Csaba; Millán-Martín, Silvia; Bones, Jonathan

2017-01-01

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.

Plasmonically amplified fluorescence bioassay with microarray format

NASA Astrophysics Data System (ADS)

Gogalic, S.; Hageneder, S.; Ctortecka, C.; Bauch, M.; Khan, I.; Preininger, Claudia; Sauer, U.; Dostalek, J.

2015-05-01

Plasmonic amplification of fluorescence signal in bioassays with microarray detection format is reported. A crossed relief diffraction grating was designed to couple an excitation laser beam to surface plasmons at the wavelength overlapping with the absorption and emission bands of fluorophore Dy647 that was used as a label. The surface of periodically corrugated sensor chip was coated with surface plasmon-supporting gold layer and a thin SU8 polymer film carrying epoxy groups. These groups were employed for the covalent immobilization of capture antibodies at arrays of spots. The plasmonic amplification of fluorescence signal on the developed microarray chip was tested by using interleukin 8 sandwich immunoassay. The readout was performed ex situ after drying the chip by using a commercial scanner with high numerical aperture collecting lens. Obtained results reveal the enhancement of fluorescence signal by a factor of 5 when compared to a regular glass chip.

Detection of Differentially Expressed Wound-Healing–Related Glycogenes in Galectin-3–Deficient Mice

PubMed Central

Saravanan, Chandrassegar; Cao, Zhiyi; Head, Steven R.; Panjwani, Noorjahan

2010-01-01

Purpose A prior study showed that exogenous galectin-3 (Gal-3) stimulates re-epithelialization of corneal wounds in wild-type (Gal-3+/+) mice but, surprisingly, not in galectin-3–deficient (Gal-3−/−) mice. In an effort to understand why the injured corneas of Gal-3−/− mice are unresponsive to exogenous Gal-3, the present study was designed to determine whether genes encoding the enzymes that regulate the synthesis of glycan ligands of Gal-3 are differentially expressed in Gal-3−/− corneas compared with the Gal-3+/+ corneas. Methods Glycogene microarray technology was used to identify differentially expressed glycosyltransferases in healing Gal-3+/+ and Gal-3−/− corneas. Results Of ~2000 glycogenes on the array, the expression of 8 was upregulated and that of 14 was downregulated more than 1.3-fold in healing Gal-3−/− corneas. A galactosyltransferase, β3GalT5, which has the ability to synthesize Gal-3 ligands was markedly downregulated in healing Gal-3−/− corneas. The genes for polypeptide galactosaminyltransferases (ppGalNAcT-3 and -7) that are known to initiate O-linked glycosylation and N-aspartyl-β-glucosaminidase, which participates in the removal of N-glycans, were found to be upregulated in healing Gal-3−/− corneas. Microarray data were validated by qRT-PCR. Conclusions Based on the known functions of the differentially expressed glycogenes, it appears that the glycan structures on glycoproteins and glycolipids, synthesized as a result of the differential glycogene expression pattern in healing Gal-3−/− corneas may lead to the downregulation of specific counterreceptors for Gal-3. This may explain, at least in part, why, unlike healing Gal-3+/+ corneas, the healing Gal-3−/− corneas are unresponsive to the stimulatory effect of exogenous Gal-3 on re-epithelialization of corneal wounds. PMID:19643959

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

PubMed

Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2017-02-10

Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

Comparison of RP-HPLC modes to analyse the N-glycome of the free-living nematode Pristionchus pacificus

PubMed Central

Yan, Shi; Wilson, Iain B. H.; Paschinger, Katharina

2015-01-01

Pristionchus pacificus is a free-living nematode increasingly used as an organism for comparison to the more familiar model Caenorhabditis elegans. In this study, we examined the N-glycans of this organism isolated after serial release with peptide:N-glycosidases F and A; after fluorescent labelling with 2-aminopyridine, chromatographic fractionation by three types of reversed-phase HPLC (with either classical C18, fused core C18 or alkylamide bonded phases) followed by mass spectrometric analyses revealed key features of its N-glycome. In addition to paucimannosidic and oligomannosidic glycans typical of invertebrates, N-glycans with two core fucose residues were detected. Furthermore, a range of glycans carrying up to three phosphorylcholine residues was observed whereas, unlike C. elegans, no tetrafucosylated N-glycans were detected. Structures with three fucose residues, unusual methylation of core α1,3-fucose or with galactosylated fucose motifs were found in low amounts; these features may correlate with a different ensemble or expression of glycosyltransferase genes as compared to C. elegans. From an analytical perspective, both the alkylamide RP-amide and fused core C18 columns, as compared to a classical C18 material, offer advantages in terms of resolution and of elution properties, as some minor pyridylamino-labelled glycans (e.g., those carrying phosphorylcholine) appear in earlier fractions and so potential losses of such structures due to insufficient gradient length can be avoided. PMID:25639343

Development and application of a robust N-glycan profiling method for heightened characterization of monoclonal antibodies and related glycoproteins.

PubMed

Shang, Tanya Q; Saati, Andrew; Toler, Kelly N; Mo, Jianming; Li, Heyi; Matlosz, Tonya; Lin, Xi; Schenk, Jennifer; Ng, Chee-Keng; Duffy, Toni; Porter, Thomas J; Rouse, Jason C

2014-07-01

A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed characterization of a marketed therapeutic mAb, Rituxan(®), is described. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Reusable conductimetric array of interdigitated microelectrodes for the readout of low-density microarrays.

PubMed

Mallén, Maria; Díaz-González, María; Bonilla, Diana; Salvador, Juan P; Marco, María P; Baldi, Antoni; Fernández-Sánchez, César

2014-06-17

Low-density protein microarrays are emerging tools in diagnostics whose deployment could be primarily limited by the cost of fluorescence detection schemes. This paper describes an electrical readout system of microarrays comprising an array of gold interdigitated microelectrodes and an array of polydimethylsiloxane microwells, which enabled multiplexed detection of up to thirty six biological events on the same substrate. Similarly to fluorescent readout counterparts, the microarray can be developed on disposable glass slide substrates. However, unlike them, the presented approach is compact and requires a simple and inexpensive instrumentation. The system makes use of urease labeled affinity reagents for developing the microarrays and is based on detection of conductivity changes taking place when ionic species are generated in solution due to the catalytic hydrolysis of urea. The use of a polydimethylsiloxane microwell array facilitates the positioning of the measurement solution on every spot of the microarray. Also, it ensures the liquid tightness and isolation from the surrounding ones during the microarray readout process, thereby avoiding evaporation and chemical cross-talk effects that were shown to affect the sensitivity and reliability of the system. The performance of the system is demonstrated by carrying out the readout of a microarray for boldenone anabolic androgenic steroid hormone. Analytical results are comparable to those obtained by fluorescent scanner detection approaches. The estimated detection limit is 4.0 ng mL(-1), this being below the threshold value set by the World Anti-Doping Agency and the European Community. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of a low-cost detection method for miRNA microarray.

PubMed

Li, Wei; Zhao, Botao; Jin, Youxin; Ruan, Kangcheng

2010-04-01

MicroRNA (miRNA) microarray is a powerful tool to explore the expression profiling of miRNA. The current detection method used in miRNA microarray is mainly fluorescence based, which usually requires costly detection system such as laser confocal scanner of tens of thousands of dollars. Recently, we developed a low-cost yet sensitive detection method for miRNA microarray based on enzyme-linked assay. In this approach, the biotinylated miRNAs were captured by the corresponding oligonucleotide probes immobilized on microarray slide; and then the biotinylated miRNAs would capture streptavidin-conjugated alkaline phosphatase. A purple-black precipitation on each biotinylated miRNA spot was produced by the enzyme catalytic reaction. It could be easily detected by a charge-coupled device digital camera mounted on a microscope, which lowers the detection cost more than 100 fold compared with that of fluorescence method. Our data showed that signal intensity of the spot correlates well with the biotinylated miRNA concentration and the detection limit for miRNAs is at least 0.4 fmol and the detection dynamic range spans about 2.5 orders of magnitude, which is comparable to that of fluorescence method.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

PubMed

Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

2017-07-05

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation.

PubMed

Balog, Crina I A; Stavenhagen, Kathrin; Fung, Wesley L J; Koeleman, Carolien A; McDonnell, Liam A; Verhoeven, Aswin; Mesker, Wilma E; Tollenaar, Rob A E M; Deelder, André M; Wuhrer, Manfred

2012-09-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ~1 million cases and an annual mortality rate of ~655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers.

N-glycosylation of Colorectal Cancer Tissues

PubMed Central

Balog, Crina I. A.; Stavenhagen, Kathrin; Fung, Wesley L. J.; Koeleman, Carolien A.; McDonnell, Liam A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

2012-01-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ∼1 million cases and an annual mortality rate of ∼655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers. PMID:22573871

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

Glycosylation of random IgG distinguishes seropositive and seronegative rheumatoid arthritis.

PubMed

Magorivska, I; Döncző, B; Dumych, T; Karmash, A; Boichuk, M; Hychka, K; Mihalj, M; Szabó, M; Csánky, E; Rech, J; Guttman, A; Vari, S G; Bilyy, R

2018-05-01

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

Structure and receptor binding preferences of recombinant hemagglutinins from avian and human H6 and H10 influenza A virus subtypes.

PubMed

Yang, Hua; Carney, Paul J; Chang, Jessie C; Villanueva, Julie M; Stevens, James

2015-04-01

During 2013, three new avian influenza A virus subtypes, A(H7N9), A(H6N1), and A(H10N8), resulted in human infections. While the A(H7N9) virus resulted in a significant epidemic in China across 19 provinces and municipalities, both A(H6N1) and A(H10N8) viruses resulted in only a few human infections. This study focuses on the major surface glycoprotein hemagglutinins from both of these novel human viruses. The detailed structural and glycan microarray analyses presented here highlight the idea that both A(H6N1) and A(H10N8) virus hemagglutinins retain a strong avian receptor binding preference and thus currently pose a low risk for sustained human infections. Human infections with zoonotic influenza virus subtypes continue to be a great public health concern. We report detailed structural analysis and glycan microarray data for recombinant hemagglutinins from A(H6N1) and A(H10N8) viruses, isolated from human infections in 2013, and compare them with hemagglutinins of avian origin. This is the first structural report of an H6 hemagglutinin, and our results should further the understanding of these viruses and provide useful information to aid in the continuous surveillance of these zoonotic influenza viruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

PubMed Central

Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

2012-01-01

We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway.

PubMed

Geisler, Christoph; Jarvis, Donald L

2012-03-02

Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the net outcome of the insect cell N-glycosylation pathway.

Microarray slide hybridization using fluorescently labeled cDNA.

PubMed

Ares, Manuel

2014-01-01

Microarray hybridization is used to determine the amount and genomic origins of RNA molecules in an experimental sample. Unlabeled probe sequences for each gene or gene region are printed in an array on the surface of a slide, and fluorescently labeled cDNA derived from the RNA target is hybridized to it. This protocol describes a blocking and hybridization protocol for microarray slides. The blocking step is particular to the chemistry of "CodeLink" slides, but it serves to remind us that almost every kind of microarray has a treatment step that occurs after printing but before hybridization. We recommend making sure of the precise treatment necessary for the particular chemistry used in the slides to be hybridized because the attachment chemistries differ significantly. Hybridization is similar to northern or Southern blots, but on a much smaller scale.

Assessing antibody microarrays for space missions: effect of long-term storage, gamma radiation, and temperature shifts on printed and fluorescently labeled antibodies.

PubMed

de Diego-Castilla, Graciela; Cruz-Gil, Patricia; Mateo-Martí, Eva; Fernández-Calvo, Patricia; Rivas, Luis A; Parro, Víctor

2011-10-01

Antibody microarrays are becoming frequently used tools for analytical purposes. A key factor for optimal performance is the stability of the immobilized (capturing) antibodies as well as those that have been fluorescently labeled to achieve the immunological test (tracers). This is especially critical for long-distance transport, field testing, or planetary exploration. A number of different environmental stresses may affect the antibody integrity, such as dryness, sudden temperature shift cycles, or, as in the case of space science, exposure to large quantities of the highly penetrating gamma radiation. Here, we report on the effect of certain stabilizing solutions for long-term storage of printed antibody microarrays under different conditions. We tested the effect of gamma radiation on printed and freeze- or vacuum-dried fluorescent antibodies at working concentrations (tracer antibodies), as well as the effect of multiple cycles of sudden and prolonged temperature shifts on the stability of fluorescently labeled tracer antibody cocktails. Our results show that (i) antibody microarrays are stable at room temperature when printed on stabilizing spotting solutions for at least 6 months, (ii) lyophilized and vacuum-dried fluorescently labeled tracer antibodies are stable for more than 9 months of sudden temperature shift cycles (-20°C to 25°C and 50°C), and (iii) both printed and freeze- or vacuum-dried fluorescent tracer antibodies are stable after several-fold excess of the dose of gamma radiation expected during a mission to Mars. Although different antibodies may exhibit different susceptibilities, we conclude that, in general, antibodies are suitable for use in planetary exploration purposes if they are properly treated and stored with the use of stabilizing substances.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution

PubMed Central

Van Damme, Els J. M.; Nakamura-Tsuruta, Sachiko; Smith, David F.; Ongenaert, Maté; Winter, Harry C.; Rougé, Pierre; Goldstein, Irwin J.; Mo, Hanqing; Kominami, Junko; Culerrier, Raphaël; Barre, Annick; Hirabayashi, Jun; Peumans, Willy J.

2007-01-01

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes. PMID:17288538

Essential role of gastric gland mucin in preventing gastric cancer in mice

PubMed Central

Karasawa, Fumitoshi; Shiota, Akira; Goso, Yukinobu; Kobayashi, Motohiro; Sato, Yoshiko; Masumoto, Junya; Fujiwara, Maiko; Yokosawa, Shuichi; Muraki, Takashi; Miyagawa, Shinichi; Ueda, Masatsugu; Fukuda, Michiko N.; Fukuda, Minoru; Ishihara, Kazuhiko; Nakayama, Jun

2012-01-01

Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt–/– mice to better understand its role in vivo. A4gnt–/– mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt–/– mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation. PMID:22307328

Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells

PubMed Central

Sumiyoshi, Mika; Ricciuto, Jessica; Tisdale, Ann; Gipson, Ilene K.; Mantelli, Flavio; Argüeso, Pablo

2008-01-01

Purpose Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal–limbal epithelial (HCLE) cells. Methods Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-α-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell–cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. Results Benzyl-α-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-α-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. Conclusions These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface–associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye). PMID:18172093

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

PubMed Central

Gulati, Shelly; Smith, David F.; Cummings, Richard D.; Couch, Robert B.; Griesemer, Sara B.; St. George, Kirsten; Webster, Robert G.; Air, Gillian M.

2013-01-01

It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population. PMID:23805213

Microarray-based screening of heat shock protein inhibitors.

PubMed

Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

Waveguide-excited fluorescence microarray

NASA Astrophysics Data System (ADS)

Sagarzazu, Gabriel; Bedu, Mélanie; Martinelli, Lucio; Ha, Khoi-Nguyen; Pelletier, Nicolas; Safarov, Viatcheslav I.; Weisbuch, Claude; Gacoin, Thierry; Benisty, Henri

2008-04-01

Signal-to-noise ratio is a crucial issue in microarray fluorescence read-out. Several strategies are proposed for its improvement. First, light collection in conventional microarrays scanners is quite limited. It was recently shown that almost full collection can be achieved in an integrated lens-free biosensor, with labelled species hybridizing practically on the surface of a sensitive silicon detector [L. Martinelli et al. Appl. Phys. Lett. 91, 083901 (2007)]. However, even with such an improvement, the ultimate goal of real-time measurements during hybridization is challenging: the detector is dazzled by the large fluorescence of labelled species in the solution. In the present paper we show that this unwanted signal can effectively be reduced if the excitation light is confined in a waveguide. Moreover, the concentration of excitation light in a waveguide results in a huge signal gain. In our experiment we realized a structure consisting of a high index sol-gel waveguide deposited on a low-index substrate. The fluorescent molecules deposited on the surface of the waveguide were excited by the evanescent part of a wave travelling in the guide. The comparison with free-space excitation schemes confirms a huge gain (by several orders of magnitude) in favour of waveguide-based excitation. An optical guide deposited onto an integrated biosensor thus combines both advantages of ideal light collection and enhanced surface localized excitation without compromising the imaging properties. Modelling predicts a negligible penalty from spatial cross-talk in practical applications. We believe that such a system would bring microarrays to hitherto unattained sensitivities.

TiO2 Nanolayer-Enhanced Fluorescence for Simultaneous Multiplex Mycotoxin Detection by Aptamer Microarrays on a Porous Silicon Surface.

PubMed

Liu, Rui; Li, Wei; Cai, Tingting; Deng, Yang; Ding, Zhi; Liu, Yan; Zhu, Xuerui; Wang, Xin; Liu, Jie; Liang, Baowen; Zheng, Tiesong; Li, Jianlin

2018-05-02

A new aptamer microarray method on the TiO 2 -porous silicon (PSi) surface was developed to simultaneously screen multiplex mycotoxins. The TiO 2 nanolayer on the surface of PSi can enhance the fluorescence intensity 14 times than that of the thermally oxidized PSi. The aptamer fluorescence signal recovery principle was performed on the TiO 2 -PSi surface by hybridization duplex strand DNA from the mycotoxin aptamer and antiaptamer, respectively, labeled with fluorescence dye and quencher. The aptamer microarray can simultaneously screen for multiplex mycotoxins with a dynamic linear detection range of 0.1-10 ng/mL for ochratoxin A (OTA), 0.01-10 ng/mL for aflatoxins B 1 (AFB 1 ), and 0.001-10 ng/mL for fumonisin B 1 (FB 1 ) and limits of detection of 15.4, 1.48, and 0.21 pg/mL for OTA, AFB 1 , and FB 1 , respectively. The newly developed method shows good specificity and recovery rates. This method can provide a simple, sensitive, and cost-efficient platform for simultaneous screening of multiplex mycotoxins and can be easily expanded to the other aptamer-based protocol.

Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

A Microwell-Printing Fabrication Strategy for the On-Chip Templated Biosynthesis of Protein Microarrays for Surface Plasmon Resonance Imaging

PubMed Central

Manuel, Gerald; Lupták, Andrej; Corn, Robert M.

2017-01-01

A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein. In the second step, the protein microwell array is used to contact print one or more protein microarrays onto nitrilotriacetic acid (NTA)-functionalized gold thin film SPRI chips for real-time SPRI surface bioaffinity adsorption measurements. Even though each microwell array element only contains approximately 40 picomoles of protein, the concentration is sufficiently high for the efficient bioaffinity adsorption and capture of the approximately 100 femtomoles of hexahistidine-tagged protein required to create each SPRI microarray element. As a first example, the protein biosynthesis process is verified with fluorescence imaging measurements of a microwell array containing His-tagged green fluorescent protein (GFP), yellow fluorescent protein (YFP) and mCherry (RFP), and then the fidelity of SPRI chips printed from this protein microwell array is ascertained by measuring the real-time adsorption of various antibodies specific to these three structurally related proteins. This greatly simplified two-step synthesis/printing fabrication methodology eliminates most of the handling, purification and processing steps normally required in the synthesis of multiple protein probes, and enables the rapid fabrication of SPRI protein microarrays from DNA templates for the study of protein-protein bioaffinity interactions. PMID:28706572

Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Proudnikov, D.; Kirillov, E.; Chumakov, K.

2000-01-01

This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements.more » Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.« less

Reverse phase protein microarrays: fluorometric and colorimetric detection.

PubMed

Gallagher, Rosa I; Silvestri, Alessandra; Petricoin, Emanuel F; Liotta, Lance A; Espina, Virginia

2011-01-01

The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.

Mass spectrometry characterization for N-glycosylation of immunoglobulin Y from hen egg yolk.

PubMed

Sheng, Long; He, Zhenjiao; Liu, Yaping; Ma, Meihu; Cai, Zhaoxia

2018-03-01

Immunoglobulin Y (IgY) is a new therapeutic antibody that exists in hen egg yolk. It is a glycoprotein, not much is known about its N-glycan structures, site occupancy and site-specific N-glycosylation. In this study, purified protein from hen egg yolk was identified as IgY based on SDS-PAGE and MALDI-TOF/TOF MS. N-glycan was released from IgY using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase treatment, and the molecular weight of IgY was calculated using the difference between the molecular weight of IgY and deglycosylated IgY. Two potential N-Glycosylation sites (ASN 308 and ASN 409 ) were detected on IgY by nanoLC-ESI MS. Sugar chains were separated using normal phase liquid chromatography after fluorescence labeling, and 17 N-glycan structures were confirmed using ESI-MS. The sugar chain pattern contained high-mannose oligosaccharide, hybrid oligosaccharide and complex oligosaccharide. These results could lead to other important information regarding IgY glycosylation. Copyright © 2017 Elsevier B.V. All rights reserved.

A procedure for Alcian blue staining of mucins on polyvinylidene difluoride membranes.

PubMed

Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

2012-10-16

The isolation and characterization of mucins are critically important for obtaining insight into the molecular pathology of various diseases, including cancers and cystic fibrosis. Recently, we developed a novel membrane electrophoretic method, supported molecular matrix electrophoresis (SMME), which separates mucins on a polyvinylidene difluoride (PVDF) membrane impregnated with a hydrophilic polymer. Alcian blue staining is widely used to visualize mucopolysaccharides and acidic mucins on both blotted membranes and SMME membranes; however, this method cannot be used to stain mucins with a low acidic glycan content. Meanwhile, periodic acid-Schiff staining can selectively visualize glycoproteins, including mucins, but is incompatible with glycan analysis, which is indispensable for mucin characterizations. Here we describe a novel staining method, designated succinylation-Alcian blue staining, for visualizing mucins on a PVDF membrane. This method can visualize mucins regardless of the acidic residue content and shows a sensitivity 2-fold higher than that of Pro-Q Emerald 488, a fluorescent periodate Schiff-base stain. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan analysis using porcine gastric mucin as a model mucin.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

PubMed Central

Scheler, Ott; Glynn, Barry; Parkel, Sven; Palta, Priit; Toome, Kadri; Kaplinski, Lauris; Remm, Maido; Maher, Majella; Kurg, Ants

2009-01-01

Background Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Results Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. Conclusion We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology. PMID:19445684

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels.

PubMed

Sevenler, Derin; Daaboul, George G; Ekiz Kanik, Fulya; Ünlü, Neşe Lortlar; Ünlü, M Selim

2018-05-21

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique's simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.

Evidence for an imidazoline by-product from glycans using tandem mass spectrometry.

PubMed

Duff, Robert J; Smith, Elaine; Li, Wenzhou; Fodor, Szilan

2017-06-09

Herein is reported the separation and identification of a previously unknown imidazoline by-product originating from the fluorescent labeling procedure when applied to enzymatically released N-linked glycans of a human IgG1. The imidazoline by-product was generated via the reductive amination procedure with either sodium cyanoborohydride or 2-picoline borane. Using ultra performance liquid chromatography (UPLC) in conjunction with hydrophilic interaction-based chromatography (HILIC), the 2-aminobenzoic acid (2-AA)-labeled glycans were well-resolved from imidazoline by-products to facilitate direct identification utilizing electrospray ionization mass spectrometry (ESI-MS) with fragmentation. It was found that this minor species (∼2%) was 18.0105u less than the neighboring peak GlcNAc 2 Man 3 GlcNAc 2 Fuc peak, abbreviated as A2G0F at 1582.5899u. While this mass loss corresponds to the mass of a water molecule, the molecular location of loss of water was not straightforward in consideration of the biantennary A2G0F structure. Model studies were carried out using A2G0F standard and N-acetyllactosamine to identify the impurity as an imidazoline ring structure located at the reducing end of the glycan as confirmed by high resolution mass fragment ions. Imidazoline content decreased when the reductant concentration was increased. To conclude, evidence for the imidazoline structure was accomplished through high resolution, high accuracy mass spectrometry (HRAM), and experiments showing chemical susceptibility and isotopically labeled tracers. This study is the first to identify these minor species which likely impact all N-acetylglucosamine-type N-linked glycans from biologics. Copyright © 2017 Elsevier B.V. All rights reserved.

A multi-method approach toward de novo glycan characterization: a Man-5 case study.

PubMed

Prien, Justin M; Prater, Bradley D; Cockrill, Steven L

2010-05-01

Regulatory agencies' expectations for biotherapeutic approval are becoming more stringent with regard to product characterization, where minor species as low as 0.1% of a given profile are typically identified. The mission of this manuscript is to demonstrate a multi-method approach toward de novo glycan characterization and quantitation, including minor species at or approaching the 0.1% benchmark. Recently, unexpected isomers of the Man(5)GlcNAc(2) (M(5)) were reported (Prien JM, Ashline DJ, Lapadula AJ, Zhang H, Reinhold VN. 2009. The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap mass spectrometry (MS). J Am Soc Mass Spectrom. 20:539-556). In the current study, quantitative analysis of these isomers found in commercial M(5) standard demonstrated that they are in low abundance (<1% of the total) and therefore an exemplary "litmus test" for minor species characterization. A simple workflow devised around three core well-established analytical procedures: (1) fluorescence derivatization; (2) online rapid resolution reversed-phase separation coupled with negative-mode sequential mass spectrometry (RRRP-(-)-MS(n)); and (3) permethylation derivatization with nanospray sequential mass spectrometry (NSI-MS(n)) provides comprehensive glycan structural determination. All methods have limitations; however, a multi-method workflow is an at-line stopgap/solution which mitigates each method's individual shortcoming(s) providing greater opportunity for more comprehensive characterization. This manuscript is the first to demonstrate quantitative chromatographic separation of the M(5) isomers and the use of a commercially available stable isotope variant of 2-aminobenzoic acid to detect and chromatographically resolve multiple M(5) isomers in bovine ribonuclease B. With this multi-method approach, we have the capabilities to comprehensively characterize a biotherapeutic's glycan array in a de novo manner, including structural isomers at >/=0.1% of the total chromatographic peak area.

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.

PubMed

Kautto, Liisa; Nguyen-Khuong, Terry; Everest-Dass, Arun; Leong, Andrea; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H; Peterson, Robyn

2016-04-01

The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with α2-3,6,8 neuraminidase (sialidase) and α1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes

PubMed Central

Kallemeijn, Wouter W.; Scheij, Saskia; Hoogendoorn, Sascha; Witte, Martin D.; Herrera Moro Chao, Daniela; van Roomen, Cindy P. A. A.; Ottenhoff, Roelof; Overkleeft, Herman S.; Boot, Rolf G.; Aerts, Johannes M. F. G.

2017-01-01

Deficiency of glucocerebrosidase (GBA) causes Gaucher disease (GD). In the common non-neuronopathic GD type I variant, glucosylceramide accumulates primarily in the lysosomes of visceral macrophages. Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s). Two recombinant GBA preparations with distinct N-linked glycans are registered in Europe for treatment of type I GD: imiglucerase (Genzyme), contains predominantly Man(3) glycans, and velaglucerase (Shire PLC) Man(9) glycans. Activity-based probes (ABPs) enable fluorescent labeling of recombinant GBA preparations through their covalent attachment to the catalytic nucleophile E340 of GBA. We comparatively studied binding and uptake of ABP-labeled imiglucerase and velaglucerase in isolated dendritic cells, cultured human macrophages and living mice, through simultaneous detection of different GBAs by paired measurements. Uptake of ABP-labeled rGBAs by dendritic cells was comparable, as well as the bio-distribution following equimolar intravenous administration to mice. ABP-labeled rGBAs were recovered largely in liver, white-blood cells, bone marrow and spleen. Lungs, brain and skin, affected tissues in severe GD types II and III, were only poorly supplemented. Small, but significant differences were noted in binding and uptake of rGBAs in cultured human macrophages, in the absence and presence of mannan. Mannan-competed binding and uptake were largest for velaglucerase, when determined with single enzymes or as equimolar mixtures of both enzymes. Vice versa, imiglucerase showed more prominent binding and uptake not competed by mannan. Uptake of recombinant GBAs by cultured macrophages seems to involve multiple receptors, including several mannose-binding lectins. Differences among cells from different donors (n = 12) were noted, but the same trends were always observed. Our study suggests that further insight in targeting and efficacy of enzyme therapy of individual Gaucher patients could be obtained by the use of recombinant GBA, trace-labeled with an ABP, preferably equipped with an infrared fluorophore or other reporter tag suitable for in vivo imaging. PMID:28207759

Maskless localized patterning of biomolecules on carbon nanotube microarray functionalized by ultrafine atmospheric pressure plasma jet using biotin-avidin system

NASA Astrophysics Data System (ADS)

Abuzairi, Tomy; Okada, Mitsuru; Purnamaningsih, Retno Wigajatri; Poespawati, Nji Raden; Iwata, Futoshi; Nagatsu, Masaaki

2016-07-01

Ultrafine plasma jet is a promising technology with great potential for nano- or micro-scale surface modification. In this letter, we demonstrated the use of ultrafine atmospheric pressure plasma jet (APPJ) for patterning bio-immobilization on vertically aligned carbon nanotube (CNT) microarray platform without a physical mask. The biotin-avidin system was utilized to demonstrate localized biomolecule patterning on the biosensor devices. Using ±7.5 kV square-wave pulses, the optimum condition of plasma jet with He/NH3 gas mixture and 2.5 s treatment period has been obtained to functionalize CNTs. The functionalized CNTs were covalently linked to biotin, bovine serum albumin (BSA), and avidin-(fluorescein isothiocyanate) FITC, sequentially. BSA was necessary as a blocking agent to protect the untreated CNTs from avidin adsorption. The localized patterning results have been evaluated from avidin-FITC fluorescence signals analyzed using a fluorescence microscope. The patterning of biomolecules on the CNT microarray platform using ultrafine APPJ provides a means for potential application of microarray biosensors based on CNTs.

Geiger mode avalanche photodiodes for microarray systems

NASA Astrophysics Data System (ADS)

Phelan, Don; Jackson, Carl; Redfern, R. Michael; Morrison, Alan P.; Mathewson, Alan

2002-06-01

New Geiger Mode Avalanche Photodiodes (GM-APD) have been designed and characterized specifically for use in microarray systems. Critical parameters such as excess reverse bias voltage, hold-off time and optimum operating temperature have been experimentally determined for these photon-counting devices. The photon detection probability, dark count rate and afterpulsing probability have been measured under different operating conditions. An active- quench circuit (AQC) is presented for operating these GM- APDs. This circuit is relatively simple, robust and has such benefits as reducing average power dissipation and afterpulsing. Arrays of these GM-APDs have already been designed and together with AQCs open up the possibility of having a solid-state microarray detector that enables parallel analysis on a single chip. Another advantage of these GM-APDs over current technology is their low voltage CMOS compatibility which could allow for the fabrication of an AQC on the same device. Small are detectors have already been employed in the time-resolved detection of fluorescence from labeled proteins. It is envisaged that operating these new GM-APDs with this active-quench circuit will have numerous applications for the detection of fluorescence in microarray systems.

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

PubMed

Brunner, C; Hoffmann, K; Thiele, T; Schedler, U; Jehle, H; Resch-Genger, U

2015-04-01

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

A microarray immunoassay for simultaneous detection of proteins and bacteria

NASA Technical Reports Server (NTRS)

Delehanty, James B.; Ligler, Frances S.

2002-01-01

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

PubMed

Inagaki, M; Muranishi, H; Yamada, K; Kakehi, K; Uchida, K; Suzuki, T; Yabe, T; Nakagomi, T; Nakagomi, O; Kanamaru, Y

2014-05-01

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A Sensitive and Rapid Method to Determin the Adhesion Capacity of Probiotics and Pathogenic Microorganisms to Human Gastrointestinal Mucins.

PubMed

Ringot-Destrez, Bélinda; D'Alessandro, Zéa; Lacroix, Jean-Marie; Mercier-Bonin, Muriel; Léonard, Renaud; Robbe-Masselot, Catherine

2018-05-29

Mucus is the habitat for the microorganisms, bacteria and yeast that form the commensal flora. Mucins, the main macromolecules of mucus, and more specifically, the glycans that cover them, play essential roles in microbial gastrointestinal colonization. Probiotics and pathogens must also colonize mucus to have lasting positive or deleterious effects. The question of which mucin-harboured glycan motifs favour the adhesion of specific microorganisms remains very poorly studied. In the current study, a simple test based on the detection of fluorescent-labeled microorganisms raised against microgram amounts of mucins spotted on nitrocellulose was developed. The adhesion of various probiotic, commensal and pathogenic microorganisms was evaluated on a panel of human purified gastrointestinal mucins and compared with that of commercially available pig gastric mucins (PGM) and of mucins secreted by the colonic cancer cell line HT29-MTX. The latter two proved to be very poor indicators of adhesion capacity on intestinal mucins. Our results show that the nature of the sialylated cores of O -glycans, determined by MALDI MS-MS analysis, potentially enables sialic acid residues to modulate the adhesion of microorganisms either positively or negatively. Other identified factors affecting the adhesion propensity were O -glycan core types and the presence of blood group motifs. This test should help to select probiotics with enhanced adhesion capabilities as well as deciphering the role of specific mucin glycotopes on microbial adhesion.

Human Group C Rotavirus VP8*s Recognize Type A Histo-Blood Group Antigens as Ligands.

PubMed

Sun, Xiaoman; Wang, Lihong; Qi, Jianxun; Li, Dandi; Wang, Mengxuan; Cong, Xin; Peng, Ruchao; Chai, Wengang; Zhang, Qing; Wang, Hong; Wen, Hongling; Gao, George F; Tan, Ming; Duan, Zhaojun

2018-06-01

Group/species C rotaviruses (RVCs) have been identified as important pathogens of acute gastroenteritis (AGE) in children, family-based outbreaks, as well as animal infections. However, little is known regarding their host-specific interaction, infection, and pathogenesis. In this study, we performed serial studies to characterize the function and structural features of a human G4P[2] RVC VP8* that is responsible for the host receptor interaction. Glycan microarrays demonstrated that the human RVC VP8* recognizes type A histo-blood group antigens (HBGAs), which was confirmed by synthetic glycan-/saliva-based binding assays and hemagglutination of red blood cells, establishing a paradigm of RVC VP8*-glycan interactions. Furthermore, the high-resolution crystal structure of the human RVC VP8* was solved, showing a typical galectin-like structure consisting of two β-sheets but with significant differences from cogent proteins of group A rotaviruses (RVAs). The VP8* in complex with a type A trisaccharide displays a novel ligand binding site that consists of a particular set of amino acid residues of the C-D, G-H, and K-L loops. RVC VP8* interacts with type A HBGAs through a unique mechanism compared with that used by RVAs. Our findings shed light on the host-virus interaction and the coevolution of RVCs and will facilitate the development of specific antivirals and vaccines. IMPORTANCE Group/species C rotaviruses (RVCs), members of Reoviridae family, infect both humans and animals, but our knowledge about the host factors that control host susceptibility and specificity is rudimentary. In this work, we characterized the glycan binding specificity and structural basis of a human RVC that recognizes type A HBGAs. We found that human RVC VP8*, the rotavirus host ligand binding domain that shares only ∼15% homology with the VP8* domains of RVAs, recognizes type A HBGA at an as-yet-unknown glycan binding site through a mechanism distinct from that used by RVAs. Our new advancements provide insights into RVC-cell attachment, the critical step of virus infection, which will in turn help the development of control and prevention strategies against RVs. Copyright © 2018 American Society for Microbiology.

Electron Detachment Dissociation (EDD) of Fluorescently Labeled Sialylated Oligosaccharides

PubMed Central

Zhou, Wen; Håkansson, Kristina

2012-01-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared to IRMPD. Neutral losses and satellite ions such as C – 2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared to 2-AA labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. PMID:22120881

Parallel confocal detection of single biomolecules using diffractive optics and integrated detector units.

PubMed

Blom, H; Gösch, M

2004-04-01

The past few years we have witnessed a tremendous surge of interest in so-called array-based miniaturised analytical systems due to their value as extremely powerful tools for high-throughput sequence analysis, drug discovery and development, and diagnostic tests in medicine (see articles in Issue 1). Terminologies that have been used to describe these array-based bioscience systems include (but are not limited to): DNA-chip, microarrays, microchip, biochip, DNA-microarrays and genome chip. Potential technological benefits of introducing these miniaturised analytical systems include improved accuracy, multiplexing, lower sample and reagent consumption, disposability, and decreased analysis times, just to mention a few examples. Among the many alternative principles of detection-analysis (e.g.chemiluminescence, electroluminescence and conductivity), fluorescence-based techniques are widely used, examples being fluorescence resonance energy transfer, fluorescence quenching, fluorescence polarisation, time-resolved fluorescence, and fluorescence fluctuation spectroscopy (see articles in Issue 11). Time-dependent fluctuations of fluorescent biomolecules with different molecular properties, like molecular weight, translational and rotational diffusion time, colour and lifetime, potentially provide all the kinetic and thermodynamic information required in analysing complex interactions. In this mini-review article, we present recent extensions aimed to implement parallel laser excitation and parallel fluorescence detection that can lead to even further increase in throughput in miniaturised array-based analytical systems. We also report on developments and characterisations of multiplexing extension that allow multifocal laser excitation together with matched parallel fluorescence detection for parallel confocal dynamical fluorescence fluctuation studies at the single biomolecule level.

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain.

PubMed

Huang, Hung-Hsiang; Hassinen, Antti; Sundaram, Subha; Spiess, Andrej-Nikolai; Kellokumpu, Sakari; Stanley, Pamela

2015-09-15

Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis.

Hydrophobic interactions in complexes of antimicrobial peptides with bacterial polysaccharides.

PubMed

Kuo, Hsin H; Chan, Celine; Burrows, Lori L; Deber, Charles M

2007-06-01

Biofilms of Pseudomonas aeruginosa are responsible for chronic lung infections in cystic fibrosis patients, where they are characterized by overproduction of the exopolysaccharide alginate and are recalcitrant to treatment with conventional antibiotics. Cationic antimicrobial peptides (CAPs) are potential alternatives for the treatment of multi-drug-resistant P. aeruginosa. However, alginate in P. aeruginosa biofilms has been proposed to bind these peptides through hydrophobic interactions, consequently reducing their activity [Chan et al., J Biol Chem 2004; 279: 38749-38754]. Here we perform biophysical analyses of the interactions of alginate with a series of novel peptide antibiotics (alpha-CAPs) of prototypic sequence KK-AAAXAAAAAXAAWAAXAAA-KKKK (where X = Phe, Trp or Leu). The hydrophobic interaction interface in alginate was investigated by examining (i) the effects of polysaccharide composition with respect to D-mannuronate and L-guluronate content; (ii) glycan chain length; (iii) alpha-CAP Trp fluorescence; and (iv) 1-anilinonaphthalene-8-sulfonate fluorescence. The results show that, while M and G residues produce equivalent effects, hydrophobic interactions between alginate and alpha-CAPs require a minimal glycan chain length. Peptide interactions with alginate are deduced to be mediated by hydrophobic microdomains comprised of pyranosyl C-H groups that are inducible upon formation of alpha-CAP-alginate complexes due to charge neutralization between the two species.

Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

DOE PAGES

da Silva, Roberta Peres; Heiss, Christian; Black, Ian; ...

2015-09-21

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) andmore » α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.« less

Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

da Silva, Roberta Peres; Heiss, Christian; Black, Ian

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) andmore » α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.« less

Identification of cell surface glycoprotein markers for glioblastoma-derived stem-like cells using a lectin microarray and LC-MS/MS approach

PubMed Central

He, Jintang; Liu, Yashu; Xie, Xiaolei; Zhu, Thant; Soules, Mary; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within two years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. Lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma. PMID:20235609

A focused microarray approach to functional glycomics: transcriptional regulation of the glycome.

PubMed

Comelli, Elena M; Head, Steven R; Gilmartin, Tim; Whisenant, Thomas; Haslam, Stuart M; North, Simon J; Wong, Nyet-Kui; Kudo, Takashi; Narimatsu, Hisashi; Esko, Jeffrey D; Drickamer, Kurt; Dell, Anne; Paulson, James C

2006-02-01

Glycosylation is the most common posttranslational modification of proteins, yet genes relevant to the synthesis of glycan structures and function are incompletely represented and poorly annotated on the commercially available arrays. To fill the need for expression analysis of such genes, we employed the Affymetrix technology to develop a focused and highly annotated glycogene-chip representing human and murine glycogenes, including glycosyltransferases, nucleotide sugar transporters, glycosidases, proteoglycans, and glycan-binding proteins. In this report, the array has been used to generate glycogene-expression profiles of nine murine tissues. Global analysis with a hierarchical clustering algorithm reveals that expression profiles in immune tissues (thymus [THY], spleen [SPL], lymph node, and bone marrow [BM]) are more closely related, relative to those of nonimmune tissues (kidney [KID], liver [LIV], brain [BRN], and testes [TES]). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N- and O-linked oligosaccharides are ubiquitously expressed, whereas glycosyltransferases that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell-type-specific glycosylation. Comparison of gene expression profiles with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) profiling of N-linked oligosaccharides suggested that the alpha1-3 fucosyltransferase 9, Fut9, is the enzyme responsible for terminal fucosylation in KID and BRN, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and Siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (CFG) (http://www.functionalglycomics.org).

Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.

PubMed

Deng, Lingquan; Bensing, Barbara A; Thamadilok, Supaporn; Yu, Hai; Lau, Kam; Chen, Xi; Ruhl, Stefan; Sullam, Paul M; Varki, Ajit

2014-12-01

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.

Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping.

PubMed

Eckard, Anahita D; Dupont, David R; Young, Johnie K

2018-01-01

N -lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N -glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography-mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N -glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N -glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area.

Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

PubMed

Chen, Huang-Han; Wu, Chih-Hsing; Tsai, Mei-Ling; Huang, Yi-Jing; Chen, Shu-Hui

2012-10-16

The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

A Chemical Biology Solution to Problems with Studying Biologically Important but Unstable 9-O-Acetyl Sialic Acids.

PubMed

Khedri, Zahra; Xiao, An; Yu, Hai; Landig, Corinna Susanne; Li, Wanqing; Diaz, Sandra; Wasik, Brian R; Parrish, Colin R; Wang, Lee-Ping; Varki, Ajit; Chen, Xi

2017-01-20

9-O-Acetylation is a common natural modification on sialic acids (Sias) that terminate many vertebrate glycan chains. This ester group has striking effects on many biological phenomena, including microbe-host interactions, complement action, regulation of immune responses, sialidase action, cellular apoptosis, and tumor immunology. Despite such findings, 9-O-acetyl sialoglycoconjugates have remained largely understudied, primarily because of marked lability of the 9-O-acetyl group to even small pH variations and/or the action of mammalian or microbial esterases. Our current studies involving 9-O-acetylated sialoglycans on glycan microarrays revealed that even the most careful precautions cannot ensure complete stability of the 9-O-acetyl group. We now demonstrate a simple chemical biology solution to many of these problems by substituting the oxygen atom in the ester with a nitrogen atom, resulting in sialic acids with a chemically and biologically stable 9-N-acetyl group. We present an efficient one-pot multienzyme method to synthesize a sialoglycan containing 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) and compare it to the one with naturally occurring 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac 2 ). Conformational resemblance of the two molecules was confirmed by computational molecular dynamics simulations. Microarray studies showed that the Neu5Ac9NAc-sialoglycan is a ligand for viruses naturally recognizing Neu5,9Ac 2 , with a similar affinity but with much improved stability in handling and study. Feeding of Neu5Ac9NAc or Neu5,9Ac 2 to mammalian cells resulted in comparable incorporation and surface expression as well as binding to 9-O-acetyl-Sia-specific viruses. However, cells fed with Neu5Ac9NAc remained resistant to viral esterases and showed a slower turnover. This simple approach opens numerous research opportunities that have heretofore proved intractable.

Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures.

PubMed

Stefanowicz, Karolina; Lannoo, Nausicaä; Proost, Paul; Van Damme, Els J M

2012-01-01

The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly-N-acetyllactosamine ([Galβ1-4GlcNAc]n) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.

Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer

PubMed Central

Miwa, Hazuki E; Koba, Wade R; Fine, Eugene J; Giricz, Orsi; Kenny, Paraic A; Stanley, Pamela

2013-01-01

Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3−/−/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3−/−/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ∼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer. PMID:24037315

Gene expression profiling of two distinct neuronal populations in the rodent spinal cord.

PubMed

Ryge, Jesper; Westerdahl, Ann-Charlotte; Alstrøm, Preben; Kiehn, Ole

2008-01-01

In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50-250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord.

Gene Expression Profiling of Two Distinct Neuronal Populations in the Rodent Spinal Cord

PubMed Central

Alstrøm, Preben; Kiehn, Ole

2008-01-01

Background In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. Methodology/Principal Findings We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50–250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. Conclusions/Significance We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord. PMID:18923679

Interlaboratory comparison of immunohistochemical testing for HER2: results of the 2004 and 2005 College of American Pathologists HER2 Immunohistochemistry Tissue Microarray Survey.

PubMed

Fitzgibbons, Patrick L; Murphy, Douglas A; Dorfman, David M; Roche, Patrick C; Tubbs, Raymond R

2006-10-01

Correct assessment of human epidermal growth factor receptor 2 (HER2) status is essential in managing patients with invasive breast carcinoma, but few data are available on the accuracy of laboratories performing HER2 testing by immunohistochemistry (IHC). To review the results of the 2004 and 2005 College of American Pathologists HER2 Immunohistochemistry Tissue Microarray Survey. The HER2 survey is designed for laboratories performing immunohistochemical staining and interpretation for HER2. The survey uses tissue microarrays, each consisting of ten 3-mm tissue cores obtained from different invasive breast carcinomas. All cases are also analyzed by fluorescence in situ hybridization. Participants receive 8 tissue microarrays (80 cases) with instructions to perform immunostaining for HER2 using the laboratory's standard procedures. The laboratory interprets the stained slides and returns results to the College of American Pathologists for analysis. In 2004 and 2005, a core was considered "graded" when at least 90% of laboratories agreed on the result--negative (0, 1+) versus positive (2+, 3+). This interlaboratory comparison survey included 102 laboratories in 2004 and 141 laboratories in 2005. Of the 160 cases in both surveys, 111 (69%) achieved 90% consensus (graded). All 43 graded cores scored as IHC-positive were fluorescence in situ hybridization-positive, whereas all but 3 of the 68 IHC-negative graded cores were fluorescence in situ hybridization-negative. Ninety-seven (95%) of 102 laboratories in 2004 and 129 (91%) of 141 laboratories in 2005 correctly scored at least 90% of the graded cores. Performance among laboratories performing HER2 IHC in this tissue microarray-based survey was excellent. Cores found to be IHC-positive or IHC-negative by participant consensus can be used as validated benchmarks for interlaboratory comparison, allowing laboratories to assess their performance and determine if improvements are needed.

Integrated Microfluidic Lectin Barcode Platform for High-Performance Focused Glycomic Profiling

NASA Astrophysics Data System (ADS)

Shang, Yuqin; Zeng, Yun; Zeng, Yong

2016-02-01

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges in analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and speed of lectin microarray. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, CA125 protein purified from ovarian cancer cell line and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated “sample-to-answer” microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glyco-biomarker development.

Integrated Microfluidic Lectin Barcode Platform for High-Performance Focused Glycomic Profiling

PubMed Central

Shang, Yuqin; Zeng, Yun; Zeng, Yong

2016-01-01

Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges in analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and speed of lectin microarray. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, CA125 protein purified from ovarian cancer cell line and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated “sample-to-answer” microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glyco-biomarker development. PMID:26831207

Development and validation of a Luminex assay for detection of a predictive biomarker for PROSTVAC-VF therapy

PubMed Central

Lucas, Julie L.; Tacheny, Erin A.; Ferris, Allison; Galusha, Michelle; Srivastava, Apurva K.; Ganguly, Aniruddha; Williams, P. Mickey; Sachs, Michael C.; Thurin, Magdalena; Tricoli, James V.; Ricker, Winnie; Gildersleeve, Jeffrey C.

2017-01-01

Cancer therapies can provide substantially improved survival in some patients while other seemingly similar patients receive little or no benefit. Strategies to identify patients likely to respond well to a given therapy could significantly improve health care outcomes by maximizing clinical benefits while reducing toxicities and adverse effects. Using a glycan microarray assay, we recently reported that pretreatment serum levels of IgM specific to blood group A trisaccharide (BG-Atri) correlate positively with overall survival of cancer patients on PROSTVAC-VF therapy. The results suggested anti-BG-Atri IgM measured prior to treatment could serve as a biomarker for identifying patients likely to benefit from PROSTVAC-VF. For continued development and clinical application of serum IgM specific to BG-Atri as a predictive biomarker, a clinical assay was needed. In this study, we developed and validated a Luminex-based clinical assay for measuring serum IgM specific to BG-Atri. IgM levels were measured with the Luminex assay and compared to levels measured using the microarray for 126 healthy individuals and 77 prostate cancer patients. This assay provided reproducible and consistent results with low %CVs, and tolerance ranges were established for the assay. IgM levels measured using the Luminex assay were found to be highly correlated to the microarray results with R values of 0.93–0.95. This assay is a Laboratory Developed Test (LDT) and is suitable for evaluating thousands of serum samples in CLIA certified laboratories that have validated the assay. In addition, the study demonstrates that discoveries made using neoglycoprotein-based microarrays can be readily migrated to a clinical assay. PMID:28771597

Dye bias correction in dual-labeled cDNA microarray gene expression measurements.

PubMed Central

Rosenzweig, Barry A; Pine, P Scott; Domon, Olen E; Morris, Suzanne M; Chen, James J; Sistare, Frank D

2004-01-01

A significant limitation to the analytical accuracy and precision of dual-labeled spotted cDNA microarrays is the signal error due to dye bias. Transcript-dependent dye bias may be due to gene-specific differences of incorporation of two distinctly different chemical dyes and the resultant differential hybridization efficiencies of these two chemically different targets for the same probe. Several approaches were used to assess and minimize the effects of dye bias on fluorescent hybridization signals and maximize the experimental design efficiency of a cell culture experiment. Dye bias was measured at the individual transcript level within each batch of simultaneously processed arrays by replicate dual-labeled split-control sample hybridizations and accounted for a significant component of fluorescent signal differences. This transcript-dependent dye bias alone could introduce unacceptably high numbers of both false-positive and false-negative signals. We found that within a given set of concurrently processed hybridizations, the bias is remarkably consistent and therefore measurable and correctable. The additional microarrays and reagents required for paired technical replicate dye-swap corrections commonly performed to control for dye bias could be costly to end users. Incorporating split-control microarrays within a set of concurrently processed hybridizations to specifically measure dye bias can eliminate the need for technical dye swap replicates and reduce microarray and reagent costs while maintaining experimental accuracy and technical precision. These data support a practical and more efficient experimental design to measure and mathematically correct for dye bias. PMID:15033598

A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

PubMed

Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

2016-03-15

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

PubMed

Ares, Manuel

2014-01-01

This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice

PubMed Central

Kaneko, Kentaro; Takamatsu, Takeshi; Inomata, Takuya; Oikawa, Kazusato; Itoh, Kimiko; Hirose, Kazuko; Amano, Maho; Nishimura, Shin-Ichiro; Toyooka, Kiminori; Matsuoka, Ken; Pozueta-Romero, Javier; Mitsui, Toshiaki

2016-01-01

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs. PMID:27335351

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

PubMed

Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

2012-05-01

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

PubMed Central

Friedman, M L; Schlueter, K T; Kirley, T L; Halsall, H B

1985-01-01

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field. PMID:4091825

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides.

PubMed

Zhou, Wen; Håkansson, Kristina

2011-12-01

We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm.

PubMed

Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

2015-01-01

DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

PubMed

Adak, Avijit K; Li, Ben-Yuan; Huang, Li-De; Lin, Ting-Wei; Chang, Tsung-Che; Hwang, Kuo Chu; Lin, Chun-Cheng

2014-07-09

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

PubMed

Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

2015-06-01

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Unique Proteins Expressed by Blood Vessels in Skeletal Sites Colonized by Breast Cancer Cells

DTIC Science & Technology

2006-08-01

fluorescent labeled acetylated LDL at an accelerated rate (3). After one week in culture BVECs and MVECs were harvested. Total RNA was extracted from...both cell types using the Qiagen RNeasy kit (Valencia, CA). Microarray labeling, hybridization and analysis was conducted on the RNA by the Penn...State University DNA Microarray Facility under the direction of Dr. Craig Praul. Briefly, RNA obtained from three separate isolations of BVECs and

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

PubMed

Dinca, V; Ranella, A; Farsari, M; Kafetzopoulos, D; Dinescu, M; Popescu, A; Fotakis, C

2008-10-01

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

PubMed Central

Ludwig, Susann K. J.; Tokarski, Christian; Lang, Stefan N.; van Ginkel, Leendert A.; Zhu, Hongying; Ozcan, Aydogan; Nielen, Michel W. F.

2015-01-01

Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this ‘protein microarray on a smartphone’-concept for on-site testing, e.g., in food safety, environment and health monitoring. PMID:26308444

Development of a DNA microarray for species identification of quarantine aphids.

PubMed

Lee, Won Sun; Choi, Hwalran; Kang, Jinseok; Kim, Ji-Hoon; Lee, Si Hyeock; Lee, Seunghwan; Hwang, Seung Yong

2013-12-01

Aphid pests are being brought into Korea as a result of increased crop trading. Aphids exist on growth areas of plants, and thus plant growth is seriously affected by aphid pests. However, aphids are very small and have several sexual morphs and life stages, so it is difficult to identify species on the basis of morphological features. This problem was approached using DNA microarray technology. DNA targets of the cytochrome c oxidase subunit I gene were generated with a fluorescent dye-labelled primer and were hybridised onto a DNA microarray consisting of specific probes. After analysing the signal intensity of the specific probes, the unique patterns from the DNA microarray, consisting of 47 species-specific probes, were obtained to identify 23 aphid species. To confirm the accuracy of the developed DNA microarray, ten individual blind samples were used in blind trials, and the identifications were completely consistent with the sequencing data of all individual blind samples. A microarray has been developed to distinguish aphid species. DNA microarray technology provides a rapid, easy, cost-effective and accurate method for identifying aphid species for pest control management. © 2013 Society of Chemical Industry.

Characterization of Bovine Serum Albumin Blocking Efficiency on Epoxy-Functionalized Substrates for Microarray Applications.

PubMed

Sun, Yung-Shin; Zhu, Xiangdong

2016-10-01

Microarrays provide a platform for high-throughput characterization of biomolecular interactions. To increase the sensitivity and specificity of microarrays, surface blocking is required to minimize the nonspecific interactions between analytes and unprinted yet functionalized surfaces. To block amine- or epoxy-functionalized substrates, bovine serum albumin (BSA) is one of the most commonly used blocking reagents because it is cheap and easy to use. Based on standard protocols from microarray manufactories, a BSA concentration of 1% (10 mg/mL or 200 μM) and reaction time of at least 30 min are required to efficiently block epoxy-coated slides. In this paper, we used both fluorescent and label-free methods to characterize the BSA blocking efficiency on epoxy-functionalized substrates. The blocking efficiency of BSA was characterized using a fluorescent scanner and a label-free oblique-incidence reflectivity difference (OI-RD) microscope. We found that (1) a BSA concentration of 0.05% (0.5 mg/mL or 10 μM) could give a blocking efficiency of 98%, and (2) the BSA blocking step took only about 5 min to be complete. Also, from real-time and in situ measurements, we were able to calculate the conformational properties (thickness, mass density, and number density) of BSA molecules deposited on the epoxy surface. © 2015 Society for Laboratory Automation and Screening.

Tuning Fluorescence Direction with Plasmonic Metal–Dielectric– Metal Substrates

PubMed Central

Choudhury, Sharmistha Dutta; Badugu, Ramachandram; Nowaczyk, Kazimierz; Ray, Krishanu; Lakowicz, Joseph R.

2013-01-01

Controlling the emission properties of fluorophores is essential for improving the performance of fluorescence-based techniques in modern biochemical research, medical diagnosis, and sensing. Fluorescence emission is isotropic in nature, which makes it difficult to capture more than a small fraction of the total emission. Metal– dielectric–metal (MDM) substrates, discussed in this Letter, convert isotropic fluorescence into beaming emission normal to the substrate. This improves fluorescence collection efficiency and also opens up new avenues for a wide range of fluorescence-based applications. We suggest that MDM substrates can be readily adapted for multiple uses, such as in microarray formats, for directional fluorescence studies of multiple probes or for molecule-specific sensing with a high degree of spatial control over the fluorescence emission. SECTION: Physical Processes in Nanomaterials and Nanostructures PMID:24013521

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

PubMed

Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

PubMed Central

Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide. PMID:20210464

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics▿

PubMed Central

Zhao, Zhengshan; Peytavi, Régis; Diaz-Quijada, Gerardo A.; Picard, Francois J.; Huletsky, Ann; Leblanc, Éric; Frenette, Johanne; Boivin, Guy; Veres, Teodor; Dumoulin, Michel M.; Bergeron, Michel G.

2008-01-01

Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations. PMID:18784318

Bivalent Carbohydrate Binding Is Required for Biological Activity of Clitocybe nebularis Lectin (CNL), the N,N′-Diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc)-specific Lectin from Basidiomycete C. nebularis*

PubMed Central

Pohleven, Jure; Renko, Miha; Magister, Špela; Smith, David F.; Künzler, Markus; Štrukelj, Borut; Turk, Dušan; Kos, Janko; Sabotič, Jerica

2012-01-01

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency. PMID:22298779

Nanoroughened plasmonic films for enhanced biosensing detection

NASA Astrophysics Data System (ADS)

LeMoal, Eric; Lévêque-Fort, Sandrine; Potier, Marie-Claude; Fort, Emmanuel

2009-06-01

Although fluorescence is the prevailing labeling technique in biosensing applications, sensitivity improvement is still a striving challenge. We show that coating standard microscope slides with nanoroughened silver films provides a high fluorescence signal enhancement due to plasmonic interactions. As a proof of concept, we applied these films with tailored plasmonic properties to DNA microarrays. Using common optical scanning devices, we achieved signal amplifications of more than 40-fold.

Restricted N-glycan conformational space in the PDB and its implication in glycan structure modeling.

PubMed

Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil

2013-01-01

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.

Solid-phase glycan isolation for glycomics analysis

PubMed Central

Yang, Shuang; Zhang, Hui

2013-01-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. PMID:23090885

Solid-phase glycan isolation for glycomics analysis.

PubMed

Yang, Shuang; Zhang, Hui

2012-12-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

PubMed Central

Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil

2013-01-01

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures. PMID:23516343

Recognition of Mannosylated Ligands and Influenza A Virus by Human Surfactant Protein D: Contributions of an Extended Site and Residue 343

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crouch, E.; Hartshorn, K; Horlacher, T

2009-01-01

Surfactant protein D (SP-D) plays important roles in antiviral host defense. Although SP-D shows a preference for glucose/maltose, the protein also recognizes d-mannose and a variety of mannose-rich microbial ligands. This latter preference prompted an examination of the mechanisms of mannose recognition, particularly as they relate to high-mannose viral glycans. Trimeric neck plus carbohydrate recognition domains from human SP-D (hNCRD) preferred ?1-2-linked dimannose (DM) over the branched trimannose (TM) core, ?1-3 or ?1-6 DM, or d-mannose. Previous studies have shown residues flanking the carbohydrate binding site can fine-tune ligand recognition. A mutant with valine at 343 (R343V) showed enhanced bindingmore » to mannan relative to wild type and R343A. No alteration in affinity was observed for d-mannose or for ?1-3- or ?1-6-linked DM; however, substantially increased affinity was observed for ?1-2 DM. Both proteins showed efficient recognition of linear and branched subdomains of high-mannose glycans on carbohydrate microarrays, and R343V showed increased binding to a subset of the oligosaccharides. Crystallographic analysis of an R343V complex with 1,2-DM showed a novel mode of binding. The disaccharide is bound to calcium by the reducing sugar ring, and a stabilizing H-bond is formed between the 2-OH of the nonreducing sugar ring and Arg349. Although hNCRDs show negligible binding to influenza A virus (IAV), R343V showed markedly enhanced viral neutralizing activity. Hydrophobic substitutions for Arg343 selectively blocked binding of a monoclonal antibody (Hyb 246-05) that inhibits IAV binding activity. Our findings demonstrate an extended ligand binding site for mannosylated ligands and the significant contribution of the 343 side chain to specific recognition of multivalent microbial ligands, including high-mannose viral glycans.« less

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples.

PubMed

Sanchis, Ana; Salvador, J-Pablo; Campbell, Katrina; Elliott, Christopher T; Shelver, Weilin L; Li, Qing X; Marco, M-Pilar

2018-07-01

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal or polyclonal antibodies raised against important families of chemical pollutants such as triazine biocide (i.e. Irgarol 1051®), sulfonamide and chloramphenicol antibiotics, polybrominated diphenyl ether flame-retardant (PBDE, i.e. BDE-47), hormone (17β-estradiol), and algae toxin (domoic acid). These contaminants were selected as model analytes, however, the platform developed has the potential to detect a broader group of compounds based on the cross-reactivity of the immunoreagents used. The microarray chip is able to simultaneously determine these families of contaminants directly in seawater samples reaching limits of detection close to the levels found in contaminated areas (Irgarol 1051®, 0.19 ± 0,06 µg L -1 ; sulfapyridine, 0.17 ± 0.07 µg L -1 ; chloramphenicol, 0.11 ± 0.03 µg L -1 ; BDE-47, 2.71 ± 1.13 µg L -1 ; 17β-estradiol, 0.94 ± 0.30 µg L -1 and domoic acid, 1.71 ± 0.30 µg L -1 ). Performance of the multiplexed microarray chip was assessed by measuring 38 blind spiked seawater samples containing either one of these contaminants or mixtures of them. The accuracy found was very good and the coefficient of variation was < 20% in all the cases. No sample pre-treatment was necessary, and the results could be obtained in just 1 h 30 min. The microarray shows high sample throughput capabilities, being able to measure simultaneously more than 68 samples and screen them for a significant number of chemical contaminants of interest in environmental screening programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and application of an algorithm to compute weighted multiple glycan alignments.

PubMed

Hosoda, Masae; Akune, Yukie; Aoki-Kinoshita, Kiyoko F

2017-05-01

A glycan consists of monosaccharides linked by glycosidic bonds, has branches and forms complex molecular structures. Databases have been developed to store large amounts of glycan-binding experiments, including glycan arrays with glycan-binding proteins. However, there are few bioinformatics techniques to analyze large amounts of data for glycans because there are few tools that can handle the complexity of glycan structures. Thus, we have developed the MCAW (Multiple Carbohydrate Alignment with Weights) tool that can align multiple glycan structures, to aid in the understanding of their function as binding recognition molecules. We have described in detail the first algorithm to perform multiple glycan alignments by modeling glycans as trees. To test our tool, we prepared several data sets, and as a result, we found that the glycan motif could be successfully aligned without any prior knowledge applied to the tool, and the known recognition binding sites of glycans could be aligned at a high rate amongst all our datasets tested. We thus claim that our tool is able to find meaningful glycan recognition and binding patterns using data obtained by glycan-binding experiments. The development and availability of an effective multiple glycan alignment tool opens possibilities for many other glycoinformatics analysis, making this work a big step towards furthering glycomics analysis. http://www.rings.t.soka.ac.jp. kkiyoko@soka.ac.jp. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

PubMed Central

2011-01-01

Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. PMID:21235785

A Versatile Microarray Platform for Capturing Rare Cells

NASA Astrophysics Data System (ADS)

Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

LS-CAP: an algorithm for identifying cytogenetic aberrations in hepatocellular carcinoma using microarray data.

PubMed

He, Xianmin; Wei, Qing; Sun, Meiqian; Fu, Xuping; Fan, Sichang; Li, Yao

2006-05-01

Biological techniques such as Array-Comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH) and affymetrix single nucleotide pleomorphism (SNP) array have been used to detect cytogenetic aberrations. However, on genomic scale, these techniques are labor intensive and time consuming. Comparative genomic microarray analysis (CGMA) has been used to identify cytogenetic changes in hepatocellular carcinoma (HCC) using gene expression microarray data. However, CGMA algorithm can not give precise localization of aberrations, fails to identify small cytogenetic changes, and exhibits false negatives and positives. Locally un-weighted smoothing cytogenetic aberrations prediction (LS-CAP) based on local smoothing and binomial distribution can be expected to address these problems. LS-CAP algorithm was built and used on HCC microarray profiles. Eighteen cytogenetic abnormalities were identified, among them 5 were reported previously, and 12 were proven by CGH studies. LS-CAP effectively reduced the false negatives and positives, and precisely located small fragments with cytogenetic aberrations.

Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation

PubMed Central

Samuelson, John; Robbins, Phillips W.

2014-01-01

Asparagine-linked glycans (N-glycans) of medically important protists have much to tell us about the evolution of N-glycosylation and of N-glycan-dependent quality control (N-glycan QC) of protein folding in the endoplasmic reticulum. While host N-glycans are built upon a dolichol-pyrophosphate-linked precursor with 14 sugars (Glc3Man9GlcNAc2), protist N-glycan precursors vary from Glc3Man9GlcNAc2 (Acanthamoeba) to Man9GlcNAc2 (Trypanosoma) to Glc3Man5GlcNAc2 (Toxoplasma) to Man5GlcNAc2 (Entamoeba, Trichomonas, and Eimeria) to GlcNAc2 (Plasmodium and Giardia) to zero (Theileria). As related organisms have differing N-glycan lengths (e.g. Toxoplasma, Eimeria, Plasmodium, and Theileria), the present N-glycan variation is based upon secondary loss of Alg genes, which encode enzymes that add sugars to the N-glycan precursor. An N-glycan precursor with Man5GlcNAc2 is necessary but not sufficient for N-glycan QC, which is predicted by the presence of the UDP-glucose:glucosyltransferase (UGGT) plus calreticulin and/or calnexin. As many parasites lack glucose in their N-glycan precursor, UGGT product may be identified by inhibition of glucosidase II. The presence of an armless calnexin in Toxoplasma suggests secondary loss of N-glycan QC from coccidia. Positive selection for N-glycan sites occurs in secreted proteins of organisms with NG-QC and is based upon an increased likelihood of threonine but not serine in the second position versus asparagine. In contrast, there appears to be selection against N-glycan length in Plasmodium and N-glycan site density in Toxoplasma. Finally, there is suggestive evidence for N-glycan-dependent ERAD in Trichomonas, which glycosylates and degrades the exogenous reporter mutant carboxypeptidase Y (CPY*). PMID:25475176

A spin column-free approach to sodium hydroxide-based glycan permethylation.

PubMed

Hu, Yueming; Borges, Chad R

2017-07-24

Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues-yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p < 0.05), but overall yield of O-glycans was similar or slightly diminished (p < 0.05 for tetrasaccharides or smaller). When the SCF procedure was employed upstream to hydrolysis, reduction and acetylation for glycan linkage analysis of pooled glycans from unfractionated blood plasma, analytical reproducibility was on par with that from previous spin column-based "glycan node" analysis results. When applied to blood plasma samples from stage III-IV breast cancer patients (n = 20) and age-matched controls (n = 20), the SCF procedure facilitated identification of three glycan nodes with significantly different distributions between the cases and controls (ROC c-statistics > 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens.

A spin column-free approach to sodium hydroxide-based glycan permethylation†

PubMed Central

Hu, Yueming; Borges, Chad R.

2018-01-01

Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues—yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p < 0.05), but overall yield of O-glycans was similar or slightly diminished (p < 0.05 for tetrasaccharides or smaller). When the SCF procedure was employed upstream to hydrolysis, reduction and acetylation for glycan linkage analysis of pooled glycans from unfractionated blood plasma, analytical reproducibility was on par with that from previous spin column-based “glycan node” analysis results. When applied to blood plasma samples from stage III–IV breast cancer patients (n = 20) and age-matched controls (n = 20), the SCF procedure facilitated identification of three glycan nodes with significantly different distributions between the cases and controls (ROC c-statistics > 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens. PMID:28635997

Conformational Heterogeneity of the HIV Envelope Glycan Shield.

PubMed

Yang, Mingjun; Huang, Jing; Simon, Raphael; Wang, Lai-Xi; MacKerell, Alexander D

2017-06-30

To better understand the conformational properties of the glycan shield covering the surface of the HIV gp120/gp41 envelope (Env) trimer, and how the glycan shield impacts the accessibility of the underlying protein surface, we performed enhanced sampling molecular dynamics (MD) simulations of a model glycosylated HIV Env protein and related systems. Our simulation studies revealed a conformationally heterogeneous glycan shield with a network of glycan-glycan interactions more extensive than those observed to date. We found that partial preorganization of the glycans potentially favors binding by established broadly neutralizing antibodies; omission of several specific glycans could increase the accessibility of other glycans or regions of the protein surface to antibody or CD4 receptor binding; the number of glycans that can potentially interact with known antibodies is larger than that observed in experimental studies; and specific glycan conformations can maximize or minimize interactions with individual antibodies. More broadly, the enhanced sampling MD simulations described here provide a valuable tool to guide the engineering of specific Env glycoforms for HIV vaccine design.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B and G

PubMed Central

Stewart-Jones, Guillaume B. E.; Soto, Cinque; Lemmin, Thomas; Chuang, Gwo-Yu; Druz, Aliaksandr; Kong, Rui; Thomas, Paul V.; Wagh, Kshitij; Zhou, Tongqing; Behrens, Anna-Janina; Bylund, Tatsiana; Choi, Chang W.; Davison, Jack R.; Georgiev, Ivelin S.; Joyce, M. Gordon; Do Kwon, Young; Pancera, Marie; Taft, Justin; Yang, Yongping; Zhang, Baoshan; Shivatare, Sachin S.; Shivatare, Vidya S.; Lee, Chang-Chun D.; Wu, Chung-Yi; Bewley, Carole A.; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Crispin, Max; Baxa, Ulrich; Korber, Bette T.; Wong, Chi-Huey; Mascola, John R.; Kwong, Peter D.

2017-01-01

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ~90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, which encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans amongst known broadly neutralizing antibodies that target the glycan-shielded trimer. PMID:27114034

Dielectrophoretic Isolation and Detection of cfc-DNA Nanoparticulate Biomarkers and Virus from Blood

PubMed Central

Sonnenberg, Avery; Marciniak, Jennifer Y.; McCanna, James; Krishnan, Rajaram; Rassenti, Laura; Kipps, Thomas J.; Heller, Michael J.

2015-01-01

Dielectrophoretic (DEP) microarray devices allow important cellular nanoparticulate biomarkers and virus to be rapidly isolated, concentrated and detected directly from clinical and biological samples. A variety of sub-micron nanoparticulate entities including cell free circulating (cfc) DNA, mitochondria and virus can be isolated into DEP high-field areas on microelectrodes, while blood cells and other micron-size entities become isolated into DEP low-field areas between the microelectrodes. The nanoparticulate entities are held in the DEP high-field areas while cells are washed away along with proteins and other small molecules which are not affected by the DEP electric fields. DEP carried out on 20 µL of whole blood obtained from Chronic Lymphocytic Leukemia (CLL) patients showed a considerable amount of SYBR Green stained DNA fluorescent material concentrated in the DEP high-field regions. Whole blood obtained from healthy individuals showed little or no fluorescent DNA materials in the DEP high-field regions. Fluorescent T7 bacteriophage virus could be isolated directly from blood samples, and fluorescently stained mitochondria could be isolated from biological buffer samples. Using newer DEP microarray devices, high molecular weight (hmw) DNA could be isolated from serum and detected at levels as low as 8–16 ng/mL. PMID:23436471

The glycan-mediated mechanism on the interactions of gp120 with CD4 and antibody: Insights from molecular dynamics simulation.

PubMed

Zhang, Yan; Niu, Yuzhen; Tian, Jiaqi; Liu, Xuewei; Yao, Xiaojun; Liu, Huanxiang

2017-12-01

N-linked glycans such as 234 and 276 gp 120 glycans are vital components of HIV evasion from humoral immunity and important for HIV-1 neutralization of many broadly neutralizing antibodies (bNAbs). However, it is unknown the action mechanism of two glycans. To investigate the roles of the glycans on the interactions of gp120 with CD4 and antibody, molecular dynamic simulations based on gp120-CD4-8ANC195 complex with 234 and 276 gp 120 glycans, 234 gp 120 glycan, 276 gp 120 glycan, and without glycan were performed. Our results reveal that 276 gp 120 glycan can enhance gp120-CD4 and gp120-antibody interactions through the formation of hydrogen bonds of the glycan with CD4 and antibody and make the binding interface of gp120, CD4 and antibody stable; 234 gp 120 glycan primarily reinforces gp120-antibody interactions and weakly affects gp120-CD4 interactions as it mainly lies between gp120 and antibody. The co-operating of two glycans can enhance gp120-CD4 and gp120-antibody associations. Through the structural analysis, it can be seen that 234 gp 120 glycan leads to moving upward of two glycans and the variable region of heavy chain, which is favorable for the interactions of gp120 with CD4 and antibody. The information obtained in this study can provide the guidance for design vaccines and small molecule inhibitors. © 2017 John Wiley & Sons A/S.

Glycan fragment database: a database of PDB-based glycan 3D structures.

PubMed

Jo, Sunhwan; Im, Wonpil

2013-01-01

The glycan fragment database (GFDB), freely available at http://www.glycanstructure.org, is a database of the glycosidic torsion angles derived from the glycan structures in the Protein Data Bank (PDB). Analogous to protein structure, the structure of an oligosaccharide chain in a glycoprotein, referred to as a glycan, can be characterized by the torsion angles of glycosidic linkages between relatively rigid carbohydrate monomeric units. Knowledge of accessible conformations of biologically relevant glycans is essential in understanding their biological roles. The GFDB provides an intuitive glycan sequence search tool that allows the user to search complex glycan structures. After a glycan search is complete, each glycosidic torsion angle distribution is displayed in terms of the exact match and the fragment match. The exact match results are from the PDB entries that contain the glycan sequence identical to the query sequence. The fragment match results are from the entries with the glycan sequence whose substructure (fragment) or entire sequence is matched to the query sequence, such that the fragment results implicitly include the influences from the nearby carbohydrate residues. In addition, clustering analysis based on the torsion angle distribution can be performed to obtain the representative structures among the searched glycan structures.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

PubMed

Stewart-Jones, Guillaume B E; Soto, Cinque; Lemmin, Thomas; Chuang, Gwo-Yu; Druz, Aliaksandr; Kong, Rui; Thomas, Paul V; Wagh, Kshitij; Zhou, Tongqing; Behrens, Anna-Janina; Bylund, Tatsiana; Choi, Chang W; Davison, Jack R; Georgiev, Ivelin S; Joyce, M Gordon; Kwon, Young Do; Pancera, Marie; Taft, Justin; Yang, Yongping; Zhang, Baoshan; Shivatare, Sachin S; Shivatare, Vidya S; Lee, Chang-Chun D; Wu, Chung-Yi; Bewley, Carole A; Burton, Dennis R; Koff, Wayne C; Connors, Mark; Crispin, Max; Baxa, Ulrich; Korber, Bette T; Wong, Chi-Huey; Mascola, John R; Kwong, Peter D

2016-05-05

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer. Copyright © 2016 Elsevier Inc. All rights reserved.

Informatic innovations in glycobiology: relevance to drug discovery.

PubMed

Mamitsuka, Hiroshi

2008-02-01

The recent development and applications of tree-based informatics on glycans have accelerated the biological analysis on glycans, particularly from structural viewpoints. We review three major aspects of recent informatics innovations on glycan structures: maturity of well-organized databases on glycan structures linking with other biological information, implementation of glycan structure matching algorithms and extensive development of methods for mining frequent patterns from glycan structures.

Glycobiology simplified: diverse roles of glycan recognition in inflammation

PubMed Central

Schnaar, Ronald L.

2016-01-01

Glycans and complementary glycan-binding proteins are essential components in the language of cell-cell interactions in immunity. The study of glycan function is the purview of glycobiology, which has often been presented as an unusually complex discipline. In fact, the human glycome, composed of all of its glycans, is built primarily from only 9 building blocks that are combined by enzymes (writers) with specific and limited biosynthetic capabilities into a tractable and increasingly accessible number of potential glycan patterns that are functionally read by several dozen human glycan-binding proteins (readers). Nowhere is the importance of glycan recognition better understood than in infection and immunity, and knowledge in this area has already led to glycan mimetic anti-infective and anti-inflammatory drugs. This review includes a brief tutorial on human glycobiology and a limited number of specific examples of glycan-binding protein-glycan interactions that initiate and regulate inflammation. Examples include representatives from different glycan-binding protein families, including the C-type lectins (E-selectin, P-selectin, dectin-1, and dectin-2), sialic acid-binding immunoglobulin-like lectins (sialic acid-binding immunoglobulin-like lectins 8 and 9), galectins (galectin-1, galectin-3, and galectin-9), as well as hyaluronic acid-binding proteins. As glycoscience technologies advance, opportunities for enhanced understanding of glycans and their roles in leukocyte cell biology provide increasing opportunities for discovery and therapeutic intervention. PMID:27004978

Hydrazinonicotinic acid derivatization for selective ionization and improved glycan structure characterization by MALDI-MS.

PubMed

Jiao, Jing; Yang, Lijun; Zhang, Ying; Lu, Haojie

2015-08-21

The analysis of glycan is important for understanding cell biology and disease processes because the glycans play a key role in many important biological behaviors, such as cell division, cellular localization, tumor immunology and inflammation. Nevertheless, it is still hard work to analyze glycans by MALDI-MS, which generally stems from the inherent low abundance and the low ionization efficiency of glycans. Moreover, the difficulty in generating informative fragmentations further hinders glycans structure characterization. In this work, hydrazinonicotinic acid (HYNIC) was used as a novel derivatized reagent for improved and selective detection of glycans. Through tagging the reducing terminus of glycans with the diazanyl group of HYNIC, significant enhancement of the ionization efficiency of glycans was achieved. After derivatization, the signal to noise ratio (S/N) of the maltoheptaose was improved by more than one order of magnitude in positive mode. HYNIC derivatization also allowed the sensitive detection of sialylated glycan in negative mode, with a 15 fold enhancement of S/N. Interestingly, it is noteworthy that the HYNIC reagent not only effectively labeled the reducing end of glycans in the presence of tryptic peptides, but also suppressed the ionization of peptides, enabling the direct detection of glycans from glycoprotein without separation. Therefore, analysis of glycans became easier due to the omission of a pre-separation step. Importantly, by using different acid reagents as the catalyst, derivatized product signals corresponding to [M + Na](+) or [M + H](+) were obtained respectively, which yield complementary fragmentation patterns for the structure elucidation of glycans. Finally, more than 40 N-glycans were successfully detected in 10 μL human serum using this method.

Regulation of glycan structures in murine embryonic stem cells: combined transcript profiling of glycan-related genes and glycan structural analysis.

PubMed

Nairn, Alison V; Aoki, Kazuhiro; dela Rosa, Mitche; Porterfield, Mindy; Lim, Jae-Min; Kulik, Michael; Pierce, J Michael; Wells, Lance; Dalton, Stephen; Tiemeyer, Michael; Moremen, Kelley W

2012-11-02

The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development.

Glycan reductive isotope labeling for quantitative glycomics.

PubMed

Xia, Baoyun; Feasley, Christa L; Sachdev, Goverdhan P; Smith, David F; Cummings, Richard D

2009-04-15

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [(12)C(6)]aniline and [(13)C(6)]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.

GLYCAN REDUCTIVE ISOTOPE LABELING (GRIL) FOR QUANTITATIVE GLYCOMICS

PubMed Central

Xia, Baoyun; Feasley, Christa L.; Sachdev, Goverdhan P.; Smith, David F.; Cummings, Richard D.

2009-01-01

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed Glycan Reductive Isotope Labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [12C6]-aniline and [13C6]-aniline. These dual-labeled aniline-tagged glycans can be recovered by reversed-phase chromatography and quantified based on UV-absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins using this method. This technique allows for linear, relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of Glycomics. PMID:19454239

The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk.

PubMed

Sheng, Long; He, Zhenjiao; Chen, Jiahui; Liu, Yaofa; Ma, Meihu; Cai, Zhaoxia

2017-03-01

Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochloride-induced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular glycopathology by capillary electrophoresis: Analysis of the N-glycome of formalin-fixed paraffin-embedded mouse tissue samples.

PubMed

Donczo, Boglarka; Szarka, Mate; Tovari, Jozsef; Ostoros, Gyorgyi; Csanky, Eszter; Guttman, Andras

2017-06-01

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A python module to normalize microarray data by the quantile adjustment method.

PubMed

Baber, Ibrahima; Tamby, Jean Philippe; Manoukis, Nicholas C; Sangaré, Djibril; Doumbia, Seydou; Traoré, Sekou F; Maiga, Mohamed S; Dembélé, Doulaye

2011-06-01

Microarray technology is widely used for gene expression research targeting the development of new drug treatments. In the case of a two-color microarray, the process starts with labeling DNA samples with fluorescent markers (cyanine 635 or Cy5 and cyanine 532 or Cy3), then mixing and hybridizing them on a chemically treated glass printed with probes, or fragments of genes. The level of hybridization between a strand of labeled DNA and a probe present on the array is measured by scanning the fluorescence of spots in order to quantify the expression based on the quality and number of pixels for each spot. The intensity data generated from these scans are subject to errors due to differences in fluorescence efficiency between Cy5 and Cy3, as well as variation in human handling and quality of the sample. Consequently, data have to be normalized to correct for variations which are not related to the biological phenomena under investigation. Among many existing normalization procedures, we have implemented the quantile adjustment method using the python computer language, and produced a module which can be run via an HTML dynamic form. This module is composed of different functions for data files reading, intensity and ratio computations and visualization. The current version of the HTML form allows the user to visualize the data before and after normalization. It also gives the option to subtract background noise before normalizing the data. The output results of this module are in agreement with the results of other normalization tools. Published by Elsevier B.V.

Simple Sugars to Complex Disease—Mucin-Type O-Glycans in Cancer

PubMed Central

Kudelka, Matthew R.; Ju, Tongzhong; Heimburg-Molinaro, Jamie; Cummings, Richard D.

2017-01-01

Mucin-type O-glycans are a class of glycans initiated with N-acetylgalactosamine (GalNAc) α-linked primarily to Ser/Thr residues within glycoproteins and often extended or branched by sugars or saccharides. Most secretory and membrane-bound proteins receive this modification, which is important in regulating many biological processes. Alterations in mucin-type O-glycans have been described across tumor types and include expression of relatively small-sized, truncated O-glycans and altered terminal structures, both of which are associated with patient prognosis. New discoveries in the identity and expression of tumor-associated O-glycans are providing new avenues for tumor detection and treatment. This chapter describes mucin-type O-glycan biosynthesis, altered mucin-type O-glycans in primary tumors, including mechanisms for structural changes and contributions to the tumor phenotype, and clinical approaches to detect and target altered O-glycans for cancer treatment and management. PMID:25727146

Structural evolution of glycan recognition by a family of potent HIV antibodies.

PubMed

Garces, Fernando; Sok, Devin; Kong, Leopold; McBride, Ryan; Kim, Helen J; Saye-Francisco, Karen F; Julien, Jean-Philippe; Hua, Yuanzi; Cupo, Albert; Moore, John P; Paulson, James C; Ward, Andrew B; Burton, Dennis R; Wilson, Ian A

2014-09-25

The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans. Copyright © 2014 Elsevier Inc. All rights reserved.

Quantitation of permethylated N-glycans through multiple-reaction monitoring (MRM) LC-MS/MS.

PubMed

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

NASA Astrophysics Data System (ADS)

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

High-density fiber-optic DNA random microsphere array.

PubMed

Ferguson, J A; Steemers, F J; Walt, D R

2000-11-15

A high-density fiber-optic DNA microarray sensor was developed to monitor multiple DNA sequences in parallel. Microarrays were prepared by randomly distributing DNA probe-functionalized 3.1-microm-diameter microspheres in an array of wells etched in a 500-microm-diameter optical imaging fiber. Registration of the microspheres was performed using an optical encoding scheme and a custom-built imaging system. Hybridization was visualized using fluorescent-labeled DNA targets with a detection limit of 10 fM. Hybridization times of seconds are required for nanomolar target concentrations, and analysis is performed in minutes.

Glycan Reader is improved to recognize most sugar types and chemical modifications in the Protein Data Bank.

PubMed

Park, Sang-Jun; Lee, Jumin; Patel, Dhilon S; Ma, Hongjing; Lee, Hui Sun; Jo, Sunhwan; Im, Wonpil

2017-10-01

Glycans play a central role in many essential biological processes. Glycan Reader was originally developed to simplify the reading of Protein Data Bank (PDB) files containing glycans through the automatic detection and annotation of sugars and glycosidic linkages between sugar units and to proteins, all based on atomic coordinates and connectivity information. Carbohydrates can have various chemical modifications at different positions, making their chemical space much diverse. Unfortunately, current PDB files do not provide exact annotations for most carbohydrate derivatives and more than 50% of PDB glycan chains have at least one carbohydrate derivative that could not be correctly recognized by the original Glycan Reader. Glycan Reader has been improved and now identifies most sugar types and chemical modifications (including various glycolipids) in the PDB, and both PDB and PDBx/mmCIF formats are supported. CHARMM-GUI Glycan Reader is updated to generate the simulation system and input of various glycoconjugates with most sugar types and chemical modifications. It also offers a new functionality to edit the glycan structures through addition/deletion/modification of glycosylation types, sugar types, chemical modifications, glycosidic linkages, and anomeric states. The simulation system and input files can be used for CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Glycan Fragment Database in GlycanStructure.Org is also updated to provide an intuitive glycan sequence search tool for complex glycan structures with various chemical modifications in the PDB. http://www.charmm-gui.org/input/glycan and http://www.glycanstructure.org. wonpil@lehigh.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

21 CFR 172.898 - Bakers yeast glycan.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

21 CFR 172.898 - Bakers yeast glycan.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

21 CFR 172.898 - Bakers yeast glycan.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Bakers yeast glycan. 172.898 Section 172.898 Food... Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and dried cell walls of the yeast...

The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.

PubMed

Kronewitter, Scott R; An, Hyun Joo; de Leoz, Maria Lorna; Lebrilla, Carlito B; Miyamoto, Suzanne; Leiserowitz, Gary S

2009-06-01

Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.

N-Linked Glycan Profiling of Mature Human Milk by High Performance Microfluidic Chip Liquid Chromatography Time-of-Flight Tandem Mass Spectrometry

PubMed Central

Dallas, David C.; Martin, William F.; Strum, John S.; Zivkovic, Angela M.; Smilowitz, Jennifer T.; Underwood, Mark A.; Affolter, Michael; Lebrilla, Carlito B.; German, J. Bruce

2015-01-01

N-linked glycans of skim human milk proteins were determined for three mothers. N-linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with Peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library, be at least two-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was comprised of N-glycans found in all three milk samples. PMID:21384928

Glycan array data management at Consortium for Functional Glycomics.

PubMed

Venkataraman, Maha; Sasisekharan, Ram; Raman, Rahul

2015-01-01

Glycomics or the study of structure-function relationships of complex glycans has reshaped post-genomics biology. Glycans mediate fundamental biological functions via their specific interactions with a variety of proteins. Recognizing the importance of glycomics, large-scale research initiatives such as the Consortium for Functional Glycomics (CFG) were established to address these challenges. Over the past decade, the Consortium for Functional Glycomics (CFG) has generated novel reagents and technologies for glycomics analyses, which in turn have led to generation of diverse datasets. These datasets have contributed to understanding glycan diversity and structure-function relationships at molecular (glycan-protein interactions), cellular (gene expression and glycan analysis), and whole organism (mouse phenotyping) levels. Among these analyses and datasets, screening of glycan-protein interactions on glycan array platforms has gained much prominence and has contributed to cross-disciplinary realization of the importance of glycomics in areas such as immunology, infectious diseases, cancer biomarkers, etc. This manuscript outlines methodologies for capturing data from glycan array experiments and online tools to access and visualize glycan array data implemented at the CFG.

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

PubMed

Anumula, Kalyan Rao

2012-07-01

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

Functional Characterization of HFR1, a High-Mannose N-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae1[C][W

PubMed Central

Subramanyam, Subhashree; Smith, David F.; Clemens, James C.; Webb, Mary A.; Sardesai, Nagesh; Williams, Christie E.

2008-01-01

We previously cloned and characterized a novel jacalin-like lectin gene from wheat (Triticum aestivum) plants that responds to infestation by Hessian fly (Mayetiola destructor) larvae, a major dipteran pest of this crop. The infested resistant plants accumulated higher levels of Hfr-1 (for Hessian fly-responsive gene 1) transcripts compared with uninfested or susceptible plants. Here, we characterize the soluble and active recombinant His6-HFR1 protein isolated from Escherichia coli. Functional characterization of the protein using hemagglutination assays revealed lectin activity. Glycan microarray-binding assays indicated strong affinity of His6-HFR1 to Manα1-6(Manα1-3)Man trisaccharide structures. Resistant wheat plants accumulated high levels of HFR1 at the larval feeding sites, as revealed by immunodetection, but the avirulent larvae were deterred from feeding and consumed only small amounts of the lectin. Behavioral studies revealed that avirulent Hessian fly larvae on resistant plants exhibited prolonged searching and writhing behaviors as they unsuccessfully attempted to establish feeding sites. During His6-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percentage mortality increased with progressively higher concentrations of His6-HFR1 in the diet. Thus, HFR1 is an antinutrient to dipteran larvae and may play a significant role in deterring Hessian fly larvae from feeding on resistant wheat plants. PMID:18467454

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

PubMed

McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

2015-07-01

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

Context-specific target definition in influenza a virus hemagglutinin-glycan receptor interactions.

PubMed

Shriver, Zachary; Raman, Rahul; Viswanathan, Karthik; Sasisekharan, Ram

2009-08-28

Protein-glycan interactions are important regulators of a variety of biological processes, ranging from immune recognition to anticoagulation. An important area of active research is directed toward understanding the role of host cell surface glycans as recognition sites for pathogen protein receptors. Recognition of cell surface glycans is a widely employed strategy for a variety of pathogens, including bacteria, parasites, and viruses. We present here a representative example of such an interaction: the binding of influenza A hemagglutinin (HA) to specific sialylated glycans on the cell surface of human upper airway epithelial cells, which initiates the infection cycle. We detail a generalizable strategy to understand the nature of protein-glycan interactions both structurally and biochemically, using HA as a model system. This strategy combines a top-down approach using available structural information to define important contacts between glycans and HA, with a bottom-up approach using data-mining and informatics approaches to identify the common motifs that distinguish glycan binders from nonbinders. By probing protein-glycan interactions simultaneously through top-down and bottom-up approaches, we can scientifically validate a series of observations. This in turn provides additional confidence and surmounts known challenges in the study of protein-glycan interactions, such as accounting for multivalency, and thus truly defines concepts such as specificity, affinity, and avidity. With the advent of new technologies for glycomics-including glycan arrays, data-mining solutions, and robust algorithms to model protein-glycan interactions-we anticipate that such combination approaches will become tractable for a wide variety of protein-glycan interactions.

Spatially selective photonic crystal enhanced fluorescence and application to background reduction for biomolecule detection assays

PubMed Central

Chaudhery, Vikram; Huang, Cheng-Sheng; Pokhriyal, Anusha; Polans, James; Cunningham, Brian T.

2011-01-01

By combining photonic crystal label-free biosensor imaging with photonic crystal enhanced fluorescence, it is possible to selectively enhance the fluorescence emission from regions of the PC surface based upon the density of immobilized capture molecules. A label-free image of the capture molecules enables determination of optimal coupling conditions of the laser used for fluorescence imaging of the photonic crystal surface on a pixel-by-pixel basis, allowing maximization of fluorescence enhancement factor from regions incorporating a biomolecule capture spot and minimization of background autofluorescence from areas between capture spots. This capability significantly improves the contrast of enhanced fluorescent images, and when applied to an antibody protein microarray, provides a substantial advantage over conventional fluorescence microscopy. Using the new approach, we demonstrate detection limits as low as 0.97 pg/ml for a representative protein biomarker in buffer. PMID:22109210

Spatially selective photonic crystal enhanced fluorescence and application to background reduction for biomolecule detection assays.

PubMed

Chaudhery, Vikram; Huang, Cheng-Sheng; Pokhriyal, Anusha; Polans, James; Cunningham, Brian T

2011-11-07

By combining photonic crystal label-free biosensor imaging with photonic crystal enhanced fluorescence, it is possible to selectively enhance the fluorescence emission from regions of the PC surface based upon the density of immobilized capture molecules. A label-free image of the capture molecules enables determination of optimal coupling conditions of the laser used for fluorescence imaging of the photonic crystal surface on a pixel-by-pixel basis, allowing maximization of fluorescence enhancement factor from regions incorporating a biomolecule capture spot and minimization of background autofluorescence from areas between capture spots. This capability significantly improves the contrast of enhanced fluorescent images, and when applied to an antibody protein microarray, provides a substantial advantage over conventional fluorescence microscopy. Using the new approach, we demonstrate detection limits as low as 0.97 pg/ml for a representative protein biomarker in buffer.

Demonstration of hydrazide tagging for O-glycans and a central composite design of experiments optimization using the INLIGHT™ reagent.

PubMed

King, Samuel R; Hecht, Elizabeth S; Muddiman, David C

2018-02-01

The INLIGHT™ strategy for N-linked glycan derivatization has been shown to overcome many of the challenges associated with glycan analysis. The hydrazide tag reacts efficiently with the glycans, increasing their non-polar surface area, allowing for reversed-phase separations and increased ionization efficiency. We have taken the INLIGHT™ strategy and adopted it for use with O-linked glycans. A central composite design was utilized to find optimized tagging conditions (45% acetic acid, 0.1 μg/μL tag concentration, 37 C, 1.75 h). Derivatization at optimized conditions was much quicker than any hydrazide derivatization strategy used previously. Human immunoglobulin A (IgA) and bovine submaxillary mucin (BSM) were then deglycosylated through hydrazinolysis and the removed glycans were tagged under optimum conditions. XIC of tagged glycans and MS2 data show successful hydrazide tagging of O-linked glycans for the first time. Graphical abstract The INLIGHT™ hydrazide tag was optimized using a central composite design for derivatization of O-linked glycans. Two glycoprotein standards were deglycosylated through hydrazinolysis and tagged at the optimized conditions. MS/MS data shows INLIGHT™ derivatization of glycans demonstrating successful hydrazide tagging of O-glycans for the first time.

The logic of automated glycan assembly.

PubMed

Seeberger, Peter H

2015-05-19

Carbohydrates are the most abundant biopolymers on earth and part of every living creature. Glycans are essential as materials for nutrition and for information transfer in biological processes. To date, in few cases a detailed correlation between glycan structure and glycan function has been established. A molecular understanding of glycan function will require pure glycans for biological, immunological, and structural studies. Given the immense structural complexity of glycans found in living organisms and the lack of amplification methods or expression systems, chemical synthesis is the only means to access usable quantities of pure glycan molecules. While the solid-phase synthesis of DNA and peptides has become routine for decades, access to glycans has been technically difficult, time-consuming and confined to a few expert laboratories. In this Account, the development of a comprehensive approach to the automated synthesis of all classes of mammalian glycans, including glycosaminoglycans and glycosylphosphatidyl inositol (GPI) anchors, as well as bacterial and plant carbohydrates is described. A conceptual advance concerning the logic of glycan assembly was required in order to enable automated execution of the synthetic process. Based on the central glycosidic bond forming reaction, a general concept for the protecting groups and leaving groups has been developed. Building blocks that can be procured on large scale, are stable for prolonged periods of time, but upon activation result in high yields and selectivities were identified. A coupling-capping and deprotection cycle was invented that can be executed by an automated synthesis instrument. Straightforward postsynthetic protocols for cleavage from the solid support as well as purification of conjugation-ready oligosaccharides have been established. Introduction of methods to install selectively a wide variety of glycosidic linkages has enabled the rapid assembly of linear and branched oligo- and polysaccharides as large as 30-mers. Fast, reliable access to defined glycans that are ready for conjugation has given rise to glycan arrays, glycan probes, and synthetic glycoconjugate vaccines. While an ever increasing variety of glycans are accessible by automated synthesis, further methodological advances in carbohydrate chemistry are needed to make all possible glycans found in nature. These tools begin to fundamentally impact the medical but also materials aspects of the glycosciences.

The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures.

PubMed

Ceroni, Alessio; Dell, Anne; Haslam, Stuart M

2007-08-07

Carbohydrates play a critical role in human diseases and their potential utility as biomarkers for pathological conditions is a major driver for characterization of the glycome. However, the additional complexity of glycans compared to proteins and nucleic acids has slowed the advancement of glycomics in comparison to genomics and proteomics. The branched nature of carbohydrates, the great diversity of their constituents and the numerous alternative symbolic notations, make the input and display of glycans not as straightforward as for example the amino-acid sequence of a protein. Every glycoinformatic tool providing a user interface would benefit from a fast, intuitive, appealing mechanism for input and output of glycan structures in a computer readable format. A software tool for building and displaying glycan structures using a chosen symbolic notation is described here. The "GlycanBuilder" uses an automatic rendering algorithm to draw the saccharide symbols and to place them on the drawing board. The information about the symbolic notation is derived from a configurable graphical model as a set of rules governing the aspect and placement of residues and linkages. The algorithm is able to represent a structure using only few traversals of the tree and is inherently fast. The tool uses an XML format for import and export of encoded structures. The rendering algorithm described here is able to produce high-quality representations of glycan structures in a chosen symbolic notation. The automated rendering process enables the "GlycanBuilder" to be used both as a user-independent component for displaying glycans and as an easy-to-use drawing tool. The "GlycanBuilder" can be integrated in web pages as a Java applet for the visual editing of glycans. The same component is available as a web service to render an encoded structure into a graphical format. Finally, the "GlycanBuilder" can be integrated into other applications to create intuitive and appealing user interfaces: an example is the "GlycoWorkbench", a software tool for assisted annotation of glycan mass spectra. The "GlycanBuilder" represent a flexible, reliable and efficient solution to the problem of input and output of glycan structures in any glycomic tool or database.

A novel surface modification approach for protein and cell microarrays

NASA Astrophysics Data System (ADS)

Kurkuri, Mahaveer D.; Driever, Chantelle; Thissen, Helmut W.; Voelcker, Nicholas H.

2007-01-01

Tissue engineering and stem cell technologies have led to a rapidly increasing interest in the control of the behavior of mammalian cells growing on tissue culture substrates. Multifunctional polymer coatings can assist research in this area in many ways, for example, by providing low non-specific protein adsorption properties and reactive functional groups at the surface. The latter can be used for immobilization of specific biological factors that influence cell behavior. In this study, glass slides were coated with copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). The coatings were prepared by three different methods based on dip and spin coating as well as polymer grafting procedures. Coatings were characterized by X-ray photoelectron spectroscopy, surface sensitive infrared spectroscopy, ellipsometry and contact angle measurements. A fluorescently labelled protein was deposited onto reactive coatings using a contact microarrayer. Printing of a model protein (fluorescein labeled bovine serum albumin) was performed at different protein concentrations, pH, temperature, humidity and using different micropins. The arraying of proteins was studied with a microarray scanner. Arrays printed at a protein concentration above 50 μg/mL prepared in pH 5 phosphate buffer at 10°C and 65% relative humidity gave the most favourable results in terms of the homogeneity of the printed spots and the fluorescence intensity.

Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.

PubMed

Lee, Patrick K H; Men, Yujie; Wang, Shanquan; He, Jianzhong; Alvarez-Cohen, Lisa

2015-02-03

Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ∼1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ∼10% and ∼1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.

Improving N-Glycan Coverage using HPLC-MS with Electrospray Ionization at Subambient Pressure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marginean, Ioan; Kronewitter, Scott R.; Moore, Ronald J.

Human serum glycan profiling with mass spectrometry (MS) has been employed to study several disease conditions and is demonstrating promise for e.g. clinical biomarker discovery. However, the poor glycan ionization efficiency and the large dynamic range of glycan concentrations in human sera hinder comprehensive profiling. In particular, large glycans are problematic because they are present at low concentrations and prone to fragmentation. Here we show that the sub-ambient pressure ionization with nanoelectrospray (SPIN)-MS can expand the serum glycome profile when compared with the conventional atmospheric pressure electrospray ionization (ESI)-MS with a heated capillary inlet. Notably, the ions generated by themore » SPIN interface were observed at higher charge states for 50% of the annotated glycans. Out of a total of 130 detected glycans, 34 were only detected with the SPIN-MS, resulting in improved coverage of glycan families as well as of glycans with larger numbers of labile monosaccharides.« less

N-glycans in liver-secreted and immunoglogulin-derived protein fractions

PubMed Central

Bekesova, S.; Kosti, O.; Chandler, K.B.; Wu, J.; Madej, H.L.; Brown, K.C.; Simonyan, V.; Goldman, R.

2013-01-01

N-glycosylation of proteins provides a rich source of information on liver disease progression because majority of serum glycoproteins, with the exception of immunoglobulins, are secreted by the liver. In this report, we present results of an optimized workflow for MALDI-TOF analysis of permethylated N-glycans detached from serum proteins and separated into liver secreted and immunoglobulin fractions. We have compared relative intensities of N-glycans in 23 healthy controls and 23 cirrhosis patients. We were able to detect 82 N-glycans associated primarily with liver secreted glycoproteins, 54 N-glycans in the protein G bound fraction and 52 N-glycans in the fraction bound to protein A. The N-glycan composition of the fractions differed substantially, independent of liver disease. The relative abundance of approximately 53% N-glycans in all fractions was significantly altered in the cirrhotic liver. The removal of immunoglobulins allowed detection of an increase in a series of high mannose and hybrid N-glycans associated with the liver secreted protein fraction. PMID:22326963

Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.

PubMed

Anumula, Kalyan Rao

2012-08-31

Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation of IgG glycans (neutral (G0, G1, and G2), mono- and di-sialylated) using simple procedures. The method should prove useful for monitoring glycan changes in clinical settings. Copyright © 2012 Elsevier B.V. All rights reserved.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

PubMed

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis

PubMed Central

2011-01-01

Background Cytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence in situ hybridization (FISH). Results Using a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23. Conclusions Our results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future. PMID:22087757

Simple and Robust N-Glycan Analysis Based on Improved 2-Aminobenzoic Acid Labeling for Recombinant Therapeutic Glycoproteins.

PubMed

Jeong, Yeong Ran; Kim, Sun Young; Park, Young Sam; Lee, Gyun Min

2018-03-21

N-glycans of therapeutic glycoproteins are critical quality attributes that should be monitored throughout all stages of biopharmaceutical development. To reduce both the time for sample preparation and the variations in analytical results, we have developed an N-glycan analysis method that includes improved 2-aminobenzoic acid (2-AA) labeling to easily remove deglycosylated proteins. Using this analytical method, 15 major 2-AA-labeled N-glycans of Enbrel ® were separated into single peaks in hydrophilic interaction chromatography mode and therefore could be quantitated. 2-AA-labeled N-glycans were also highly compatible with in-line quadrupole time-of-flight mass spectrometry (MS) for structural identification. The structures of 15 major and 18 minor N-glycans were identified from their mass values determined by quadrupole time-of-flight MS. Furthermore, the structures of 14 major N-glycans were confirmed by interpreting the MS/MS data of each N-glycan. This analytical method was also successfully applied to neutral N-glycans of Humira ® and highly sialylated N-glycans of NESP ® . Furthermore, the analysis data of Enbrel ® that were accumulated for 2.5 years demonstrated the high-level consistency of this analytical method. Taken together, the results show that a wide repertoire of N-glycans of therapeutic glycoproteins can be analyzed with high efficiency and consistency using the improved 2-AA labeling-based N-glycan analysis method. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Controlling false-negative errors in microarray differential expression analysis: a PRIM approach.

PubMed

Cole, Steve W; Galic, Zoran; Zack, Jerome A

2003-09-22

Theoretical considerations suggest that current microarray screening algorithms may fail to detect many true differences in gene expression (Type II analytic errors). We assessed 'false negative' error rates in differential expression analyses by conventional linear statistical models (e.g. t-test), microarray-adapted variants (e.g. SAM, Cyber-T), and a novel strategy based on hold-out cross-validation. The latter approach employs the machine-learning algorithm Patient Rule Induction Method (PRIM) to infer minimum thresholds for reliable change in gene expression from Boolean conjunctions of fold-induction and raw fluorescence measurements. Monte Carlo analyses based on four empirical data sets show that conventional statistical models and their microarray-adapted variants overlook more than 50% of genes showing significant up-regulation. Conjoint PRIM prediction rules recover approximately twice as many differentially expressed transcripts while maintaining strong control over false-positive (Type I) errors. As a result, experimental replication rates increase and total analytic error rates decline. RT-PCR studies confirm that gene inductions detected by PRIM but overlooked by other methods represent true changes in mRNA levels. PRIM-based conjoint inference rules thus represent an improved strategy for high-sensitivity screening of DNA microarrays. Freestanding JAVA application at http://microarray.crump.ucla.edu/focus

Nanotechnology: moving from microarrays toward nanoarrays.

PubMed

Chen, Hua; Li, Jun

2007-01-01

Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

Reductive chemical release of N-glycans as 1-amino-alditols and subsequent 9-fluorenylmethyloxycarbonyl labeling for MS and LC/MS analysis.

PubMed

Wang, Chengjian; Qiang, Shan; Jin, Wanjun; Song, Xuezheng; Zhang, Ying; Huang, Linjuan; Wang, Zhongfu

2018-06-06

Glycoproteins play pivotal roles in a series of biological processes and their glycosylation patterns need to be structurally and functionally characterized. However, the lack of versatile methods to release N-glycans as functionalized forms has been undermining glycomics studies. Here a novel method is developed for dissociation of N-linked glycans from glycoproteins for analysis by MS and online LC/MS. This new method employs aqueous ammonia solution containing NaBH 3 CN as the reaction medium to release glycans from glycoproteins as 1-amino-alditol forms. The released glycans are conveniently labeled with 9-fluorenylmethyloxycarbonyl (Fmoc) and analyzed by ESI-MS and online LC/MS. Using the method, the neutral and acidic N-glycans were successfully released without peeling degradation of the core α-1,3-fucosylated structure or detectable de-N-acetylation, revealing its general applicability to various types of N-glycans. The Fmoc-derivatized N-glycans derived from chicken ovalbumin, Fagopyrum esculentum Moench Pollen and FBS were successfully analyzed by online LC/MS to distinguish isomers. The 1-amino-alditols were also permethylated to form quaternary ammonium cations at the reducing end, which enhance the MS sensitivity and are compatible with sequential multi-stage mass spectrometry (MS n ) fragmentation for glycan sequencing. The Fmoc-labeled N-glycans were further permethylated to produce methylated carbamates for determination of branches and linkages by sequential MS n fragmentation. N-Glycosylation represents one of the most common post-translational modification forms and plays pivotal roles in the structural and functional regulation of proteins in various biological activities, relating closely to human health and diseases. As a type of informational molecule, the N-glycans of glycoproteins participate directly in the molecular interactions between glycan epitopes and their corresponding protein receptors. Detailed structural and functional characterization of different types of N-glycans is essential for understanding the functional mechanisms of many biological activities and the pathologies of many diseases. Here we describe a simple, versatile method to indistinguishably release all types of N-glycans as functionalized forms without remarkable side reactions, enabling convenient, rapid analysis and preparation of released N-glycans from various complex biological samples. It is very valuable for studies on the complicated structure-function relationship of N-glycans, as well as for the search of N-glycan biomarkers of some major diseases and N-glycan related targets of some drugs. Copyright © 2018. Published by Elsevier B.V.

Structural features of N-glycans linked to glycoproteins expressed in three kinds of water plants: Predominant occurrence of the plant complex type N-glycans bearing Lewis a epitope.

PubMed

Maeda, Megumi; Tani, Misato; Yoshiie, Takeo; Vavricka, Christopher J; Kimura, Yoshinobu

2016-11-29

The Japanese cedar pollen allergen (Cry j1) and the mountain cedar pollen allergen (Jun a1) are glycosylated with plant complex type N-glycans bearing Lewis a epitope(s) (Galβ1-3[Fucα1-4]GlcNAc-). The biological significance of Lewis a type plant N-glycans and their effects on the human immune system remain to be elucidated. Since a substantial amount of such plant specific N-glycans are required to evaluate immunological activity, we have searched for good plant-glycan sources to characterize Lewis a epitope-containing plant N-glycans. In this study, we have found that three water plants, Elodea nuttallii, Egeria densa, and Ceratophyllum demersum, produce glycoproteins bearing Lewis a units. Structural analysis of the N-glycans revealed that almost all glycoproteins expressed in these three water plants predominantly carry plant complex type N-glycans including the Lewis a type, suggesting that these water plants are good sources for preparation of Lewis a type plant N-glycans in substantial amounts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans.

PubMed

Tsukimura, Wataru; Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Matsuda, Akio; Takegawa, Kaoru; Furukawa, Kiyoshi; Shirai, Takashi

2017-12-01

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.

Glycan gimmickry by parasitic helminths: a strategy for modulating the host immune response?

PubMed

van Die, Irma; Cummings, Richard D

2010-01-01

Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/ secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le(X) (Galbeta1-4[Fucalpha1-3]GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-), and Tn (GalNAcalpha1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry," in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry," which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.

Characterization of Pharmaceutical IgG and Biosimilars Using Miniaturized Platforms and LC-MS/MS

PubMed Central

Wooding, Kerry M.; Peng, Wenjing; Mechref, Yehia

2016-01-01

Therapeutic monoclonal antibodies (mAbs) have made a tremendous impact in treating patients with various diseases. MAbs are designed to target specifically a cell and illicit a response from the immune system to destroy the cell. As originator mAb drug patents are coming to an end, generic pharmaceutical companies are poised to replicate and produce so-called biosimilar drugs. MAbs are significantly more complicated than small drugs to analyze and produce. The mAb proteoform and glycoform must be as similar to the original drug as possible to be a viable replacement. The mAb proteoform is well characterized but can be altered through various undesirable reactions such as deamidation. The mAb glycoform is harder to replicate as the glycan formation is a complicated template-less one; it is proving difficult for the originator companies to produce a homogenous population of mAbs from batch to batch. Severe side-effects have occurred in patients taking mAbs with immunogenic glycans, highlighting the importance of quality control mechanisms. The complex nature of mAbs requires sensitive and robust tools amenable to the high-throughput analysis required by a manufacturing setting. Miniaturized analytical platforms for complex biosimilar analysis are still in their infancy but have shown great promise for sample preparation. Capillary electrophoresis-laser induced fluorescence remains a powerful and fast technique for routine glycan analysis. Mass spectrometry is the method of choice for the analysis of mAb proteoforms and is emerging as a powerful tool for glycoform analysis. PMID:27033511

Chemoenzymatic assembly of mammalian O-mannose glycans.

PubMed

Cao, Hongzhi; Meng, Caicai; Sasmal, Aniruddha; Zhang, Yan; Gao, Tian; Liu, Chang-Cheng; Khan, Naazneen; Varki, Ajit; Wang, Fengshan

2018-05-26

O-Mannose glycans account up to 30% of total O-glycans in brain. Previous synthesis and functional studies only focused on the Core M3 O-mannose glycans of α-dystroglycan which are a causative factor for various muscular diseases. In this study, a highly efficient chemoenzymatic strategy was developed that enabled the first collective synthesis of 63 Core M1 and Core M2 O-mannose glycans. This chemoenzymatic strategy features the gram-scale chemical synthesis of 5 judiciously designed core structures, and the diversity-oriented modification of the core structures with 3 enzyme modules to provide 58 complex O-mannose glycans in a linear sequence that does not exceed 4 steps. The binding profiles of synthetic O-mannose glycans with a panel of lectins, antibodies and brain proteins were also explored using the printed O-mannose glycan array. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reciprocal Prioritization to Dietary Glycans by Gut Bacteria in a Competitive Environment Promotes Stable Coexistence

PubMed Central

Tuncil, Yunus E.; Xiao, Yao; Porter, Nathan T.; Reuhs, Bradley L.

2017-01-01

ABSTRACT When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome. PMID:29018117

Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications

NASA Astrophysics Data System (ADS)

Geissner, Andreas; Seeberger, Peter H.

2016-06-01

A major branch of glycobiology and glycan-focused biomedicine studies the interaction between carbohydrates and other biopolymers, most importantly, glycan-binding proteins. Today, this research into glycan-biopolymer interaction is unthinkable without glycan arrays, tools that enable high-throughput analysis of carbohydrate interaction partners. Glycan arrays offer many applications in basic biochemical research, for example, defining the specificity of glycosyltransferases and lectins such as immune receptors. Biomedical applications include the characterization and surveillance of influenza strains, identification of biomarkers for cancer and infection, and profiling of immune responses to vaccines. Here, we review major applications of glycan arrays both in basic and applied research. Given the dynamic nature of this rapidly developing field, we focus on recent findings.

Glycomics: revealing the dynamic ecology and evolution of sugar molecules.

PubMed

Springer, Stevan A; Gagneux, Pascal

2016-03-01

Sugars are the most functionally and structurally diverse molecules in the biological world. Glycan structures range from tiny single monosaccharide units to giant chains thousands of units long. Some glycans are branched, their monosaccharides linked together in many different combinations and orientations. Some exist as solitary molecules; others are conjugated to proteins and lipids and alter their collective functional properties. In addition to structural and storage roles, glycan molecules participate in and actively regulate physiological and developmental processes. Glycans also mediate cellular interactions within and between individuals. Their roles in ecology and evolution are pivotal, but not well studied because glycan biochemistry requires different methods than standard molecular biology practice. The properties of glycans are in some ways convenient, and in others challenging. Glycans vary on organismal timescales, and in direct response to physiological and ecological conditions. Their mature structures are physical records of both genetic and environmental influences during maturation. We describe the scope of natural glycan variation and discuss how studying glycans will allow researchers to further integrate the fields of ecology and evolution. Copyright © 2015 Elsevier B.V. All rights reserved.

Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems—The taliglucerase alfa story

PubMed Central

Rup, Bonita; Alon, Sari; Amit-Cohen, Bat-Chen; Brill Almon, Einat; Chertkoff, Raul; Rudd, Pauline M.

2017-01-01

Plants are a promising alternative for the production of biotherapeutics. Manufacturing in-planta adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies. PMID:29088235

Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems-The taliglucerase alfa story.

PubMed

Rup, Bonita; Alon, Sari; Amit-Cohen, Bat-Chen; Brill Almon, Einat; Chertkoff, Raul; Tekoah, Yoram; Rudd, Pauline M

2017-01-01

Plants are a promising alternative for the production of biotherapeutics. Manufacturing in-planta adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies.

Advances in the Biology and Chemistry of Sialic Acids

PubMed Central

Chen, Xi; Varki, Ajit

2010-01-01

Sialic acids are a subset of nonulosonic acids, which are nine-carbon alpha-keto aldonic acids. Natural existing sialic acid-containing structures are presented in different sialic acid forms, various sialyl linkages, and on diverse underlying glycans. They play important roles in biological, pathological, and immunological processes. Sialobiology has been a challenging and yet attractive research area. Recent advances in chemical and chemoenzymatic synthesis as well as large-scale E. coli cell-based production have provided a large library of sialoside standards and derivatives in amounts sufficient for structure-activity relationship studies. Sialoglycan microarrays provide an efficient platform for quick identification of preferred ligands for sialic acid-binding proteins. Future research on sialic acid will continue to be at the interface of chemistry and biology. Research efforts will not only lead to a better understanding of the biological and pathological importance of sialic acids and their diversity, but could also lead to the development of therapeutics. PMID:20020717

Oligonucleotide microarray for subtyping of influenza A viruses

NASA Astrophysics Data System (ADS)

Klotchenko, S. A.; Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Chervyakova, O. V.; Smirnova, E. A.; Kushnareva, E. V.; Strochkov, V. M.; Taylakova, E. T.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Sansyzbay, A. R.

2012-02-01

Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

A Universal Protocol for Photochemical Covalent Immobilization of Intact Carbohydrates for the Preparation of Carbohydrate Microarrays

PubMed Central

Wang, Huibin; Zhang, Yiming; Yuan, Xun; Chen, Yi; Yan, Mingdi

2010-01-01

A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, non-reducing sugars such as alditols and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging. PMID:21138274

A universal protocol for photochemical covalent immobilization of intact carbohydrates for the preparation of carbohydrate microarrays.

PubMed

Wang, Huibin; Zhang, Yiming; Yuan, Xun; Chen, Yi; Yan, Mingdi

2011-01-19

A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, nonreducing sugars such as alditols, and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose, and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging.

Development of Rare Bacterial Monosaccharide Analogs for Metabolic Glycan Labeling in Pathogenic Bacteria.

PubMed

Clark, Emily L; Emmadi, Madhu; Krupp, Katharine L; Podilapu, Ananda R; Helble, Jennifer D; Kulkarni, Suvarn S; Dube, Danielle H

2016-12-16

Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.

Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): Characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans

PubMed Central

Herget, Stephan; Toukach, Philip V; Ranzinger, René; Hull, William E; Knirel, Yuriy A; von der Lieth, Claus-Wilhelm

2008-01-01

Background There are considerable differences between bacterial and mammalian glycans. In contrast to most eukaryotic carbohydrates, bacterial glycans are often composed of repeating units with diverse functions ranging from structural reinforcement to adhesion, colonization and camouflage. Since bacterial glycans are typically displayed at the cell surface, they can interact with the environment and, therefore, have significant biomedical importance. Results The sequence characteristics of glycans (monosaccharide composition, modifications, and linkage patterns) for the higher bacterial taxonomic classes have been examined and compared with the data for mammals, with both similarities and unique features becoming evident. Compared to mammalian glycans, the bacterial glycans deposited in the current databases have a more than ten-fold greater diversity at the monosaccharide level, and the disaccharide pattern space is approximately nine times larger. Specific bacterial subclasses exhibit characteristic glycans which can be distinguished on the basis of distinctive structural features or sequence properties. Conclusion For the first time a systematic database analysis of the bacterial glycome has been performed. This study summarizes the current knowledge of bacterial glycan architecture and diversity and reveals putative targets for the rational design and development of therapeutic intervention strategies by comparing bacterial and mammalian glycans. PMID:18694500

Dual Modifications Strategy to Quantify Neutral and Sialylated N-Glycans Simultaneously by MALDI-MS

PubMed Central

2015-01-01

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-13[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans. PMID:24766348

Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS.

PubMed

Zhou, Hui; Warren, Peter G; Froehlich, John W; Lee, Richard S

2014-07-01

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays

PubMed Central

Goodman, Corey W.; Major, Heather J.; Walls, William D.; Sheffield, Val C.; Casavant, Thomas L.; Darbro, Benjamin W.

2016-01-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. PMID:25595567

Biological roles of glycans

PubMed Central

Varki, Ajit

2017-01-01

Abstract Simple and complex carbohydrates (glycans) have long been known to play major metabolic, structural and physical roles in biological systems. Targeted microbial binding to host glycans has also been studied for decades. But such biological roles can only explain some of the remarkable complexity and organismal diversity of glycans in nature. Reviewing the subject about two decades ago, one could find very few clear-cut instances of glycan-recognition-specific biological roles of glycans that were of intrinsic value to the organism expressing them. In striking contrast there is now a profusion of examples, such that this updated review cannot be comprehensive. Instead, a historical overview is presented, broad principles outlined and a few examples cited, representing diverse types of roles, mediated by various glycan classes, in different evolutionary lineages. What remains unchanged is the fact that while all theories regarding biological roles of glycans are supported by compelling evidence, exceptions to each can be found. In retrospect, this is not surprising. Complex and diverse glycans appear to be ubiquitous to all cells in nature, and essential to all life forms. Thus, >3 billion years of evolution consistently generated organisms that use these molecules for many key biological roles, even while sometimes coopting them for minor functions. In this respect, glycans are no different from other major macromolecular building blocks of life (nucleic acids, proteins and lipids), simply more rapidly evolving and complex. It is time for the diverse functional roles of glycans to be fully incorporated into the mainstream of biological sciences. PMID:27558841

Property Graph vs RDF Triple Store: A Comparison on Glycan Substructure Search

PubMed Central

Alocci, Davide; Mariethoz, Julien; Horlacher, Oliver; Bolleman, Jerven T.; Campbell, Matthew P.; Lisacek, Frederique

2015-01-01

Resource description framework (RDF) and Property Graph databases are emerging technologies that are used for storing graph-structured data. We compare these technologies through a molecular biology use case: glycan substructure search. Glycans are branched tree-like molecules composed of building blocks linked together by chemical bonds. The molecular structure of a glycan can be encoded into a direct acyclic graph where each node represents a building block and each edge serves as a chemical linkage between two building blocks. In this context, Graph databases are possible software solutions for storing glycan structures and Graph query languages, such as SPARQL and Cypher, can be used to perform a substructure search. Glycan substructure searching is an important feature for querying structure and experimental glycan databases and retrieving biologically meaningful data. This applies for example to identifying a region of the glycan recognised by a glycan binding protein (GBP). In this study, 19,404 glycan structures were selected from GlycomeDB (www.glycome-db.org) and modelled for being stored into a RDF triple store and a Property Graph. We then performed two different sets of searches and compared the query response times and the results from both technologies to assess performance and accuracy. The two implementations produced the same results, but interestingly we noted a difference in the query response times. Qualitative measures such as portability were also used to define further criteria for choosing the technology adapted to solving glycan substructure search and other comparable issues. PMID:26656740

Quantitative O-glycomics based on improvement of the one-pot method for nonreductive O-glycan release and simultaneous stable isotope labeling with 1-(d0/d5)phenyl-3-methyl-5-pyrazolone followed by mass spectrometric analysis.

PubMed

Wang, Chengjian; Zhang, Ping; Jin, Wanjun; Li, Lingmei; Qiang, Shan; Zhang, Ying; Huang, Linjuan; Wang, Zhongfu

2017-01-06

Rapid, simple and versatile methods for quantitative analysis of glycoprotein O-glycans are urgently required for current studies on protein O-glycosylation patterns and the search for disease O-glycan biomarkers. Relative quantitation of O-glycans using stable isotope labeling followed by mass spectrometric analysis represents an ideal and promising technique. However, it is hindered by the shortage of reliable nonreductive O-glycan release methods as well as the too large or too small inconstant mass difference between the light and heavy isotope form derivatives of O-glycans, which results in difficulties during the recognition and quantitative analysis of O-glycans by mass spectrometry. Herein we report a facile and versatile O-glycan relative quantification strategy, based on an improved one-pot method that can quantitatively achieve nonreductive release and in situ chromophoric labeling of intact mucin-type O-glycans in one step. In this study, the one-pot method is optimized and applied for quantitative O-glycan release and tagging with either non-deuterated (d 0 -) or deuterated (d 5 -) 1-phenyl-3-methyl-5-pyrazolone (PMP). The obtained O-glycan derivatives feature a permanent 10-Da mass difference between the d 0 - and d 5 -PMP forms, allowing complete discrimination and comparative quantification of these isotopically labeled O-glycans by mass spectrometric techniques. Moreover, the d 0 - and d 5 -PMP derivatives of O-glycans also have a relatively high hydrophobicity as well as a strong UV adsorption, especially suitable for high-resolution separation and high-sensitivity detection by RP-HPLC-UV. We have refined the conditions for the one-pot reaction as well as the corresponding sample purification approach. The good quantitation feasibility, reliability and linearity of this strategy have been verified using bovine fetuin and porcine stomach mucin as model O-glycoproteins. Additionally, we have also successfully applied this method to the quantitative O-glycomic comparison between perch and salmon eggs by ESI-MS, MS/MS and online RP-HPLC-UV-ESI-MS/MS, demonstrating its excellent applicability to various complex biological samples. O-Linked glycoproteins, generated via a widely existing glycosylation modification process on serine (Ser) or threonine (Thr) residues of nascent proteins, play essential roles in a series of biological processes. As a type of informational molecule, the O-glycans of these glycoproteins participate directly in these biological mechanisms. Thus, the characteristic differences or changes of O-glycans in expression level usually relate to pathologies of many diseases and represent an important opportunity to uncover the functional mechanisms of various glycoprotein O-glycans. The novel strategy introduced here provides a simple and versatile analytical method for the precise quantitation of glycoprotein O-glycans by mass spectrometry, enabling rapid evaluation of the differences or changes of O-glycans in expression level. It is attractive for the field of quantitative/comparative O-glycomics, which has great significance for exploring the complex structure-function relationship of O-glycans, as well as for the search of O-glycan biomarkers of some major diseases and O-glycan related targets of some drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

Mass spectrometric profiling reveals association of N-glycan patterns with epithelial ovarian cancer progression.

PubMed

Chen, Huanhuan; Deng, Zaian; Huang, Chuncui; Wu, Hongmei; Zhao, Xia; Li, Yan

2017-07-01

Aberrant changes of N-glycan modifications on proteins have been linked to various diseases including different cancers, suggesting possible avenue for exploring their etiologies based on N-glycomic analysis. Changes in N-glycan patterns during epithelial ovarian cancer development have so far been investigated mainly using serum, plasma, ascites, and cell lines. However, changes in patterns of N-glycans in tumor tissues during epithelial ovarian cancer progression have remained largely undefined. To investigate whether changes in N-glycan patterns correlate with oncogenesis and progression of epithelial ovarian cancer, we profiled N-glycans from formalin-fixed paraffin-embedded tissue slides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitatively compared among different pathological grades of epithelial ovarian cancer and healthy controls. Our results show that among the 80 compositions of N-glycan detected, expression levels of high-mannose type were higher in epithelial ovarian cancer samples than that observed in healthy controls, accompanied by reduced levels of hybrid-type glycans. By applying receiver operating characteristic analysis, we show that a combined panel composed of four high-mannose and three fucosylated neutral complex N-glycans allows for good discrimination of epithelial ovarian cancer from healthy controls. Furthermore, using a statistical analysis of variance assay, we found that different N-glycan patterns, including 2 high-mannose-type, 2 fucosylated and sialylated complex structures, and 10 fucosylated neutral complex N-glycans, exhibited specific changes in N-glycan abundance across epithelial ovarian cancer grades. Together, our results provide strong evidence that N-glycomic changes are a strong indicator for epithelial ovarian cancer pathological grades and should provide avenues to identify novel biomarkers for epithelial ovarian cancer diagnosis and monitoring.

Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite

PubMed Central

Prasanphanich, Nina Salinger; Luyai, Anthony E; Song, Xuezheng; Heimburg-Molinaro, Jamie; Mandalasi, Msano; Mickum, Megan; Smith, David F; Nyame, A Kwame; Cummings, Richard D

2014-01-01

Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. PMID:24727440

Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement.

PubMed

Kallemeijn, Wouter W; Scheij, Saskia; Voorn-Brouwer, Tineke M; Witte, Martin D; Verhoek, Marri; Overkleeft, Hermen S; Boot, Rolf G; Aerts, Johannes M F G

2016-09-15

β-Glucoside-configured cyclophellitols are activity-based probes (ABPs) that allow sensitive detection of β-glucosidases. Their applicability to detect proteins fused with β-glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M-777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4-methylumbelliferyl β-d-lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre-blocking with conduritol β-epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β-glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high-resolution detection moieties) should assist further research in living cells and organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

PubMed Central

Liu, Yan; Blaum, Bärbel S.; Reiter, Dirk M.; Feizi, Ten; Dermody, Terence S.; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus. PMID:23236285

The GM2 glycan serves as a functional coreceptor for serotype 1 reovirus.

PubMed

Reiss, Kerstin; Stencel, Jennifer E; Liu, Yan; Blaum, Bärbel S; Reiter, Dirk M; Feizi, Ten; Dermody, Terence S; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valentine, Jenny L.; Chen, Linxiao; Perregaux, Emily C.

The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced highmore » titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.« less

Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.

PubMed

Wu, Zhengliang L; Person, Anthony D; Anderson, Matthew; Burroughs, Barbara; Tatge, Timothy; Khatri, Kshitij; Zou, Yonglong; Wang, Lianchun; Geders, Todd; Zaia, Joseph; Sackstein, Robert

2018-02-01

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans. © The Author(s) 2017. Published by Oxford University Press.

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

DOE PAGES

Valentine, Jenny L.; Chen, Linxiao; Perregaux, Emily C.; ...

2016-06-23

The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced highmore » titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.« less

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission.

PubMed

Mathys, Leen; Balzarini, Jan

2015-01-01

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

PubMed Central

Mathys, Leen; Balzarini, Jan

2015-01-01

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission. PMID:26121645

Functional analysis of PGRP-LA in Drosophila immunity.

PubMed

Gendrin, Mathilde; Zaidman-Rémy, Anna; Broderick, Nichole A; Paredes, Juan; Poidevin, Mickaël; Roussel, Alain; Lemaitre, Bruno

2013-01-01

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.

Xylose Migration During Tandem Mass Spectrometry of N-Linked Glycans

NASA Astrophysics Data System (ADS)

Hecht, Elizabeth S.; Loziuk, Philip L.; Muddiman, David C.

2017-04-01

Understanding the rearrangement of gas-phase ions via tandem mass spectrometry is critical to improving manual and automated interpretation of complex datasets. N-glycan analysis may be carried out under collision induced (CID) or higher energy collision dissociation (HCD), which favors cleavage at the glycosidic bond. However, fucose migration has been observed in tandem MS, leading to the formation of new bonds over four saccharide units away. In the following work, we report the second instance of saccharide migration ever to occur for N-glycans. Using horseradish peroxidase as a standard, the beta-1,2 xylose was observed to migrate from a hexose to a glucosamine residue on the (Xyl)Man3GlcNac2 glycan. This investigation was followed up in a complex N-linked glycan mixture derived from stem differentiating xylem tissue, and the rearranged product ion was observed for 75% of the glycans. Rearrangement was not favored in isomeric glycans with a core or antennae fucose and unobserved in glycans predicted to have a permanent core-fucose modification. As the first empirical observation of this rearrangement, this work warrants dissemination so it may be searched in de novo sequencing glycan workflows.

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes.

PubMed

Kim, Yeong Hun; Kang, Ji-Yeon; Gil, Jin Young; Kim, Sang-Yoon; Shin, Keun Koo; Kang, Hyun Ah; Kim, Jeong-Yoon; Kwon, Ohsuk; Oh, Doo-Byoung

2017-04-01

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

High-temperature LC-MS/MS of permethylated glycans derived from glycoproteins.

PubMed

Zhou, Shiyue; Hu, Yunli; Mechref, Yehia

2016-06-01

Various glycomic analysis methods have been developed due to the essential roles of glycans in biological processes as well as the potential application of glycomics in biomarker discovery in many diseases. Permethylation is currently considered to be one of the most common derivatization methods in MS-based glycomic analysis. Permethylation not only improves ionization efficiency and stability of sialylated glycans in positive mode but also allows for enhanced separation performance on reversed-phase liquid chromatography (RPLC). Recently, RPLC-MS analysis of permethylated glycans exhibited excellent performance in sensitivity and reproducibility and became a widely-applied comprehensive strategy in glycomics. However, separating permethylated glycans by RPLC always suffers from peak broadening for high-molecular-weight branched glycans, which probably due to the low exchange rate between the stationary phase and mobile phase limited by intermolecular interactions of the methyl groups associated with the branching of the glycan structures. In this study, we employed high separation temperature conditions for RPLC of permethylated glycans, thus achieving enhanced peak capacity, improving peak shape, and enhancing separation efficiency. Additionally, partial isomeric separation were observed in RPLC of permethylated glycans at high-temperature. Mathematical processing of the correlation between retention time and molecular weight also revealed the advantage of high-temperature LC method for both manual and automatic glycan identification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry.

PubMed

Szabo, Zoltan; Thayer, James R; Agroskin, Yury; Lin, Shanhua; Liu, Yan; Srinivasan, Kannan; Saba, Julian; Viner, Rosa; Huhmer, Andreas; Rohrer, Jeff; Reusch, Dietmar; Harfouche, Rania; Khan, Shaheer H; Pohl, Christopher

2017-05-01

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS 2 in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS 2 permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.

Extracting Both Peptide Sequence and Glycan Structural Information by 157 nm Photodissociation of N-Linked Glycopeptides

PubMed Central

Zhang, Liangyi; Reilly, James P.

2009-01-01

157 nm photodissociation of N-linked glycopeptides was investigated in MALDI tandem time-of-flight (TOF) and linear ion trap mass spectrometers. Singly-charged glycopeptides yielded abundant peptide and glycan fragments. The peptide fragments included a series of x-, y-, v- and w- ions with the glycan remaining intact. These provide information about the peptide sequence and the glycosylation site. In addition to glycosidic fragments, abundant cross-ring glycan fragments that are not observed in low-energy CID were detected. These fragments provide insight into the glycan sequence and linkages. Doubly-charged glycopeptides generated by nanospray in the linear ion trap mass spectrometer also yielded peptide and glycan fragments. However, the former were dominated by low-energy fragments such as b- and y- type ions while glycan was primarily cleaved at glycosidic bonds. PMID:19113943

Sensitive Detection of Protein and miRNA Cancer Biomarkers using Silicon-Based Photonic Crystals and A Resonance Coupling Laser Scanning Platform

PubMed Central

George, Sherine; Chaudhery, Vikram; Lu, Meng; Takagi, Miki; Amro, Nabil; Pokhriyal, Anusha; Tan, Yafang; Ferreira, Placid; Cunningham, Brian T.

2013-01-01

Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by >8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels (“enhanced excitation”) and the spatially biased funneling of fluorophore emissions through coupling to PC resonances (“enhanced extraction”), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couples fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region <100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in <1 minute. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM. PMID:23963502

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

PubMed Central

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-01-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan structures was determined to be 30% while it was found to be 35% for either fucosylated or sialylated structures The optimum CE for mannose and complex type N-glycan structures was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan structures in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these structures was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitudes. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan structures enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples. PMID:25698222

Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion.

PubMed

Mancera-Arteu, Montserrat; Giménez, Estela; Barbosa, José; Sanz-Nebot, Victòria

2016-10-12

In this study, a ZIC-HILIC-MS methodology for the analysis of N-glycan isomers was optimized to obtain greater detection sensitivity and thus identify more glycan structures in hAGP. In a second step, this method was combined with glycan reductive isotope labelling (GRIL) through [(12)C6]/[(13)C6]-aniline and exoglycosidase digestion to characterize the different glycan isomers. The GRIL method allows the peak areas resulting from two different labelled samples to be compared, since neither retention time shifts nor variations in the ionization of glycans between these samples are obtained. First, sialic acid linkage assignations were performed for most hAGP glycan isomers with α2-3 sialidase digestion. Bi-, tri- and tetraantennary glycan isomers with different terminal sialic acid linkages to galactose (α2-3 or α2-6) were assigned, and the potential of this technique for the structural characterization of isobaric isomers was therefore demonstrated. Furthermore, fucose linkage isomers of hAGP glycans were also characterized using this isotope-labelling approach in combination with α1-3,4 fucosidase and β1-4 galactosidase digestion. α1-3 antennary fucoses and α1-6 core fucosylation were detected in hAGP fucosylated glycans. These established methodologies can be extremely useful for patho-glycomic studies to characterize glycoproteins of biomedical interest and find novel glycan isomers that could be used as biomarkers in cancer research. Copyright © 2016 Elsevier B.V. All rights reserved.

Autoantibodies to aberrantly glycosylated MUC1 in early stage breast cancer are associated with a better prognosis

PubMed Central

2011-01-01

Introduction Detection of serum biomarkers for early diagnosis of breast cancer remains an important goal. Changes in the structure of O-linked glycans occur in all breast cancers resulting in the expression of glycoproteins that are antigenically distinct. Indeed, the serum assay widely used for monitoring disease progression in breast cancer (CA15.3), detects a glycoprotein (MUC1), but elevated levels of the antigen cannot be detected in early stage patients. However, since the immune system acts to amplify the antigenic signal, antibodies can be detected in sera long before the antigen. We have exploited the change in O-glycosylation to measure autoantibody responses to cancer-associated glycoforms of MUC1 in sera from early stage breast cancer patients. Methods We used a microarray platform of 60mer MUC1 glycopeptides, to confirm the presence of autoantibodies to cancer associated glycoforms of MUC1 in a proportion of early breast cancer patients (54/198). Five positive sera were selected for detailed definition of the reactive epitopes using on chip glycosylation technology and a panel of glycopeptides based on a single MUC1 tandem repeat carrying specific glycans at specific sites. Based on these results, larger amounts of an extended repertoire of defined MUC1 glycopeptides were synthesised, printed on microarrays, and screened with sera from a large cohort of breast cancer patients (n = 395), patients with benign breast disease (n = 108) and healthy controls (n = 99). All sera were collected in the 1970s and 1980s and complete clinical follow-up of breast cancer patients is available. Results The presence and level of autoantibodies was significantly higher in the sera from cancer patients compared with the controls, and a highly significant correlation with age was observed. High levels of a subset of autoantibodies to the core3MUC1 (GlcNAcβ1-3GalNAc-MUC1) and STnMUC1 (NeuAcα2,6GalNAc-MUC1) glycoforms were significantly associated with reduced incidence and increased time to metastasis. Conclusions Autoantibodies to specific cancer associated glycoforms of MUC1 are found more frequently and at higher levels in early stage breast cancer patients than in women with benign breast disease or healthy women. Association of strong antibody response with reduced rate and delay in metastases suggests that autoantibodies can affect disease progression. PMID:21385452

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

PubMed Central

Groves, J T; Wülfing, C; Boxer, S G

1996-01-01

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8913608

Comparative Glycoprofiling of HIV gp120 Immunogens by Capillary Electrophoresis and MALDI Mass Spectrometry

PubMed Central

Guttman, Miklós; Váradi, Csaba; Lee, Kelly K.; Guttman, András

2015-01-01

The Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) is the primary antigenic feature on the surface of the virus and is of key importance in HIV vaccinology. Vaccine trials with the gp120 subunit of Env are ongoing with the recent RV144 trial showing moderate efficacy. gp120 is densely covered with N-linked glycans that are thought to help evade the host's humoral immune response. To assess how the global glycosylation patterns vary between gp120 constructs, the glycan profiles of several gp120s were examined by capillary electrophoresis with laser induced fluorescence detection and MALDI-MS. The glycosylation profiles were found to be similar for chronic vs. transmitter/founder isolates and only varied moderately between gp120s from different clades. This study revealed that the addition of specific tags, such as the gD tag used in the RV144 trial, had significant effects on the overall glycosylation patterns. Such effects are likely to influence the immunogenicity of various Env immunogens and should be considered for future vaccine strategies, emphasizing the importance of the glycosylation analysis approach described in this paper. PMID:25809283

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

PubMed

Gehring, Andrew G; Brewster, Jeffrey D; He, Yiping; Irwin, Peter L; Paoli, George C; Simons, Tawana; Tu, Shu-I; Uknalis, Joseph

2015-12-04

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 10⁵ cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

Why human anti-Galα1-4Galβ1-4Glc natural antibodies do not recognize the trisaccharide on erythrocyte membrane? Molecular dynamics and immunochemical investigation.

PubMed

Volynsky, Pavel; Efremov, Roman; Mikhalev, Ilya; Dobrochaeva, Kira; Tuzikov, Alexander; Korchagina, Elena; Obukhova, Polina; Rapoport, Evgenia; Bovin, Nicolai

2017-10-01

Human blood contains a big variety of natural antibodies, circulating throughout life at constant concentration. Previously, we have found natural antibodies capable of binding to trisaccharide Galα1-4Galβ1-4Glc (P k ) practically in all humans. Intriguingly, the same trisaccharide is a key fragment of glycosphingolipid globotriaosylceramide (Gb3Cer) - normal component of erythrocyte and endothelial cell membrane, i.e. the antibodies and their cognate antigen coexist without any immunological reaction. To explain the inertness of human anti-P k antibodies towards own cells. We used a combination of immunochemical and molecular dynamics (MD) experiments. Antibodies were isolated using affinity media with P k trisaccharide, their epitope specificity was characterized using ELISA (enzyme-linked immunosorbent assay) with a set of synthetic glycans related to P k synthetic glycans and FACS (Fluorescence-Activated Cell Sorting) analysis of cells with inserted natural Gb3Cer and its synthetic analogue. Conformations and clustering of glycolipids immersed into a lipid bilayer were studied using MD simulations. Isolated specific antibodies were completely unable to bind natural Gb3Cer both inserted into cells and in artificial membrane, whereas strong interaction took place with synthetic analogue differing by the presence of a spacer between trisaccharide and lipid part. MD simulations revealed: i) although membrane-bound glycans do not form stable long-living aggregates, their transient packing is more compact in natural Gb3 as compared with the synthetic analog, ii) similar conformation of P k glycan in composition of the glycolipids, iii) no effect on the mentioned above results when cholesterol was inserted into membrane, and iv) better accessibility of the synthetic version for interaction with proteins. Both immunochemical and molecular dynamics data argue that the reason of the "tolerance" of natural anti-P k antibodies towards cell-bound Gb3Cer is the spatial inaccessibility of P k glycotope for interaction. We can conclude that the antibodies are not related to the blood group P system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural analysis of N-linked glycans in Caenorhabditis elegans.

PubMed

Natsuka, Shunji; Adachi, Jiro; Kawaguchi, Masahumi; Nakakita, Shin-ichi; Hase, Sumihiro; Ichikawa, Akira; Ikura, Koji

2002-06-01

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics.

PubMed

Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L

2010-02-15

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

Endoplasmic reticulum stress in diabetic mouse or glycated LDL-treated endothelial cells: protective effect of Saskatoon berry powder and cyanidin glycans.

PubMed

Zhao, Ruozhi; Xie, Xueping; Le, Khuong; Li, Wende; Moghadasian, Mohammed H; Beta, Trust; Shen, Garry X

2015-11-01

Endoplasmic reticulum (ER) stress is associated with insulin resistance and diabetic cardiovascular complications, and mechanism or remedy for ER stress remains to be determined. The results of the present study demonstrated that the levels of ER stress or unfolded protein response (UPR) markers, the intensity of thioflavin T (ThT) fluorescence and the abundances of GRP78/94, XBP-1 and CHOP proteins were elevated in cardiovascular tissue of diabetic leptin receptor-deficient (db/db) mice. Cyanidin-3-glucoside (C3G) and cyanidin-3-galactoside (C3Ga) are major anthocyanins in Saskatoon berry (SB) powder. The administration of 5% SB powder for 4 weeks attenuated ThT fluorescence and the UPR markers in hearts and aortae of wild-type and db/db mice. Treatment with glycated low-density lipoprotein (gLDL) increased ThT intensity in human umbilical vein endothelial cells (ECs). Elevated UPR markers were detected in gLDL-treated EC compared to control cultures. The involvement of ER stress in gLDL-treated EC was supported by that the addition of 4-phenyl butyrate acid (a known ER stress antagonist) inhibited gLDL-induced increases in ER stress or UPR markers. C3G at 30 μM or C3Ga at 100 μM reached their maximal inhibition on gLDL-induced increases in ThT, GRP78/94, XBP-1 and CHOP in EC. The results demonstrated that ER stress was enhanced in cardiovascular tissue of db/db mice or gLDL-treated EC. SB powder or cyanidin glycans prevented the abnormal increases in ER stress and UPR markers in cardiovascular tissue of diabetic db/db mice or gLDL-treated EC. Copyright © 2015 Elsevier Inc. All rights reserved.

Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

DOE PAGES

Hu, Liya; Ramani, Sasirekha; Czako, Rita; ...

2015-09-30

We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

Structural basis of glycan specificity in neonate-specific bovine-human reassortant rotavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Liya; Ramani, Sasirekha; Czako, Rita

We report that strain-dependent variation of glycan recognition during initial cell attachment of viruses is a critical determinant of host specificity, tissue-tropism and zoonosis. Rotaviruses (RVs), which cause life-threatening gastroenteritis in infants and children, display significant genotype-dependent variations in glycan recognition resulting from sequence alterations in the VP8* domain of the spike protein VP4. The structural basis of this genotype-dependent glycan specificity, particularly in human RVs, remains poorly understood. Here, from crystallographic studies, we show how genotypic variations configure a novel binding site in the VP8* of a neonate-specific bovine-human reassortant to uniquely recognize either type I or type IImore » precursor glycans, and to restrict type II glycan binding in the bovine counterpart. In conclusion, such a distinct glycan-binding site that allows differential recognition of the precursor glycans, which are developmentally regulated in the neonate gut and abundant in bovine and human milk provides a basis for age-restricted tropism and zoonotic transmission of G10P[11] rotaviruses.« less

Analytical Scheme Leading to Integrated High-Sensitivity Profiling of Glycosphingolipids Together with N- and O-Glycans from One Sample

NASA Astrophysics Data System (ADS)

Benktander, John D.; Gizaw, Solomon T.; Gaunitz, Stefan; Novotny, Milos V.

2018-05-01

Glycoconjugates are directly or indirectly involved in many biological processes. Due to their complex structures, the structural elucidation of glycans and the exploration of their role in biological systems have been challenging. Glycan pools generated through release from glycoprotein or glycolipid mixtures can often be very complex. For the sake of procedural simplicity, many glycan profiling studies choose to concentrate on a single class of glycoconjugates. In this paper, we demonstrate it feasible to cover glycosphingolipids, N-glycans, and O-glycans isolated from the same sample. Small volumes of human blood serum and ascites fluid as well as small mouse brain tissue samples are sufficient to profile sequentially glycans from all three classes of glycoconjugates and even positively identify some mixture components through MALDI-MS and LC-ESI-MS. The results show that comprehensive glycan profiles can be obtained from the equivalent of 500-μg protein starting material or possibly less. These methodological improvements can help accelerating future glycomic comprehensive studies, especially for precious clinical samples.

N-Glycan Structure Annotation of Glycopeptides Using a Linearized Glycan Structure Database (GlyDB)

PubMed Central

Ren, Jian Min; Rejtar, Tomas; Li, Lingyun; Karger, Barry L.

2008-01-01

While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation. PMID:17625816

Simplified Microarray Technique for Identifying mRNA in Rare Samples

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings. Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps: 1. Purification of cellular RNA, 2. Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR), 3. Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye), 4. Hybridization to a microarray chip, 5. Fluorescence scanning the array(s) with dual excitation wavelengths, and 6. Analysis of the resulting images. This six-step procedure must be performed in a laboratory because it requires bulky equipment.

A DNA microarray for identification of selected Korean birds based on mitochondrial cytochrome c oxidase I gene sequences.

PubMed

Chung, In-Hyuk; Yoo, Hye Sook; Eah, Jae-Yong; Yoon, Hyun-Kyu; Jung, Jin-Wook; Hwang, Seung Yong; Kim, Chang-Bae

2010-10-01

DNA barcoding with the gene encoding cytochrome c oxidase I (COI) in the mitochondrial genome has been proposed as a standard marker to identify and discover animal species. Some migratory wild birds are suspected of transmitting avian influenza and pose a threat to aircraft safety because of bird strikes. We have previously reported the COI gene sequences of 92 Korean bird species. In the present study, we developed a DNA microarray to identify 17 selected bird species on the basis of nucleotide diversity. We designed and synthesized 19 specific oligonucleotide probes; these probes were arrayed on a silylated glass slide. The length of the probes was 19-24 bps. The COI sequences amplified from the tissues of the selected birds were labeled with a fluorescent probe for microarray hybridization, and unique hybridization patterns were detected for each selected species. These patterns may be considered diagnostic patterns for species identification. This microarray system will provide a sensitive and a high-throughput method for identification of Korean birds.

Relative Quantification and Higher-Order Modeling of the Plasma Glycan Cancer Burden Ratio in Ovarian Cancer Case-Control Samples

PubMed Central

Hecht, Elizabeth S.; Scholl, Elizabeth H.; Walker, S. Hunter; Taylor, Amber D.; Cliby, William A.; Motsinger-Reif, Alison A.; Muddiman, David C.

2016-01-01

An early-stage, population-wide biomarker for ovarian cancer (OVC) is essential to reverse its high mortality rate. Aberrant glycosylation by OVC has been reported, but studies have yet to identify an N-glycan with sufficiently high specificity. We curated a human biorepository of 82 case-control plasma samples, with 27%, 12%, 46%, and 15% falling across stages I–IV, respectively. For relatve quantitation, glycans were analyzed by the individuality normalization when labeling with glycan hydrazide tags (INLIGHT) strategy for enhanced electrospray ionization, MS/MS analysis. Sixty-three glycan cancer burden ratios (GBRs), defined as the log10 ratio of the case-control extracted ion chromatogram abundances, were calculated above the limit of detection. The final GBR models, built using stepwise forward regression, included three significant terms: OVC stage, normalized mean GBR, and tag chemical purity; glycan class, fucosylation, or sialylation were not significant variables. After Bonferroni correction, seven N-glycans were identified as significant (p < 0.05), and after false discovery rate correction, an additional four glycans were determined to be significant (p < 0.05), with one borderline (p = 0.05). For all N-glycans, the vectors of the effects from stages II–IV were sequentially reversed, suggesting potential biological changes in OVC morphology or in host response. PMID:26347193

Role of Glycans in Cholesteryl Ester Transfer Protein revealed by MD simulation.

PubMed

Hao, Dongxiao; Yang, Zhiwei; Gao, Teng; Tian, Zhiqi; Zhang, Lei; Zhang, Shengli

2018-05-03

Current cholesteryl ester transfer protein (CETP) inhibitors are designed based on the unglycosylated crystal structure, and most of them have failed to cure cardiovascular disease (CVD). It is particularly important for us to investigate the glycosylation structure of CETP (CETP-G) and effect of glycans on the structure and function of CETP. Here, we used a total of 3.0-μs molecular dynamics trajectories of nascent structure of CETP (CETP-N) and CETP-G to study their structural differentiations, to shed new light on the CETP-mediated lipid exchange. In accordance with our simulations and previous mutation studies, relative to CETP-N, CETP-G adopts a more stretched shape with higher hydrophobic and hydrophilic SASA of N-terminal oscillating with larger amplitude, in which Glycan88 provides partial assistance for CEs through the N-terminal. Glycan341 reduces the flexibility of neck flap, with the interference of CEs through the neck region. Besides, Glycan240 reduces the flexibility of Helix-X to interfere the CEs transfer. Glycan396 decreases the flexibility and increases the hydrophobic SASA of C-terminal. Overall, these glycans affect the dynamics and structure of CETP through forming H-bonds with surrounding residues, and the sampled conformations of glycan is also affected by its surrounding residues. Thus, glycans are an integral part of CETP, further studies on the CETP inhibition and treatment of CVD should fully consider the effect of glycans. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products.

PubMed

Yuen, Chun-Ting; Zhou, Yong; Wang, Qing-Zhou; Hou, Ji-Feng; Bristow, Adrian; Wang, Jun-Zhi

2011-11-01

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear. Copyright © 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Discrimination of Isomers of Released N- and O-Glycans Using Diagnostic Product Ions in Negative Ion PGC-LC-ESI-MS/MS

NASA Astrophysics Data System (ADS)

Ashwood, Christopher; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

2018-03-01

Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers. [Figure not available: see fulltext.

Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kronewitter, Scott R.; Marginean, Ioan; Cox, Jonathan T.

The N-glycan diversity of human serum glycoproteins, i.e. the human blood serum N-glycome, is complex due to the range of glycan structures potentially synthesizable by human glycosylation enzymes. The reported glycome, however, is limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report, several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to provide an improved view of glycan diversity. Sample preparation improvements include acidified, microwave-accelerated, PNGase F N-glycan release, and sodium borohydride reduction were optimized to improvemore » quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased the sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. On-line separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient which provides additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) has been utilized. When method improvements are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described1 these technologies demonstrate the ability to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrate application of these advances in the context of the human serum glycome, and for which our initial observations include detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.« less

The multiple roles of epidermal growth factor repeat O-glycans in animal development

PubMed Central

Haltom, Amanda R; Jafar-Nejad, Hamed

2015-01-01

The epidermal growth factor (EGF)-like repeat is a common, evolutionarily conserved motif found in secreted proteins and the extracellular domain of transmembrane proteins. EGF repeats harbor six cysteine residues which form three disulfide bonds and help generate the three-dimensional structure of the EGF repeat. A subset of EGF repeats harbor consensus sequences for the addition of one or more specific O-glycans, which are initiated by O-glucose, O-fucose or O-N-acetylglucosamine. These glycans are relatively rare compared to mucin-type O-glycans. However, genetic experiments in model organisms and cell-based assays indicate that at least some of the glycosyltransferases involved in the addition of O-glycans to EGF repeats play important roles in animal development. These studies, combined with state-of-the-art biochemical and structural biology experiments have started to provide an in-depth picture of how these glycans regulate the function of the proteins to which they are linked. In this review, we will discuss the biological roles assigned to EGF repeat O-glycans and the corresponding glycosyltransferases. Since Notch receptors are the best studied proteins with biologically-relevant O-glycans on EGF repeats, a significant part of this review is devoted to the role of these glycans in the regulation of the Notch signaling pathway. We also discuss recently identified proteins other than Notch which depend on EGF repeat glycans to function properly. Several glycosyltransferases involved in the addition or elongation of O-glycans on EGF repeats are mutated in human diseases. Therefore, mechanistic understanding of the functional roles of these carbohydrate modifications is of interest from both basic science and translational perspectives. PMID:26175457

Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS.

PubMed

Shubhakar, Archana; Kozak, Radoslaw P; Reiding, Karli R; Royle, Louise; Spencer, Daniel I R; Fernandes, Daryl L; Wuhrer, Manfred

2016-09-06

Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.

GlyQ-IQ: Glycomics Quintavariate-Informed Quantification with High-Performance Computing and GlycoGrid 4D Visualization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kronewitter, Scott R.; Slysz, Gordon W.; Marginean, Ioan

2014-05-31

Dense LC-MS datasets have convoluted extracted ion chromatograms with multiple chromatographic peaks that cloud the differentiation between intact compounds with their overlapping isotopic distributions, peaks due to insource ion fragmentation, and noise. Making this differentiation is critical in glycomics datasets because chromatographic peaks correspond to different intact glycan structural isomers. The GlyQ-IQ software is targeted chromatography centric software designed for chromatogram and mass spectra data processing and subsequent glycan composition annotation. The targeted analysis approach offers several key advantages to LC-MS data processing and annotation over traditional algorithms. A priori information about the individual target’s elemental composition allows for exactmore » isotope profile modeling for improved feature detection and increased sensitivity by focusing chromatogram generation and peak fitting on the isotopic species in the distribution having the highest intensity and data quality. Glycan target annotation is corroborated by glycan family relationships and in source fragmentation detection. The GlyQ-IQ software is developed in this work (Part 1) and was used to profile N-glycan compositions from human serum LC-MS Datasets. The companion manuscript GlyQ-IQ Part 2 discusses developments in human serum N-glycan sample preparation, glycan isomer separation, and glycan electrospray ionization. A case study is presented to demonstrate how GlyQ-IQ identifies and removes confounding chromatographic peaks from high mannose glycan isomers from human blood serum. In addition, GlyQ-IQ was used to generate a broad N-glycan profile from a high resolution (100K/60K) nESI-LS-MS/MS dataset including CID and HCD fragmentation acquired on a Velos Pro Mass spectrometer. 101 glycan compositions and 353 isomer peaks were detected from a single sample. 99% of the GlyQ-IQ glycan-feature assignments passed manual validation and are backed with high resolution mass spectra and mass accuracies less than 7 ppm.« less

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays.

PubMed

Goodman, Corey W; Major, Heather J; Walls, William D; Sheffield, Val C; Casavant, Thomas L; Darbro, Benjamin W

2015-04-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

Single molecule characterization of DNA binding and strand displacement reactions on lithographic DNA origami microarrays.

PubMed

Scheible, Max B; Pardatscher, Günther; Kuzyk, Anton; Simmel, Friedrich C

2014-03-12

The combination of molecular self-assembly based on the DNA origami technique with lithographic patterning enables the creation of hierarchically ordered nanosystems, in which single molecules are positioned at precise locations on multiple length scales. Based on a hybrid assembly protocol utilizing DNA self-assembly and electron-beam lithography on transparent glass substrates, we here demonstrate a DNA origami microarray, which is compatible with the requirements of single molecule fluorescence and super-resolution microscopy. The spatial arrangement allows for a simple and reliable identification of single molecule events and facilitates automated read-out and data analysis. As a specific application, we utilize the microarray to characterize the performance of DNA strand displacement reactions localized on the DNA origami structures. We find considerable variability within the array, which results both from structural variations and stochastic reaction dynamics prevalent at the single molecule level.

Reciprocal Prioritization to Dietary Glycans by Gut Bacteria in a Competitive Environment Promotes Stable Coexistence.

PubMed

Tuncil, Yunus E; Xiao, Yao; Porter, Nathan T; Reuhs, Bradley L; Martens, Eric C; Hamaker, Bruce R

2017-10-10

When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron , we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome. IMPORTANCE The microorganisms that reside in the human colon fulfill their energy requirements mainly from diet- and host-derived complex carbohydrates. Members of this ecosystem possess poorly understood strategies to prioritize and compete for these nutrients. Based on direct carbohydrate measurements and corresponding transcriptional analyses, our findings showed that individual bacterial species exhibit different preferences for the same set of glycans and that this prioritization is maintained in a competitive environment, which may promote stable coexistence. Such understanding of gut bacterial glycan utilization will be essential to eliciting predictable changes in the gut microbiota to improve health through the diet. Copyright © 2017 Tuncil et al.

ConA-based glucose sensing using the long-lifetime azadioxatriangulenium fluorophore

NASA Astrophysics Data System (ADS)

Cummins, Brian; Simpson, Jonathan; Gryczynski, Zygmunt; Sørensen, Thomas Just; Laursen, Bo W.; Graham, Duncan; Birch, David; Coté, Gerard

2014-02-01

Fluorescent glucose sensing technologies have been identified as possible alternatives to current continuous glucose monitoring approaches. We have recently introduced a new, smart fluorescent ligand to overcome the traditional problems of ConA-based glucose sensors. For this assay to be translated into a continuous glucose monitoring device where both components are free in solution, the molecular weight of the smart fluorescent ligand must be increased. We have identified ovalbumin as a naturally-occurring glycoprotein that could serve as the core-component of a 2nd generation smart fluorescent ligand. It has a single asparagine residue that is capable of displaying an N-linked glycan and a similar isoelectric point to ConA. Thus, binding between ConA and ovalbumin can potentially be monovalent and sugar specific. This work is the preliminary implementation of fluorescently-labeled ovalbumin in the ConA-based assay. We conjugate the red-emitting, long-lifetime azadioxatriangulenium (ADOTA+) dye to ovalbumin, as ADOTA have many advantageous properties to track the equilibrium binding of the assay. The ADOTA-labeled ovalbumin is paired with Alexa Fluor 647-labeled ConA to create a Förster Resonance Energy Transfer (FRET) assay that is glucose dependent. The assay responds across the physiologically relevant glucose range (0-500 mg/dL) with increasing intensity from the ADOTA-ovalbumin, showing that the strategy may allow for the translation of the smart fluorescent ligand concept into a continuous glucose monitoring device.

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Glycan antagonists and inhibitors: a fount for drug discovery.

PubMed

Brown, Jillian R; Crawford, Brett E; Esko, Jeffrey D

2007-01-01

Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.

Expanding probe repertoire and improving reproducibility in human genomic hybridization

PubMed Central

Dorman, Stephanie N.; Shirley, Ben C.; Knoll, Joan H. M.; Rogan, Peter K.

2013-01-01

Diagnostic DNA hybridization relies on probes composed of single copy (sc) genomic sequences. Sc sequences in probe design ensure high specificity and avoid cross-hybridization to other regions of the genome, which could lead to ambiguous results that are difficult to interpret. We examine how the distribution and composition of repetitive sequences in the genome affects sc probe performance. A divide and conquer algorithm was implemented to design sc probes. With this approach, sc probes can include divergent repetitive elements, which hybridize to unique genomic targets under higher stringency experimental conditions. Genome-wide custom probe sets were created for fluorescent in situ hybridization (FISH) and microarray genomic hybridization. The scFISH probes were developed for detection of copy number changes within small tumour suppressor genes and oncogenes. The microarrays demonstrated increased reproducibility by eliminating cross-hybridization to repetitive sequences adjacent to probe targets. The genome-wide microarrays exhibited lower median coefficients of variation (17.8%) for two HapMap family trios. The coefficients of variations of commercial probes within 300 nt of a repetitive element were 48.3% higher than the nearest custom probe. Furthermore, the custom microarray called a chromosome 15q11.2q13 deletion more consistently. This method for sc probe design increases probe coverage for FISH and lowers variability in genomic microarrays. PMID:23376933

Integrated in silico and biological validation of the blocking effect of Cot-1 DNA on Microarray-CGH.

PubMed

Kang, Seung-Hui; Park, Chan Hee; Jeung, Hei Cheul; Kim, Ki-Yeol; Rha, Sun Young; Chung, Hyun Cheol

2007-06-01

In array-CGH, various factors may act as variables influencing the result of experiments. Among them, Cot-1 DNA, which has been used as a repetitive sequence-blocking agent, may become an artifact-inducing factor in BAC array-CGH. To identify the effect of Cot-1 DNA on Microarray-CGH experiments, Cot-1 DNA was labeled directly and Microarray-CGH experiments were performed. The results confirmed that probes which hybridized more completely with Cot-1 DNA had a higher sequence similarity to the Alu element. Further, in the sex-mismatched Microarray-CGH experiments, the variation and intensity in the fluorescent signal were reduced in the high intensity probe group in which probes were better hybridized with Cot-1 DNA. Otherwise, those of the low intensity probe group showed no alterations regardless of Cot-1 DNA. These results confirmed by in silico methods that Cot-1 DNA could block repetitive sequences in gDNA and probes. In addition, it was confirmed biologically that the blocking effect of Cot-1 DNA could be presented via its repetitive sequences, especially Alu elements. Thus, in contrast to BAC-array CGH, the use of Cot-1 DNA is advantageous in controlling experimental variation in Microarray-CGH.

Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays.

PubMed

Steemers, F J; Ferguson, J A; Walt, D R

2000-01-01

We have developed a randomly ordered fiber-optic gene array for rapid, parallel detection of unlabeled DNA targets with surface immobilized molecular beacons (MB) that undergo a conformational change accompanied by a fluorescence change in the presence of a complementary DNA target. Microarrays are prepared by randomly distributing MB-functionalized 3-microm diameter microspheres in an array of wells etched in a 500-microm diameter optical imaging fiber. Using several MBs, each designed to recognize a different target, we demonstrate the selective detection of genomic cystic fibrosis related targets. Positional registration and fluorescence response monitoring of the microspheres was performed using an optical encoding scheme and an imaging fluorescence microscope system.

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

PubMed Central

Li, Xiang; Harwood, Valerie J.; Nayak, Bina

2016-01-01

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment. PMID:26729716

Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds.

PubMed

Di Patrizi, Lisa; Rosati, Floriana; Guerranti, Roberto; Pagani, Roberto; Gerwig, Gerrit J; Kamerling, Johannis P

2006-11-01

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N (4)-(N-acetyl-beta -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and (1)H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), and two xylosylated structures, Xyl(1)Man(3)GlcNAc(2) and Xyl(1)Man(4)GlcNAc(2). The PNGase A-sensitive N-glycans, containing (alpha 1-3)-linked fucose, were identified as Fuc(1)Xyl(1)Man(2)GlcNAc(2) and Fuc(1)Xyl(1)Man(3)GlcNAc(2). In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (beta 1-2)-linked xylose- and core alpha (1-3)-linked fucose-modified N-glycan chains.

Zwitterionic-hydrophilic interaction capillary liquid chromatography coupled to tandem mass spectrometry for the characterization of human alpha-acid-glycoprotein N-glycan isomers.

PubMed

Mancera-Arteu, Montserrat; Giménez, Estela; Barbosa, José; Peracaula, Rosa; Sanz-Nebot, Victòria

2017-10-23

In this work, a μZIC-HILIC-MS/MS methodology was established in negative ion mode for the characterization of glycan isomers. The possibility to separate the glycan isomers by the μZIC-HILIC strategy coupled to a high resolution tandem mass spectrometry detection permitted us to obtain valuable information about each glycan structure. The most important diagnostic ion fragments previously described to characterize structural features of glycans, were evaluated in this study using hAGP as model glycoprotein. The assignation of hAGP glycan isomers performed in our previous work using the GRIL strategy in combination with exoglycosidase digestion [1] was used in this paper to confirm or discard some ion fragments reported in the literature and delve into the structural characterization of glycan isomers. Sialic acid as well as fucose linkage-type glycan isomers were assigned using this approach and daughter ions with higher diagnostic value were determined. The location of α2-3/α2-6 sialic acids on antennas and a deeper characterization of several highly sialylated tri- and tetraantennary glycans was also possible using the established MS/MS method. Moreover, relying on the characterization performed in Ref. [1], core and antenna fucosylation were differentiated in this work using specific ion fragments obtained in the tandem mass spectra. This methodology was also applied to hAGP purified from control and pathological serum samples, which corroborated its robustness and its potential for finding novel glycan-based biomarkers in patho-glycomic studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family.

PubMed

Stone, Jacquelyn A; Nicola, Anthony V; Baum, Linda G; Aguilar, Hector C

2016-02-01

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.

Qualitative and Quantitative Analysis of Carbohydrate Modification on Glycoproteins from Seeds of Ginkgo biloba.

PubMed

Wang, Ting; Hu, Xiao-Chun; Cai, Zhi-Peng; Voglmeir, Josef; Liu, Li

2017-09-06

Recent progress in the relationship between carbohydrate cross-reactive determinants (CCDs) and allergic response highlights the importance of carbohydrate moieties in the innate immune system. Previous research pointed out that the protein allergen in Ginkgo biloba seeds is glycosylated, and the oligosaccharides conjugated to these proteins might also contribute to the allergy. The aim of this study was to analyze carbohydrate moieties, especially N-linked glycans, of glycoproteins from Ginkgo seeds originating from different places for detailed structures, to enable further research on the role played by N-glycans in Ginkgo-caused allergy. Results of monosaccharide composition and immunoblotting assays indicated the existence of N-glycans. Detailed structural elucidation of the N-glycans was further carried out by means of hydrophilic interaction ultraperformance liquid chromatography (HILIC-UPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 14 out of 16 structures detected by UPLC were confirmed by MALDI-TOF-MS and tandem mass spectrometry, among which complex-type N-glycans bearing Lewis A determinants and high-mannose-type N-glycans were identified from Ginkgo seeds for the first time. Precise quantification of N-glycans was performed by use of an external standard, and both the absolute amount of each N-glycan and the percentage of different types of N-glycan showed significant diversity among the samples without any pattern of geographic variation.

Glycosylation with ribitol-phosphate in mammals: New insights into the O-mannosyl glycan.

PubMed

Manya, Hiroshi; Endo, Tamao

2017-10-01

O-mannosyl glycans have been found in a limited number of glycoproteins of the brain, nerves, and skeletal muscles, particularly in α-dystroglycan (α-DG). Defects in O-mannosyl glycan on α-DG are the primary cause of a group of congenital muscular dystrophies, which are collectively termed α-dystroglycanopathy. Recent studies have revealed various O-mannosyl glycan structures, which can be classified as core M1, core M2, and core M3 glycans. Although many dystroglycanopathy genes are involved in core M3 processing, the structure and biosynthesis of core M3 glycan remains only partially understood. This review presents recent findings about the structure, biosynthesis, and pathology of O-mannosyl glycans. Recent studies have revealed that the entire structure of core M3 glycan, including ribitol-5-phosphate, is a novel structure in mammals; its unique biosynthetic pathway has been elucidated by the identification of new causative genes for α-dystroglycanopathies and their functions. O-mannosyl glycan has a novel, unique structure that is important for the maintenance of brain and muscle functions. These findings have opened up a new field in glycoscience. These studies will further contribute to the understanding of the pathomechanism of α-dystroglycanopathy and the development of glycotherapeutics. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancing glycan isomer separations with metal ions and positive and negative polarity ion mobility spectrometry-mass spectrometry analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Xueyun; Zhang, Xing; Schocker, Nathaniel S.

Glycomics has become an increasingly important field of research since glycans play critical roles in biology processes ranging from molecular recognition and signaling to cellular communication. Glycans often conjugate with other biomolecules such as proteins and lipids, and alter their properties and functions, so understanding the effect glycans have on cellular systems is essential. However the analysis of glycans is extremely difficult due to their complexity and structural diversity (i.e., the number and identity of monomer units, and configuration of their glycosidic linkages and connectivities). In this work, we coupled ion mobility spectrometry with mass spectrometry (IMS-MS) to characterize glycanmore » standards and biologically important isomers of synthetic αGal-containing O-glycans including glycotopes of the protozoan parasite Trypanosoma cruzi, which is the causative agent of Chagas disease. IMS-MS results showed significant differences for the glycan structural isomers when analyzed in positive and negative polarity and complexed with different metal cations. These results suggest specific metal ions or ion polarities could be used to target and baseline separate glycan isomers of interest with IMS-MS.« less

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

PubMed Central

Castilho, Alexandra; Gruber, Clemens; Thader, Andreas; Oostenbrink, Chris; Pechlaner, Maria; Steinkellner, Herta; Altmann, Friedrich

2015-01-01

We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF3, and GnGnF6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies. PMID:26067753

Alterations in expressed prostate secretion-urine PSA N-glycosylation discriminate prostate cancer from benign prostate hyperplasia

PubMed Central

Sun, Chenxia; Wen, Fuping; Wang, Haifeng; Guo, Huaizu; Gao, Xu; Xu, Chuanliang; Xu, Chuanliang; Yang, Chenghua; Sun, Yinghao

2017-01-01

The prostate specific antigen (PSA) test is widely used for early diagnosis of prostate cancer (PCa). However, its limited sensitivity has led to over-diagnosis and over-treatment of PCa. Glycosylation alteration is a common phenomenon in cancer development. Different PSA glycan subforms have been proposed as diagnostic markers to better differentiate PCa from benign prostate hyperplasia (BPH). In this study, we purified PSA from expressed prostate secretions (EPS)-urine samples from 32 BPH and 30 PCa patients and provided detailed PSA glycan profiles in Chinese population. We found that most of the PSA glycans from EPS-urine were complex type biantennary glycans. We observed two major patterns in PSA glycan profiles. Overall there was no distinct separation of PSA glycan profiles between BPH and PCa patients. However, we detected a significant increase of glycan FA2 and FM5A2G2S1 in PCa when compared with BPH patients. Furthermore, we observed that the composition of FA2 glycan increased significantly in advanced PCa with Gleason score ≥8, which potentially could be translated to clinic as a marker for aggressive PCa. PMID:29100363

Alterations in expressed prostate secretion-urine PSA N-glycosylation discriminate prostate cancer from benign prostate hyperplasia.

PubMed

Jia, Gaozhen; Dong, Zhenyang; Sun, Chenxia; Wen, Fuping; Wang, Haifeng; Guo, Huaizu; Gao, Xu; Xu, Chuanliang; Xu, Chuanliang; Yang, Chenghua; Sun, Yinghao

2017-09-29

The prostate specific antigen (PSA) test is widely used for early diagnosis of prostate cancer (PCa). However, its limited sensitivity has led to over-diagnosis and over-treatment of PCa. Glycosylation alteration is a common phenomenon in cancer development. Different PSA glycan subforms have been proposed as diagnostic markers to better differentiate PCa from benign prostate hyperplasia (BPH). In this study, we purified PSA from expressed prostate secretions (EPS)-urine samples from 32 BPH and 30 PCa patients and provided detailed PSA glycan profiles in Chinese population. We found that most of the PSA glycans from EPS-urine were complex type biantennary glycans. We observed two major patterns in PSA glycan profiles. Overall there was no distinct separation of PSA glycan profiles between BPH and PCa patients. However, we detected a significant increase of glycan FA2 and FM5A2G2S1 in PCa when compared with BPH patients. Furthermore, we observed that the composition of FA2 glycan increased significantly in advanced PCa with Gleason score ≥8, which potentially could be translated to clinic as a marker for aggressive PCa.

Sialoglycoproteins and N-glycans from secreted exosomes of ovarian carcinoma cells.

PubMed

Escrevente, Cristina; Grammel, Nicolas; Kandzia, Sebastian; Zeiser, Johannes; Tranfield, Erin M; Conradt, Harald S; Costa, Júlia

2013-01-01

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.

Neutral glycans from sandfish skin can reduce friction of polymers

PubMed Central

Vihar, Boštjan; Hanisch, Franz Georg; Baumgartner, Werner

2016-01-01

The lizard Scincus scincus, also known as sandfish, can move through aeolian desert sand in a swimming-like manner. A prerequisite for this ability is a special integument, i.e. scales with a very low friction for sand and a high abrasion resistance. Glycans in the scales are causally related to the low friction. Here, we analysed the glycans and found that neutral glycans with five to nine mannose residues are important. If these glycans were covalently bound to acrylic polymers like poly(methyl methacrylate) or acrylic car coatings at a density of approximately one molecule per 4 nm², friction for and adhesion of sand particles could be reduced to levels close to those observed with sandfish scales. This was also found true, if the glycans were isolated from sources other than sandfish scales like plants such as almonds or mistletoe. We speculate that these neutral glycans act as low density spacers separating sand particles from the dense scales thereby reducing van der Waals forces. PMID:27030038

Glycan Reader: Automated Sugar Identification and Simulation Preparation for Carbohydrates and Glycoproteins

PubMed Central

Jo, Sunhwan; Song, Kevin C.; Desaire, Heather; MacKerell, Alexander D.; Im, Wonpil

2011-01-01

Understanding how glycosylation affects protein structure, dynamics, and function is an emerging and challenging problem in biology. As a first step toward glycan modeling in the context of structural glycobiology, we have developed Glycan Reader and integrated it into the CHARMM-GUI, http://www.charmm-gui.org/input/glycan. Glycan Reader greatly simplifies the reading of PDB structure files containing glycans through (i) detection of carbohydrate molecules, (ii) automatic annotation of carbohydrates based on their three-dimensional structures, (iii) recognition of glycosidic linkages between carbohydrates as well as N-/O-glycosidic linkages to proteins, and (iv) generation of inputs for the biomolecular simulation program CHARMM with the proper glycosidic linkage setup. In addition, Glycan Reader is linked to other functional modules in CHARMM-GUI, allowing users to easily generate carbohydrate or glycoprotein molecular simulation systems in solution or membrane environments and visualize the electrostatic potential on glycoprotein surfaces. These tools are useful for studying the impact of glycosylation on protein structure and dynamics. PMID:21815173

Glycan Engineering for Cell and Developmental Biology.

PubMed

Griffin, Matthew E; Hsieh-Wilson, Linda C

2016-01-21

Cell-surface glycans are a diverse class of macromolecules that participate in many key biological processes, including cell-cell communication, development, and disease progression. Thus, the ability to modulate the structures of glycans on cell surfaces provides a powerful means not only to understand fundamental processes but also to direct activity and elicit desired cellular responses. Here, we describe methods to sculpt glycans on cell surfaces and highlight recent successes in which artificially engineered glycans have been employed to control biological outcomes such as the immune response and stem cell fate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comprehensive functional analysis of N-linked glycans on Ebola virus GP1.

PubMed

Lennemann, Nicholas J; Rhein, Bethany A; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

2014-01-28

Ebola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca(2+)-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency. Filovirus outbreaks occur sporadically throughout central Africa, causing high fatality rates among the general public and health care workers. These unpredictable hemorrhagic fever outbreaks are caused by multiple species of Ebola viruses, as well as Marburg virus. While filovirus vaccines and therapeutics are being developed, there are no licensed products. The sole viral envelope glycoprotein, which is a principal immunogenic target, contains a heavy shield of glycans surrounding the conserved receptor-binding domain. We find that disruption of this shield through targeted mutagenesis leads to an increase in cell entry, protease sensitivity, and antiserum/antibody sensitivity but is not sufficient to allow virion binding to the intracellular receptor NPC1. Therefore, our studies provide evidence that filoviruses maintain glycoprotein glycosylation to protect against proteases and antibody neutralization at the expense of efficient entry. Our results unveil interesting insights into the unique entry process of filoviruses and potential immune evasion tactics of the virus.

BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE.

PubMed

Rao, Archana N; Grainger, David W

2014-04-01

Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA's persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools.

BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

PubMed Central

Rao, Archana N.; Grainger, David W.

2014-01-01

Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA’s persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools. PMID:24765522

High-sensitivity O-glycomic analysis of mice deficient in core 2 β1,6-N-acetylglucosaminyltransferases

PubMed Central

Ismail, Mohd Nazri; Stone, Erica L; Panico, Maria; Lee, Seung Ho; Luu, Ying; Ramirez, Kevin; Ho, Samuel B; Fukuda, Minoru; Marth, Jamey D; Haslam, Stuart M; Dell, Anne

2011-01-01

Core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT), which exists in three isoforms, C2GnT1, C2GnT2 and C2GnT3, is one of the key enzymes in the O-glycan biosynthetic pathway. These isoenzymes produce core 2 O-glycans and have been correlated with the biosynthesis of core 4 O-glycans and I-branches. Previously, we have reported mice with single and multiple deficiencies of C2GnT isoenzyme(s) and have evaluated the biological and structural consequences of the loss of core 2 function. We now present more comprehensive O-glycomic analyses of neutral and sialylated glycans expressed in the colon, small intestine, stomach, kidney, thyroid/trachea and thymus of wild-type, C2GnT2 and C2GnT3 single knockouts and the C2GnT1–3 triple knockout mice. Very high-quality data have emerged from our mass spectrometry techniques with the capability of detecting O-glycans up to at least 3500 Da. We were able to unambiguously elucidate the types of O-glycan core, branching location and residue linkages, which allowed us to exhaustively characterize structural changes in the knockout tissues. The C2GnT2 knockout mice suffered a major loss of core 2 O-glycans as well as glycans with I-branches on core 1 antennae especially in the stomach and the colon. In contrast, core 2 O-glycans still dominated the O-glycomic profile of most tissues in the C2GnT3 knockout mice. Analysis of the C2GnT triple knockout mice revealed a complete loss of both core 2 O-glycans and branched core 1 antennae, confirming that the three known isoenzymes are entirely responsible for producing these structures. Unexpectedly, O-linked mannosyl glycans are upregulated in the triple deficient stomach. In addition, our studies have revealed an interesting terminal structure detected on O-glycans of the colon tissues that is similar to the RM2 antigen from glycolipids. PMID:20855471

Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells.

PubMed

Stanley, Pamela; Sundaram, Subha

2014-06-03

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis. Copyright © 2014 John Wiley & Sons, Inc.

Selected Reaction Monitoring to Differentiate and Relatively Quantitate Isomers of Sulfated and Unsulfated Core 1 O-Glycans from Salivary MUC7 Protein in Rheumatoid Arthritis*

PubMed Central

Flowers, Sarah A.; Ali, Liaqat; Lane, Catherine S.; Olin, Magnus; Karlsson, Niclas G.

2013-01-01

Rheumatoid arthritis is a common and debilitating systemic inflammatory condition affecting up to 1% of the world's population. This study aimed to investigate the immunological significance of O-glycans in chronic arthritis at a local and systemic level. O-Glycans released from synovial glycoproteins during acute and chronic arthritic conditions were compared and immune-reactive glycans identified. The sulfated core 1 O-glycan (Galβ1–3GalNAcol) was immune reactive, showing a different isomeric profile in the two conditions. From acute reactive arthritis, three isomers could be sequenced, but in patients with chronic rheumatoid arthritis, only a single 3-Gal sulfate-linked isomer could be identified. The systemic significance of this glycan epitope was investigated using the salivary mucin MUC7 in patients with rheumatoid arthritis and normal controls. To analyze this low abundance glycan, a selected reaction monitoring (SRM) method was developed to differentiate and relatively quantitate the core 1 O-glycan and the sulfated core 1 O-glycan Gal- and GalNAc-linked isomers. The acquisition of highly sensitive full scan linear ion trap MS/MS spectra in addition to quantitative SRM data allowed the 3- and 6-linked Gal isomers to be differentiated. The method was used to relatively quantitate the core 1 glycans from MUC7 to identify any systemic changes in this carbohydrate epitope. A statistically significant increase in sulfation was identified in salivary MUC7 from rheumatoid arthritis patients. This suggests a potential role for this epitope in chronic inflammation. This study was able to develop an SRM approach to specifically identify and relatively quantitate sulfated core 1 isomers and the unsulfated structure. The expansion of this method may afford an avenue for the high throughput investigation of O-glycans. PMID:23457413

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients

PubMed Central

Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V.; Hacker, Neville F.; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-01-01

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2–6 vs. α2–3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers. PMID:27764122

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients.

PubMed

Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-01-01

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2-6 vs. α2-3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers.

LC-MS/MS Analysis of Permethylated Free Oligosaccharides and N-glycans Derived from Human, Bovine, and Goat Milk Samples

PubMed Central

Dong, Xue; Zhou, Shiyue; Mechref, Yehia

2016-01-01

Oligosaccharides in milk not only provide nutrition to the infants, but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat and human milk using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat and human milk samples (without isomeric consideration) were 11, 8 and 11 respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by PGC LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using PGC column. Permethylation of the glycan structures facilitated the interpretation of tandem MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. PMID:26959529

Naturally Occurring Structural Isomers in Serum IgA1 O-Glycosylation

PubMed Central

Takahashi, Kazuo; Smith, Archer D.; Poulsen, Knud; Kilian, Mogens; Julian, Bruce A.; Mestecky, Jiri; Novak, Jan; Renfrow, Matthew B.

2013-01-01

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of O-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr225, Thr228, Ser230, Ser232 and Thr236 have been identified as the predominant sites of O-glycan attachment. The range and distribution of O-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 O-glycan heterogeneity by use of high resolution LC/MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 O-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the O-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable O-glycan chains attached to Ser230, Thr233 or Thr236 produce the predominant positional isomers, including O-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a single sample, and have implications for defining epitopes based on clustered O-glycan variability. PMID:22067045

A novel core 1 O-linked glycan-specific binding lectin from the fruiting body of Hericium erinaceus.

PubMed

Kim, Seonghun

2018-02-01

Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galβ(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays. Copyright © 2017 Elsevier B.V. All rights reserved.

LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples.

PubMed

Dong, Xue; Zhou, Shiyue; Mechref, Yehia

2016-06-01

Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Time-resolved UV-excited microarray reader for fluorescence energy transfer (FRET) measurements

NASA Astrophysics Data System (ADS)

Orellana, Adelina; Hokkanen, Ari P.; Pastinen, Tomi; Takkinen, Kristina; Soderlund, Hans

2001-05-01

Analytical systems based on immunochemistry are largely used in medical diagnostics and in biotechnology. There is a significant pressure to develop the present assay formats to become easier to use, faster, and less reagent consuming. Further developments towards high density array--like multianalyte measurement systems would be valuable. To this aim we have studied the applicability of fluorescence resonance energy transfer and time-resolved fluorescence resonance energy transfer in immunoassays on microspots and in microwells. We have used engineered recombinant antibodies detecting the pentameric protein CRP as a model analyte system, and tested different assay formats. We describe also the construction of a time-resolved scanning epifluorometer with which we could measure the FRET interaction between the slow fluorescence decay from europium chelates and its energy transfer to the rapidly decaying fluorophore Cy5.

Comprehensive analysis of the N-glycan biosynthetic pathway using bioinformatics to generate UniCorn: A theoretical N-glycan structure database.

PubMed

Akune, Yukie; Lin, Chi-Hung; Abrahams, Jodie L; Zhang, Jingyu; Packer, Nicolle H; Aoki-Kinoshita, Kiyoko F; Campbell, Matthew P

2016-08-05

Glycan structures attached to proteins are comprised of diverse monosaccharide sequences and linkages that are produced from precursor nucleotide-sugars by a series of glycosyltransferases. Databases of these structures are an essential resource for the interpretation of analytical data and the development of bioinformatics tools. However, with no template to predict what structures are possible the human glycan structure databases are incomplete and rely heavily on the curation of published, experimentally determined, glycan structure data. In this work, a library of 45 human glycosyltransferases was used to generate a theoretical database of N-glycan structures comprised of 15 or less monosaccharide residues. Enzyme specificities were sourced from major online databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan, Consortium for Functional Glycomics (CFG), Carbohydrate-Active enZymes (CAZy), GlycoGene DataBase (GGDB) and BRENDA. Based on the known activities, more than 1.1 million theoretical structures and 4.7 million synthetic reactions were generated and stored in our database called UniCorn. Furthermore, we analyzed the differences between the predicted glycan structures in UniCorn and those contained in UniCarbKB (www.unicarbkb.org), a database which stores experimentally described glycan structures reported in the literature, and demonstrate that UniCorn can be used to aid in the assignment of ambiguous structures whilst also serving as a discovery database. Copyright © 2016 Elsevier Ltd. All rights reserved.

Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

PubMed

Borges, Chad R; Rehder, Douglas S

2016-09-15

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential N-Glycosylation Patterns in Lung Adenocarcinoma Tissue

PubMed Central

Ruhaak, L. Renee; Taylor, Sandra L.; Stroble, Carol; Nguyen, Uyen Thao; Parker, Evan A.; Song, Ting; Lebrilla, Carlito B.; Rom, William N.; Pass, Harvey; Kim, Kyoungmi; Kelly, Karen; Miyamoto, Suzanne

2015-01-01

To decrease the mortality of lung cancer, better screening and diagnostic tools as well as treatment options are needed. Protein glycosylation is one of the major post-translational modifications that is altered in cancer, but it is not exactly clear which glycan structures are affected. A better understanding of the glycan structures that are differentially regulated in lung tumor tissue is highly desirable and will allow us to gain greater insight into the underlying biological mechanisms of aberrant glycosylation in lung cancer. Here, we assess differential glycosylation patterns of lung tumor tissue and nonmalignant tissue at the level of individual glycan structures using nLC–chip–TOF–MS. Using tissue samples from 42 lung adenocarcinoma patients, 29 differentially expressed (FDR < 0.05) glycan structures were identified. The levels of several oligomannose type glycans were upregulated in tumor tissue. Furthermore, levels of fully galactosylated glycans, some of which were of the hybrid type and mostly without fucose, were decreased in cancerous tissue, whereas levels of non- or low-galactosylated glycans mostly with fucose were increased. To further assess the regulation of the altered glycosylation, the glycomics data was compared to publicly available gene expression data from lung adenocarcinoma tissue compared to nonmalignant lung tissue. The results are consistent with the possibility that the observed N-glycan changes have their origin in differentially expressed glycosyltransferases. These results will be used as a starting point for the further development of clinical glycan applications in the fields of imaging, drug targeting, and biomarkers for lung cancer. PMID:26322380

Protein-Linked Glycan Degradation in Infants Fed Human Milk

PubMed Central

Dallas, David C.; Sela, David; Underwood, Mark A.; German, J. Bruce; Lebrilla, Carlito

2014-01-01

Many human milk proteins are glycosylated. Glycosylation is important in protecting bioactive proteins and peptide fragments from digestion. Protein-linked glycans have a variety of functions; however, there is a paucity of information on protein-linked glycan degradation in either the infant or the adult digestive system. Human digestive enzymes can break down dietary disaccharides and starches, but most of the digestive enzymes required for complex protein-linked glycan degradation are absent from both human digestive secretions and the external brush border membrane of the intestinal lining. Indeed, complex carbohydrates remain intact throughout their transit through the stomach and small intestine, and are undegraded by in vitro incubation with either adult pancreatic secretions or intact intestinal brush border membranes. Human gastrointestinal bacteria, however, produce a wide variety of glycosidases with regio- and anomeric specificities matching those of protein-linked glycan structures. These bacteria degrade a wide array of complex carbohydrates including various protein-linked glycans. That bacteria possess glycan degradation capabilities, whereas the human digestive system, perse, does not, suggests that most dietary protein-linked glycan breakdown will be of bacterial origin. In addition to providing a food source for specific bacteria in the colon, protein-linked glycans from human milk may act as decoys for pathogenic bacteria to prevent invasion and infection of the host. The composition of the intestinal microbiome may be particularly important in the most vulnerable humans-the elderly, the immunocompromised, and infants (particularly premature infants). PMID:24533224

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

PubMed Central

Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

2008-01-01

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. PMID:18456721

Sensitive genotyping of foodborne-associated human noroviruses and hepatitis A virus using an array-based platform

USDA-ARS?s Scientific Manuscript database

The viral pathogens, human norovirus (NoV) and hepatitis A virus (HAV), are significant contributors of foodborne associated outbreaks. To develop a typing tool for foodborne viruses, a focused, low-density DNA microarray was developed in conjunction with a rapid and high-throughput fluorescent meth...

Autoinducer Sensing Microarrays by Reporter Bacteria Encapsulated in Hybrid Supramolecular-Polysaccharide Hydrogels.

PubMed

Li, Ping; Dou, Xiaoqiu; Müller, Mareike; Feng, Chuanliang; Chang, Matthew Wook; Frettlöh, Martin; Schönherr, Holger

2017-11-01

A generally applicable strategy to obtain mechanically robust hydrogels for the incorporation and containment of functional reporter bacteria for the microarray and microparticle-based detection and signaling of N-acyl homoserine lactone autoinducers (3OC 12 HSL) at relevant concentrations is reported. For reinforcing hydrogels of 1,4-bi(phenylalanine-diglycol)-benzene (PDB), a hybrid hydrogel is formed by the combination of PDB self-assembly with Ca 2+ mediated alginate crosslinking. The different assembly mechanisms are shown not to interfere with each other and despite the more than four-fold increased moduli of the hydrogels, diffusion of autoinducers into the gels remains efficient and Escherichia coli pLuxR-green fluorescent protein (GFP) reporter bacteria are proliferating. Templating affords reporter bacteria-loaded hydrogels with controllable shape and size. Upon exposure to 3OC 12 HSL, the embedded bacteria exhibit an up to 12 ± 3 times increase in fluorescence intensity due to autoinducer-triggered GFP expression. This approach can serve as a potentially generally applicable strategy to sensitively detect bacteria via their secreted autoinducers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

PubMed

Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

2016-07-01

The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

Prenatal diagnosis of fetuses with increased nuchal translucency using an approach based on quantitative fluorescent polymerase chain reaction and genomic microarray.

PubMed

Pan, Min; Han, Jin; Zhen, Li; Yang, Xin; Li, Ru; Liao, Can; Li, Dong-Zhi

2016-02-01

To assess the clinical value of prenatal diagnosis of fetuses with increased nuchal translucency (NT) using an approach based on quantitative fluorescent polymerase chain reaction (QF-PCR) and chromosomal microarray (CMA). From January 2013 to October 2014, we included 175 pregnancies with fetal NT ≥ 3.5mm at 11-13 weeks' gestation who received chorionic villus sampling. QF-PCR was first used to rapidly detect common aneuploidies. The cases with a normal QF-PCR result were analyzed by CMA. Of the 175 cases, common aneuploidies were detected by QF-PCR in 53 (30.2%) cases (30 cases of trisomy 21, 12 cases of monosomy X, 7 cases of trisomy 18, 3 cases of trisomy 13 and 1 case of 47, XXY). Among the 122 cases with a normal QF-PCR result, microarray detected additional pathogenic copy number variants (CNVs) in 5.7% (7/122) of cases. Four cases would have expected to be detectable by conventional karyotyping because of large deletions/duplications (>10 Mb), leaving three cases (2.5%; 3/118) with pathogenic CNVs only detectable by CMA. It is rational to use a diagnostic strategy in which CMA is preceded by the less expensive, rapid, QF-PCR to detect common aneuploidies. CMA allows detection of a number of pathogenic chromosomal aberrations in fetuses with a high NT. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A Novel Endo-β-N-Acetylglucosaminidase Releases Specific N-Glycans Depending on Different Reaction Conditions

PubMed Central

De Moura Bell, Juliana Maria Leite Nobrega; Frese, Steven A.; Liu, Yan; Mills, David A.; Block, David E.; Barile, Daniela

2015-01-01

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant micro-biome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N-glycans in protein functionality. Endo-β-N-acetylglucosaminidase (EndoBI-1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat-stable enzyme that cleaves the N-N′-diacetyl chitobiose moiety found in the N-glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI-1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI-1 is able to cleave a high diversity of N-glycan structures. Nano-LC-Chip–Q-TOF MS data also revealed that different reaction conditions resulted in different N-glycan compositions released, thus modifying the relative abundance of N-glycan types. In general, more sialylated N-glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI-1 is able to release a wide variety of N-glycans, whose compositions can be selectively manipulated using different processing conditions. PMID:26101185

Systematic Comparison of Reverse Phase and Hydrophilic Interaction Liquid Chromatography Platforms for the Analysis of N-linked Glycans

PubMed Central

Walker, S. Hunter; Carlisle, Brandon C.; Muddiman, David C.

2013-01-01

Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that through an efficient hydrazone formation derivatization of N-linked glycans (∼4 hr of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of ‘multi-omic’ biological approaches. PMID:22954204

ANALYSIS OF GLYCANS DERIVED FROM GLYCOCONJUGATES BY CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

PubMed Central

Mechref, Yehia

2012-01-01

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful mass spectrometric (MS) and tandem MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high order tandem MS (MSn). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and gas chromatography (GC). However, capillary electrophoresis (CE) and microfluidics capillary electrophoresis (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to mass spectrometry. PMID:22180203

Quantification of the Impact of the HIV-1-Glycan Shield on Antibody Elicitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Tongqing; Doria-Rose, Nicole A.; Cheng, Cheng

While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralizationmore » character). To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID50) proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.« less

Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

PubMed Central

Stencel-Baerenwald, Jennifer; Reiss, Kerstin; Blaum, Bärbel S.; Colvin, Daniel; Li, Xiao-Nan; Abel, Ty; Boyd, Kelli; Stehle, Thilo

2015-01-01

ABSTRACT Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease. PMID:25736887

A chicken influenza virus recognizes fucosylated α2,3 sialoglycan receptors on the epithelial cells lining upper respiratory tracts of chickens.

PubMed

Hiono, Takahiro; Okamatsu, Masatoshi; Nishihara, Shoko; Takase-Yoden, Sayaka; Sakoda, Yoshihiro; Kida, Hiroshi

2014-05-01

Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SAα2,3Gal glycans were detected, but not linear SAα2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SAα2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucose-branched SAα2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR. Copyright © 2014 Elsevier Inc. All rights reserved.

Microfluidic bioassay system based on microarrays of hydrogel sensing elements entrapping quantum dot-enzyme conjugates.

PubMed

Jang, Eunji; Kim, Sinyoung; Koh, Won-Gun

2012-01-15

This paper presents a simple method to fabricate a microfluidic biosensor that is able to detect substrates for H(2)O(2)-generating oxidase. The biosensor consists of three components (quantum dot-enzyme conjugates, hydrogel microstructures, and a set of microchannels) that were hierarchically integrated into a microfluidic device. The quantum dot (QD)-enzyme conjugates were entrapped within the poly(ethylene glycol) (PEG)-based hydrogel microstructures that were fabricated within the microchannels by a photopatterning process. Glucose oxidase (GOX) and alcohol oxidase (AOX) were chosen as the model oxidase enzymes, conjugated to carboxyl-terminated CdSe/ZnS QDs, and entrapped within the hydrogel microstructures, which resulted in a fluorescent hydrogel microarray that was responsive to glucose or alcohol. The hydrogel-entrapped GOX and AOX were able to perform enzyme-catalyzed oxidation of glucose and alcohol, respectively, to produce H(2)O(2), which subsequently quenched the fluorescence of the conjugated QDs. The fluorescence intensity of the hydrogel microstructures decreased as the glucose and alcohol concentrations increased, and the detection limits of this system were found to be 50 μM of glucose and 70 μM of alcohol. Because each microchannel was able to carry out different assays independently, the simultaneous detection of glucose and alcohol was possible using our novel microfluidic device composed of multiple microchannels. Copyright © 2011 Elsevier B.V. All rights reserved.

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics.

PubMed

Levine, Peter M; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L

2009-03-15

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

Electronic hybridization detection in microarray format and DNA genotyping

NASA Astrophysics Data System (ADS)

Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

2014-02-01

We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.

Chromosomal Microarray Testing in 42 Korean Patients with Unexplained Developmental Delay, Intellectual Disability, Autism Spectrum Disorders, and Multiple Congenital Anomalies.

PubMed

Lee, Sun Ho; Song, Wung Joo

2017-09-01

Chromosomal microarray (CMA) is a high-resolution, high-throughput method of identifying submicroscopic genomic copy number variations (CNVs). CMA has been established as the first-line diagnostic test for individuals with developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASDs), and multiple congenital anomalies (MCAs). CMA analysis was performed in 42 Korean patients who had been diagnosed with unexplained DD, ID, ASDs, and MCAs. Clinically relevant CNVs were discovered in 28 patients. Variants of unknown significance were detected in 13 patients. The diagnostic yield was high (66.7%). CMA is a superior diagnostic tool compared with conventional karyotyping and fluorescent in situ hybridization.

Electronic hybridization detection in microarray format and DNA genotyping

PubMed Central

Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

2014-01-01

We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device. PMID:24569823

Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

PubMed

Carlson, Ruth I; Cattet, Marc R L; Sarauer, Bryan L; Nielsen, Scott E; Boulanger, John; Stenhouse, Gordon B; Janz, David M

2016-01-01

A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.

Defining Putative Glycan Cancer Biomarkers by Mass Spectrometry

PubMed Central

Mechref, Yehia; Hu, Yunli; Garcia, Aldo; Hussein, Ahmed

2013-01-01

Summary For decades, the association between aberrant glycosylation and many types of cancers has been shown. However, defining the changes of glycan structures has not been demonstrated until recently. This has been facilitated by the major advances in mass spectrometry and separation science which allowed the detailed characterization of glycan changes associated with cancer. Mass spectrometry glycomics methods have been successfully employed to compare the glycomic profiles of different human specimen collected from disease-free individuals and patients with cancer. Additionally, comparing the glycomic profiles of glycoproteins purified from specimen collected from disease-free individuals and patients with cancer has also been performed. These types of glycan analyses employing mass spectrometry or liquid-chromatography mass spectrometry allowed the characterization of native, labeled, and permethylated glycans. This review discusses the different glycomic and glycoproteomic methods employed for defining glycans as cancer biomarkers of different organs, including breast, colon, esophagus, liver, lung, ovarian, pancreas and prostate. PMID:23157355

Infection's Sweet Tooth: How Glycans Mediate Infection and Disease Susceptibility.

PubMed

Taylor, Steven L; McGuckin, Michael A; Wesselingh, Steve; Rogers, Geraint B

2018-02-01

Glycans form a highly variable constituent of our mucosal surfaces and profoundly affect our susceptibility to infection and disease. The diversity and importance of these surface glycans can be seen in individuals who lack a functional copy of the fucosyltransferase gene, FUT2. Representing around one-fifth of the population, these individuals have an altered susceptibility to many bacterial and viral infections and diseases. The mediation of host-pathogen interactions by mucosal glycans, such as those added by FUT2, is poorly understood. We highlight, with specific examples, important mechanisms by which host glycans influence infection dynamics, including by: acting as pathogen receptors (or receptor-decoys), promoting microbial stability, altering the physical characteristics of mucus, and acting as immunological markers. We argue that the effect glycans have on infection dynamics has profound implications for many aspects of healthcare and policy, including clinical management, outbreak control, and vaccination policy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Harnessing glycomics technologies: integrating structure with function for glycan characterization

PubMed Central

Robinson, Luke N.; Artpradit, Charlermchai; Raman, Rahul; Shriver, Zachary H.; Ruchirawat, Mathuros; Sasisekharan, Ram

2013-01-01

Glycans, or complex carbohydrates, are a ubiquitous class of biological molecules which impinge on a variety of physiological processes ranging from signal transduction to tissue development and microbial pathogenesis. In comparison to DNA and proteins, glycans present unique challenges to the study of their structure and function owing to their complex and heterogeneous structures and the dominant role played by multivalency in their sequence-specific biological interactions. Arising from these challenges, there is a need to integrate information from multiple complementary methods to decode structure-function relationships. Focusing on acidic glycans, we describe here key glycomics technologies for characterizing their structural attributes, including linkage, modifications, and topology, as well as for elucidating their role in biological processes. Two cases studies, one involving sialylated branched glycans and the other sulfated glycosaminoglycans, are used to highlight how integration of orthogonal information from diverse datasets enables rapid convergence of glycan characterization for development of robust structure-function relationships. PMID:22522536

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

PubMed Central

Karav, Sercan; Le Parc, Annabelle; Leite Nobrega de Moura Bell, Juliana Maria; Frese, Steven A.; Kirmiz, Nina; Block, David E.; Barile, Daniela

2016-01-01

ABSTRACT Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides. IMPORTANCE It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip–quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate. PMID:27084007

Host specific glycans are correlated with susceptibility to infection by lagoviruses, but not with their virulence.

PubMed

Lopes, Ana M; Breiman, Adrien; Lora, Mónica; Le Moullac-Vaidye, Béatrice; Galanina, Oxana; Nyström, Kristina; Marchandeau, Stephane; Le Gall-Reculé, Ghislaine; Strive, Tanja; Neimanis, Aleksija; Bovin, Nicolai V; Ruvoën-Clouet, Nathalie; Esteves, Pedro J; Abrantes, Joana; Le Pendu, Jacques

2017-11-29

The rabbit hemorrhagic disease virus (RHDV) and the European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus , respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged and many non-pathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, non-pathogenic lagoviruses and EBHSV potentially play a role in determining host range and virulence of lagoviruses. We observed binding to A, B or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits ( Oryctolagus cuniculus ), that have recently been classified as GI strains. Yet, we could not explain the emergence of virulence since similar glycan specificities were found between several pathogenic and non-pathogenic strains. By contrast, EBHSV, recently classified as GII.1, bound to terminal β-linked N-acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species ( Oryctolagus cuniculus, Lepus europaeus and Sylvilagus floridanus ) showed species-specific patterns regarding the susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagoviruses host specificity and range. IMPORTANCE Lagoviruses constitute a genus of the Caliciviridae family, comprising highly pathogenic viruses, RHDV and EBHSV, which infect rabbits and hares, respectively. Recently, non-pathogenic strains were discovered and new pathogenic strains have emerged. In addition, host jumps between lagomorphs are observed. The mechanisms responsible for the emergence of pathogenicity and host-species range are unknown. Previous studies showed that RHDV strains attach to glycans expressed in the upper respiratory and digestive tracts of rabbits, the likely doors of virus entry. Here we studied the glycan-binding properties of novel pathogenic and non-pathogenic strains looking for a link between glycan-binding and virulence or between glycan specificity and host range. We found that glycan binding did not correlate with virulence. However, expression of glycan motifs in the upper respiratory and digestive tracts of lagomorphs revealed species-specific patterns associated with the host range of the virus strains, suggesting that glycan diversity contributes to lagoviruses' host range. Copyright © 2017 American Society for Microbiology.

Recent Advances in Immobilization Strategies for Glycosidases

PubMed Central

Karav, Sercan; Cohen, Joshua L.; Barile, Daniela; de Moura Bell, Juliana Maria Leite Nobrega

2017-01-01

Glycans play important biological roles in cell-to-cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large-scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo-β-N-acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI-1) that cleaves N-N′-diacetyl chitobiose moieties found in the N-linked glycan (N-glycan) core of high mannose, hybrid, and complex N-glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N-glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state-of-the-art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. PMID:27718339

Subtle Differences in Symbiont Cell Surface Glycan Profiles Do Not Explain Species-Specific Colonization Rates in a Model Cnidarian-Algal Symbiosis

PubMed Central

Parkinson, John E.; Tivey, Trevor R.; Mandelare, Paige E.; Adpressa, Donovon A.; Loesgen, Sandra; Weis, Virginia M.

2018-01-01

Mutualisms between cnidarian hosts and dinoflagellate endosymbionts are foundational to coral reef ecosystems. These symbioses are often re-established every generation with high specificity, but gaps remain in our understanding of the cellular mechanisms that control symbiont recognition and uptake dynamics. Here, we tested whether differences in glycan profiles among different symbiont species account for the different rates at which they initially colonize aposymbiotic polyps of the model sea anemone Aiptasia (Exaiptasia pallida). First, we used a lectin array to characterize the glycan profiles of colonizing Symbiodinium minutum (ITS2 type B1) and noncolonizing Symbiodinium pilosum (ITS2 type A2), finding subtle differences in the binding of lectins Euonymus europaeus lectin (EEL) and Urtica dioica agglutinin lectin (UDA) that distinguish between high-mannoside and hybrid-type protein linked glycans. Next, we enzymatically cleaved glycans from the surfaces of S. minutum cultures and followed their recovery using flow cytometry, establishing a 48–72 h glycan turnover rate for this species. Finally, we exposed aposymbiotic host polyps to cultured S. minutum cells masked by EEL or UDA lectins for 48 h, then measured cell densities the following day. We found no effect of glycan masking on symbiont density, providing further support to the hypothesis that glycan-lectin interactions are more important for post-phagocytic persistence of specific symbionts than they are for initial uptake. We also identified several methodological and biological factors that may limit the utility of studying glycan masking in the Aiptasia system. PMID:29765363

Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile

PubMed Central

Thanabalasingham, Gaya; Huffman, Jennifer E.; Kattla, Jayesh J.; Novokmet, Mislav; Rudan, Igor; Gloyn, Anna L.; Hayward, Caroline; Adamczyk, Barbara; Reynolds, Rebecca M.; Muzinic, Ana; Hassanali, Neelam; Pucic, Maja; Bennett, Amanda J.; Essafi, Abdelkader; Polasek, Ozren; Mughal, Saima A.; Redzic, Irma; Primorac, Dragan; Zgaga, Lina; Kolcic, Ivana; Hansen, Torben; Gasperikova, Daniela; Tjora, Erling; Strachan, Mark W.J.; Nielsen, Trine; Stanik, Juraj; Klimes, Iwar; Pedersen, Oluf B.; Njølstad, Pål R.; Wild, Sarah H.; Gyllensten, Ulf; Gornik, Olga; Wilson, James F.; Hastie, Nicholas D.; Campbell, Harry; McCarthy, Mark I.; Rudd, Pauline M.; Owen, Katharine R.; Lauc, Gordan; Wright, Alan F.

2013-01-01

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction. PMID:23274891

Anti-GM1 antibodies as a model of the immune response to self-glycans.

PubMed

Nores, Gustavo A; Lardone, Ricardo D; Comín, Romina; Alaniz, María E; Moyano, Ana L; Irazoqui, Fernando J

2008-03-01

Glycans are a class of molecules with high structural variability, frequently found in the plasma membrane facing the extracellular space. Because of these characteristics, glycans are often considered as recognition molecules involved in cell social functions, and as targets of pathogenic factors. Induction of anti-glycan antibodies is one of the early events in immunological defense against bacteria that colonize the body. Because of this natural infection, antibodies recognizing a variety of bacterial glycans are found in sera of adult humans and animals. The immune response to glycans is restricted by self-tolerance, and no antibodies to self-glycans should exist in normal subjects. However, antibodies recognizing structures closely related to self-glycans do exist, and can lead to production of harmful anti-self antibodies. Normal human sera contain low-affinity anti-GM1 IgM-antibodies. Similar antibodies with higher affinity or different isotype are found in some neuropathy patients. Two hypotheses have been developed to explain the origin of disease-associated anti-GM1 antibodies. According to the "molecular mimicry" hypothesis, similarity between GM1 and Campylobacter jejuni lipopolysaccharide carrying a GM1-like glycan is the cause of Guillain-Barré syndrome associated with anti-GM1 IgG-antibodies. According to the "binding site drift" hypothesis, IgM-antibodies associated with disease originate through changes in the binding site of normally occurring anti-GM1 antibodies. We now present an "integrated" hypothesis, combining the "mimicry" and "drift" concepts, which satisfactorily explains most of the published data on anti-GM1 antibodies.

A hybrid approach to device integration on a genetic analysis platform

NASA Astrophysics Data System (ADS)

Brennan, Des; Jary, Dorothee; Kurg, Ants; Berik, Evgeny; Justice, John; Aherne, Margaret; Macek, Milan; Galvin, Paul

2012-10-01

Point-of-care (POC) systems require significant component integration to implement biochemical protocols associated with molecular diagnostic assays. Hybrid platforms where discrete components are combined in a single platform are a suitable approach to integration, where combining multiple device fabrication steps on a single substrate is not possible due to incompatible or costly fabrication steps. We integrate three devices each with a specific system functionality: (i) a silicon electro-wetting-on-dielectric (EWOD) device to move and mix sample and reagent droplets in an oil phase, (ii) a polymer microfluidic chip containing channels and reservoirs and (iii) an aqueous phase glass microarray for fluorescence microarray hybridization detection. The EWOD device offers the possibility of fully integrating on-chip sample preparation using nanolitre sample and reagent volumes. A key challenge is sample transfer from the oil phase EWOD device to the aqueous phase microarray for hybridization detection. The EWOD device, waveguide performance and functionality are maintained during the integration process. An on-chip biochemical protocol for arrayed primer extension (APEX) was implemented for single nucleotide polymorphism (SNiP) analysis. The prepared sample is aspirated from the EWOD oil phase to the aqueous phase microarray for hybridization. A bench-top instrumentation system was also developed around the integrated platform to drive the EWOD electrodes, implement APEX sample heating and image the microarray after hybridization.

N-glycan signatures identified in tumor interstitial fluid and serum of breast cancer patients: association with tumor biology and clinical outcome.

PubMed

Terkelsen, Thilde; Haakensen, Vilde D; Saldova, Radka; Gromov, Pavel; Hansen, Merete Kjaer; Stöckmann, Henning; Lingjaerde, Ole Christian; Børresen-Dale, Anne-Lise; Papaleo, Elena; Helland, Åslaug; Rudd, Pauline M; Gromova, Irina

2018-06-01

Particular N-glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N-glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N-glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N-glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor-related N-glycan patterns in blood of patients with breast cancer. The segregation of N-linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N-glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N-glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N-glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross-validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N-glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N-glycans from proximal breast tumor fluids is a promising strategy for determining tumor-derived glyco-signature(s) in the blood. N-glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Glycosyltransferase Gene Expression Profiles Classify Cancer Types and Propose Prognostic Subtypes

NASA Astrophysics Data System (ADS)

Ashkani, Jahanshah; Naidoo, Kevin J.

2016-05-01

Aberrant glycosylation in tumours stem from altered glycosyltransferase (GT) gene expression but can the expression profiles of these signature genes be used to classify cancer types and lead to cancer subtype discovery? The differential structural changes to cellular glycan structures are predominantly regulated by the expression patterns of GT genes and are a hallmark of neoplastic cell metamorphoses. We found that the expression of 210 GT genes taken from 1893 cancer patient samples in The Cancer Genome Atlas (TCGA) microarray data are able to classify six cancers; breast, ovarian, glioblastoma, kidney, colon and lung. The GT gene expression profiles are used to develop cancer classifiers and propose subtypes. The subclassification of breast cancer solid tumour samples illustrates the discovery of subgroups from GT genes that match well against basal-like and HER2-enriched subtypes and correlates to clinical, mutation and survival data. This cancer type glycosyltransferase gene signature finding provides foundational evidence for the centrality of glycosylation in cancer.

Thiol-ene immobilisation of carbohydrates onto glass slides as a simple alternative to gold-thiol monolayers, amines or lipid binding.

PubMed

Biggs, Caroline I; Edmondson, Steve; Gibson, Matthew I

2015-01-01

Carbohydrate arrays are a vital tool in studying infection, probing the mechanisms of bacterial, viral and toxin adhesion and the development of new treatments, by mimicking the structure of the glycocalyx. Current methods rely on the formation of monolayers of carbohydrates that have been chemically modified with a linker to enable interaction with a functionalised surface. This includes amines, biotin, lipids or thiols. Thiol-addition to gold to form self-assembled monolayers is perhaps the simplest method for immobilisation as thiolated glycans are readily accessible from reducing carbohydrates in a single step, but are limited to gold surfaces. Here we have developed a quick and versatile methodology which enables the use of thiolated carbohydrates to be immobilised as monolayers directly onto acrylate-functional glass slides via a 'thiol-ene'/Michael-type reaction. By combining the ease of thiol chemistry with glass slides, which are compatible with microarray scanners this offers a cost effective, but also useful method to assemble arrays.

Dissecting the polysaccharide-rich grape cell wall matrix using recombinant pectinases during winemaking.

PubMed

Gao, Yu; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A; Moore, John P

2016-11-05

The effectiveness of enzyme-mediated-maceration in red winemaking relies on the use of an optimum combination of specific enzymes. A lack of information on the relevant enzyme activities and the corresponding polysaccharide-rich berry cell wall structure is a major limitation. This study used different combinations of purified recombinant pectinases with cell wall profiling tools to follow the deconstruction process during winemaking. Multivariate data analysis of the glycan microarray (CoMPP) and gas chromatography (GC) results revealed that pectin lyase performed almost as effectively in de-pectination as certain commercial enzyme mixtures. Surprisingly the combination of endo-polygalacturonase and pectin-methyl-esterase only unraveled the cell walls without de-pectination. Datasets from the various combinations used confirmed pectin-rich and xyloglucan-rich layers within the grape pomace. These data support a proposed grape cell wall model which can serve as a foundation to evaluate testable hypotheses in future studies aimed at developing tailor-made enzymes for winemaking scenarios. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fucosylation is a common glycosylation type in pancreatic cancer stem cell-like phenotypes.

PubMed

Terao, Naoko; Takamatsu, Shinji; Minehira, Tomomi; Sobajima, Tomoaki; Nakayama, Kotarosumitomo; Kamada, Yoshihiro; Miyoshi, Eiji

2015-04-07

To evaluate/isolate cancer stem cells (CSCs) from tissue or cell lines according to various definitions and cell surface markers. Lectin microarray analysis was conducted on CSC-like fractions of the human pancreatic cancer cell line Panc1 by establishing anti-cancer drug-resistant cells. Changes in glycan structure of CSC-like cells were also investigated in sphere-forming cells as well as in CSC fractions obtained from overexpression of CD24 and CD44. Several types of fucosylation were increased under these conditions, and the expression of fucosylation regulatory genes such as fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporters were dramatically enhanced in CSC-like cells. These changes were significant in gemcitabine-resistant cells and sphere cells of a human pancreatic cancer cell line, Panc1. However, downregulation of cellular fucosylation by knockdown of the GDP-fucose transporter did not alter gemcitabine resistance, indicating that increased cellular fucosylation is a result of CSC-like transformation. Fucosylation might be a biomarker of CSC-like cells in pancreatic cancer.

Divergent Synthesis of Heparan Sulfate Oligosaccharides

PubMed Central

2015-01-01

Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis. PMID:26574650

A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs.

PubMed

Gavrovic-Jankulovic, Marija; Poulsen, Knud; Brckalo, Tamara; Bobic, Sonja; Lindner, Buko; Petersen, Arnd

2008-01-01

Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. The immunomodulatory potential of natural BanLec was recognized by a strong immunoglobulin G4 antibody response and T cell mitogen activity in humans. To explore its applicability in glycoproteomics and its modulatory potential, the gene of banana lectin was cloned, sequenced and a recombinant protein was produced in Escherichia coli. The obtained cDNA revealed a novel banana lectin isoform, with an open reading frame of 426 nucleotides, encoding a cytoplasmatic protein of 141 amino acids. The molecular mass of rBanLec determined by ESI FT-MS and N-terminal sequencing confirmed the cDNA at the protein level. The specificity of rBanLec for detection glycan structures was the same as for natural BanLec as examined with five protein extracts rich in glycoprotein content, as well as with horseradish peroxidase glycoprotein. Besides, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics and lectin microarrays, with a potential for modulation of the immune response.

Studying the Effects of Reproductive Hormones and Bacterial Vaginosis on the Glycome of Lavage Samples from the Cervicovaginal Cavity

PubMed Central

Wang, Linlin; Koppolu, Sujeethraj; Chappell, Catherine; Moncla, Bernard J.; Hillier, Sharon L.; Mahal, Lara K.

2015-01-01

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system. PMID:25993513

Studying the effects of reproductive hormones and bacterial vaginosis on the glycome of lavage samples from the cervicovaginal cavity.

PubMed

Wang, Linlin; Koppolu, Sujeethraj; Chappell, Catherine; Moncla, Bernard J; Hillier, Sharon L; Mahal, Lara K

2015-01-01

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.

Automated Glycan Assembly of Oligosaccharides Related to Arabinogalactan Proteins.

PubMed

Bartetzko, Max P; Schuhmacher, Frank; Hahm, Heung Sik; Seeberger, Peter H; Pfrengle, Fabian

2015-09-04

Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the arabinogalactan protein function. Synthetic chemistry is key to the procurement of molecular probes for plant biologists. Described is the automated glycan assembly of 14 oligosaccharides from four monosaccharide building blocks. These linear and branched glycans represent key structural features of natural type-II arabinogalactans and will serve as tools for arabinogalactan biology.

Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells.

PubMed

Srikrishna, G; Panneerselvam, K; Westphal, V; Abraham, V; Varki, A; Freeze, H H

2001-04-01

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Hemocytes and Plasma of the Eastern Oyster (Crassostrea virginica) Display a Diverse Repertoire of Sulfated and Blood Group A-modified N-Glycans*

PubMed Central

Kurz, Simone; Jin, Chunsheng; Hykollari, Alba; Gregorich, Daniel; Giomarelli, Barbara; Vasta, Gerardo R.; Wilson, Iain B. H.; Paschinger, Katharina

2013-01-01

The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal β1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1. PMID:23824194

Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

NASA Astrophysics Data System (ADS)

Everest-Dass, Arun V.; Abrahams, Jodie L.; Kolarich, Daniel; Packer, Nicolle H.; Campbell, Matthew P.

2013-06-01

Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure-function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

Mechanistic Study on Electron Capture Dissociation of the Oligosaccharide-Mg2+ Complex

NASA Astrophysics Data System (ADS)

Huang, Yiqun; Pu, Yi; Yu, Xiang; Costello, Catherine E.; Lin, Cheng

2014-08-01

Electron capture dissociation (ECD) has shown great potential in structural characterization of glycans. However, our current understanding of the glycan ECD process is inadequate for accurate interpretation of the complex glycan ECD spectra. Here, we present the first comprehensive theoretical investigation on the ECD fragmentation behavior of metal-adducted glycans, using the cellobiose-Mg2+ complex as the model system. Molecular dynamics simulation was carried out to determine the typical glycan-Mg2+ binding patterns and the lowest-energy conformer identified was used as the initial geometry for density functional theory-based theoretical modeling. It was found that the electron is preferentially captured by Mg2+ and the resultant Mg+• can abstract a hydroxyl group from the glycan moiety to form a carbon radical. Subsequent radical migration and α-cleavage(s) result in the formation of a variety of product ions. The proposed hydroxyl abstraction mechanism correlates well with the major features in the ECD spectrum of the Mg2+-adducted cellohexaose. The mechanism presented here also predicts the presence of secondary, radical-induced fragmentation pathways. These secondary fragment ions could be misinterpreted, leading to erroneous structural determination. The present study highlights an urgent need for continuing investigation of the glycan ECD mechanism, which is imperative for successful development of bioinformatics tools that can take advantage of the rich structural information provided by ECD of metal-adducted glycans.

N-Glycosylation analysis of yeast Carboxypeptidase Y reveals the ultimate removal of phosphate from glycans at Asn368.

PubMed

B S, Gnanesh Kumar; Surolia, Avadhesha

2017-05-01

Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y 1 ions (peptide+HexNAc) +n revealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man 11-15 GlcNAc 2 at Asn 13 , Man 8-12 GlcNAc 2 at Asn 87 , Man 9-14 GlcNAc 2 at Asn 168 and phosphorylated Man 12-17 GlcNAc 2 as well as Man 11-16 GlcNAc 2 at Asn 368 . The presence of N-glycans with Man <18 GlcNAc 2 indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn 13 or Asn 168 followed by at Asn 368 . However, glycans at Asn 87 which comprises Man 8-12 residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn 87 with the polypeptide chain implicating it in the folding of the protein. Copyright © 2017 Elsevier B.V. All rights reserved.

Introducing N-glycans into natural products through a chemoenzymatic approach.

PubMed

Huang, Wei; Ochiai, Hirofumi; Zhang, Xinyu; Wang, Lai-Xi

2008-11-24

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-beta-N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific, and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural products may bestow novel properties onto these natural products for drug discovery and development.

High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time of flight mass spectrometry

PubMed Central

Gil, Geun-Cheol; Iliff, Bryce; Cerny, Ron; Velander, William H.; Van Cott, Kevin E.

2010-01-01

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method. PMID:20586471

Assessment of a direct hybridization microarray strategy for comprehensive monitoring of genetically modified organisms (GMOs).

PubMed

Turkec, Aydin; Lucas, Stuart J; Karacanli, Burçin; Baykut, Aykut; Yuksel, Hakki

2016-03-01

Detection of GMO material in crop and food samples is the primary step in GMO monitoring and regulation, with the increasing number of GM events in the world market requiring detection solutions with high multiplexing capacity. In this study, we test the suitability of a high-density oligonucleotide microarray platform for direct, quantitative detection of GMOs found in the Turkish feed market. We tested 1830 different 60nt probes designed to cover the GM cassettes from 12 different GM cultivars (3 soya, 9 maize), as well as plant species-specific and contamination controls, and developed a data analysis method aiming to provide maximum throughput and sensitivity. The system was able specifically to identify each cultivar, and in 10/12 cases was sensitive enough to detect GMO DNA at concentrations of ⩽1%. These GMOs could also be quantified using the microarray, as their fluorescence signals increased linearly with GMO concentration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.

PubMed

Michael, Claudia; Rizzi, Andreas M

2015-07-15

Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively. N-Glycans were labeled via reductive amination using (12)C6-aniline and (13)C6-aniline as isotope-coded labeling reagents. The differently labeled glycans were merged and separated into various species using a porous graphitic carbon (PGC) stationary phase. Identification of structural features of separated isobaric isomers was performed by CID-based tandem mass spectrometry (MS/MS) carried out in a quadrupole time-of-flight (QqTOF) or a quadrupole ion-trap (IT) mass spectrometer. Working in the negative ion mode, new diagnostic CID fragment ions could be found that are indicative for the α2,6-type linkage of sialic acids. Other diagnostic ions, identified before as being indicative for the substitution of the 6-antenna, could be confirmed as being of relevance also in the case of aniline labeling. In the positive ion mode, CID fragment ions indicative for the structure of short neutral N-glycans were identified. One new diagnostic ion specific for the linkage position of the terminal sialic acids and one for the presence of bisecting GlcNAc in N-glycans were identified. The aniline label introduced for improved relative quantitation in MS(1) was found not to significantly alter the CID fragmentation patterns that were reported previously by other authors for unlabeled/reduced glycans or for glycans with more polar labels. Copyright © 2015 John Wiley & Sons, Ltd.

Highly Tunable Aptasensing Microarrays with Graphene Oxide Multilayers

NASA Astrophysics Data System (ADS)

Jung, Yun Kyung; Lee, Taemin; Shin, Eeseul; Kim, Byeong-Su

2013-11-01

A highly tunable layer-by-layer (LbL)-assembled graphene oxide (GO) array has been devised for high-throughput multiplex protein sensing. In this array, the fluorescence of different target-bound aptamers labeled with dye is efficiently quenched by GO through fluorescence resonance energy transfer (FRET), and simultaneous multiplex target detection is performed by recovering the quenched fluorescence caused by specific binding between an aptamer and a protein. Thin GO films consisting of 10 bilayers displayed a high quenching ability, yielding over 85% fluorescence quenching with the addition of a 2 μM dye-labeled aptamer. The limit for human thrombin detection in the 6- and 10-bilayered GO array is estimated to be 0.1 and 0.001 nM, respectively, indicating highly tunable nature of LbL assembled GO multilayers in controlling the sensitivity of graphene-based FRET aptasensor. Furthermore, the GO chip could be reused up to four times simply by cleaning it with distilled water.

Long term storage of virus templated fluorescent materials for sensing applications

NASA Astrophysics Data System (ADS)

Seetharam, Raviraja N.; Szuchmacher Blum, Amy; Soto, Carissa M.; Whitley, Jessica L.; Sapsford, Kim E.; Chatterji, Anju; Lin, Tianwei; Johnson, John E.; Guerra, Charles; Satir, Peter; Ratna, Banahalli R.

2008-03-01

Wild type, mutant, and chemically modified Cowpea mosaic viruses (CPMV) were studied for long term preservation in the presence and absence of cryoprotectants. Viral complexes were reconstituted and tested via fluorescence spectroscopy and a UV/vis-based RNase assay for structural integrity. When viruses lyophilized in the absence of cryoprotectant were rehydrated and RNase treated, UV absorption increased, indicating that the capsids were damaged. The addition of trehalose during lyophilization protected capsid integrity for at least 7 weeks. Measurements of the fluorescence peak maximum of CPMV lyophilized with trehalose and reconstituted also indicate that the virus remained intact. Microarray binding assays indicated that CPMV particles chemically modified for use as a fluorescent tracer were intact and retained binding specificity after lyophilization in the presence of trehalose. Thus, we demonstrate that functionalized CPMV nanostructures can be stored for the long term, enabling their use in practical sensing applications.

The HIV glycan shield as a target for broadly neutralizing antibodies.

PubMed

Doores, Katie J

2015-12-01

The HIV envelope glycoprotein (Env) is the sole target for HIV broadly neutralizing antibodies (bnAbs). HIV Env is one of the most heavily glycosylated proteins known, with approximately half of its mass consisting of host-derived N-linked glycans. The high density of glycans creates a shield that impedes antibody recognition but, critically, some of the most potent and broadly active bnAbs have evolved to recognize epitopes formed by these glycans. Although the virus hijacks the host protein synthesis and glycosylation machinery to generate glycosylated HIV Env, studies have shown that HIV Env glycosylation diverges from that typically observed on host-derived glycoproteins. In particular, the high density of glycans leads to a nonself motif of underprocessed oligomannose-type glycans that forms the target of some of the most broad and potent HIV bnAbs. This review discusses the changing perception of the HIV glycan shield, and summarizes the protein-directed and cell-directed factors controlling HIV Env glycosylation that impact on HIV bnAb recognition and HIV vaccine design strategies. © The Author. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Fragmentation of negative ions from N-linked carbohydrates, part 4. Fragmentation of complex glycans lacking substitution on the 6-antenna.

PubMed

Harvey, David J; Jaeken, Jaak; Butler, Mike; Armitage, Alison J; Rudd, Pauline M; Dwek, Raymond A

2010-05-01

Negative ion CID spectra of N-linked glycans released from glycoproteins contain many ions that are diagnostic for specific structural features such as the detailed arrangement of antennae and the location of fucose residues. Identification of such ions requires reference glycans that are often difficult to acquire in a pure state. The recent acquisition of a sample of N-glycans from a patient lacking the enzyme N-acetylglucosaminyltransferase-2 provided an opportunity to investigate fragmentation of glycans lacking a 6-antenna. These glycans contained one or two galactose-N-acetylglucosamine-chains attached to the 3-linked mannose residue of the trimannosyl-chitobiose core with and without fucose substitution. The spectra from the patient sample clearly defined the antenna distribution and showed striking differences from the spectra of isomeric compounds obtained from normal subjects. Furthermore, they provided additional information on previously identified antenna-specific fragment ions and indicated the presence of additional ions that were diagnostic of fucose substitution. Glycans obtained from such enzyme-deficient patients can, thus, be a valuable way of obtaining spectra of specific isomers in a relatively pure state for interpretation of mass spectra. 2010 John Wiley & Sons, Ltd.

O-mannosylation and N-glycosylation: two coordinated mechanisms regulating the tumour suppressor functions of E-cadherin in cancer

PubMed Central

Bartels, Markus F.; Miyoshi, Eiji; Pierce, Michael; Taniguchi, Naoyuki; Carneiro, Fátima; Seruca, Raquel; Reis, Celso A.; Strahl, Sabine; Pinho, Salomé S.

2016-01-01

Dysregulation of tumor suppressor protein E-cadherin is an early molecular event in cancer. O-mannosylation profile of E-cadherin is a newly-described post-translational modification crucial for its adhesive functions in homeostasis. However, the role of O-mannosyl glycans in E-cadherin-mediated cell adhesion in cancer and their interplay with N-glycans remains largely unknown. We herein demonstrated that human gastric carcinomas exhibiting a non-functional E-cadherin display a reduced expression of O-mannosyl glycans concomitantly with increased modification with branched complex N-glycans. Accordingly, overexpression of MGAT5-mediated branched N-glycans both in gastric cancer cells and transgenic mice models led to a significant decrease of O-mannosyl glycans attached to E-cadherin that was associated with impairment of its tumour suppressive functions. Importantly, overexpression of protein O-mannosyltransferase 2 (POMT2) induced a reduced expression of branched N-glycans which led to a protective effect of E-cadherin biological functions. Overall, our results reveal a newly identified mechanism of (dys)regulation of E-cadherin that occur through the interplay between O-mannosylation and N-glycosylation pathway. PMID:27533452

Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.

PubMed

Arata-Kawai, Hanayo; Singer, Mark S; Bistrup, Annette; Zante, Annemieke van; Wang, Yang-Qing; Ito, Yuki; Bao, Xingfeng; Hemmerich, Stefan; Fukuda, Minoru; Rosen, Steven D

2011-01-01

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Kinetic characterization of a novel endo-β-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

PubMed

Karav, Sercan; Parc, Annabelle Le; de Moura Bell, Juliana Maria Leite Nobrega; Rouquié, Camille; Mills, David A; Barile, Daniela; Block, David E

2015-09-01

EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans. Copyright © 2015. Published by Elsevier Inc.

The Impact of O-Glycan Chemistry on the Stability of Intrinsically Disordered Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beckham, Gregg T; Prates, Erica T; Crowley, Michael F

2018-03-02

Protein glycosylation is a diverse post-translational modification that serves myriad biological functions. O-linked glycans in particular vary widely in extent and chemistry in eukaryotes, with secreted proteins from fungi and yeast commonly exhibiting O-mannosylation in intrinsically disordered regions of proteins, likely for proteolysis protection, among other functions. However, it is not well understood why mannose is often the preferred glycan, and more generally, if the neighboring protein sequence and glycan have coevolved to protect against proteolysis in glycosylated intrinsically disordered proteins (IDPs). Here, we synthesized variants of a model IDP, specifically a natively O-mannosylated linker from a fungal enzyme, withmore » a-O-linked mannose, glucose, and galactose moieties, along with a non-glycosylated linker. Upon exposure to thermolysin, O-mannosylation, by far, provides the highest extent of proteolysis protection. To explain this observation, extensive molecular dynamics simulations were conducted, revealing that the axial configuration of the C2-hydroxyl group (2-OH) of a-mannose adjacent to the glycan-peptide bond strongly influences the conformational features of the linker. Specifically, a-mannose restricts the torsions of the IDP main chain more than other glycans whose equatorial 2-OH groups exhibit interactions that favor perpendicular glycan-protein backbone orientation. We suggest that IDP stiffening due to O-mannosylation impairs protease action, with contributions from protein-glycan interactions, protein flexibility, and protein stability. Our results further imply that resistance to proteolysis is an important driving force for evolutionary selection of a-mannose in eukaryotic IDPs, and more broadly, that glycan motifs for proteolysis protection likely coevolve with the protein sequence to which they attach.« less

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.

PubMed

Yago, Tadayuki; Fu, Jianxin; McDaniel, J Michael; Miner, Jonathan J; McEver, Rodger P; Xia, Lijun

2010-05-18

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

Ion Mobility Mass Spectrometry for Extracting Spectra of N-Glycans Directly from Incubation Mixtures Following Glycan Release: Application to Glycans from Engineered Glycoforms of Intact, Folded HIV gp120

NASA Astrophysics Data System (ADS)

Harvey, David J.; Sobott, Frank; Crispin, Max; Wrobel, Antoni; Bonomelli, Camille; Vasiljevic, Snezana; Scanlan, Christopher N.; Scarff, Charlotte A.; Thalassinos, Konstantinos; Scrivens, James H.

2011-03-01

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/ m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.

Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells.

PubMed

Yang, Xiaojing; Tao, Shujuan; Orlando, Ron; Brockhausen, Inka; Kan, Frederick W K

2012-09-01

Oviduct-specific glycoprotein (OVGP1) is a major mucin-like glycoprotein synthesized and secreted exclusively by non-ciliated secretory cells of mammalian oviduct. In vitro functional studies showed that OVGP1 plays important roles during fertilization and early embryo development. We have recently produced recombinant human oviduct-specific glycoprotein (rhOVGP1) in human embryonic kidney 293 (HEK293) cells. The present study was undertaken to characterize the structures and determine the biosynthetic pathways of the N- and O-glycans of rhOVGP1. Treatment of the stable rhOVGP1-expressing HEK293 cells with either GalNAcα-Bn to block O-glycan extension, tunicamycin to block N-glycosylation, or neuraminidase increased the electrophoretic mobility of rhOVGP1. A detailed analysis of O- and N-linked glycans of rhOVGP1 by mass spectrometry showed a broad range of many simple and complex glycan structures. In order to identify the enzymes involved in the glycosylation of rhOVGP1, we assayed glycosyltransferase activities involved in the assembly of O- and N-glycans in HEK293 cells, and compared these to those from the immortalized human oviductal cells (OE-E6/E7). Our results demonstrate that HEK293 and OE-E6/E7 cells exhibit a similar spectrum of glycosyltransferase activities that can synthesize elongated and sialylated O-glycans with core 1 and 2 structures, as well as complex multiantennary N-glycans. It is anticipated that the knowledge gained from the present study will facilitate future studies of the role of the glycans of human OVGP1 in fertilization and early embryo development. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evidence for Differential Glycosylation of Trophoblast Cell Types*

PubMed Central

Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E.; Longtine, Mark S.; Schust, Danny J.; Haslam, Stuart M.; Blois, Sandra M.; Dell, Anne; Clark, Gary F.

2016-01-01

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3–4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2–3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2–3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. PMID:26929217

Evidence for Differential Glycosylation of Trophoblast Cell Types.

PubMed

Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E; Longtine, Mark S; Schust, Danny J; Haslam, Stuart M; Blois, Sandra M; Dell, Anne; Clark, Gary F

2016-06-01

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3-4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2-3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2-3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts.

PubMed

Yoon, Seon-Joo; Utkina, Natalia; Sadilek, Martin; Yagi, Hirokazu; Kato, Koichi; Hakomori, Sen-itiroh

2013-07-01

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.

Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules.

PubMed

Hong, Senlian; Chen, Tao; Zhu, Yuntao; Li, Ang; Huang, Yanyi; Chen, Xing

2014-06-02

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action.

PubMed

Upton, Rosie; Bell, Leonard; Guy, Colin; Caldwell, Paul; Estdale, Sian; Barran, Perdita E; Firth, David

2016-10-18

In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.

Glycomics meets artificial intelligence - Potential of glycan analysis for identification of seropositive and seronegative rheumatoid arthritis patients revealed.

PubMed

Chocholova, Erika; Bertok, Tomas; Jane, Eduard; Lorencova, Lenka; Holazova, Alena; Belicka, Ludmila; Belicky, Stefan; Mislovicova, Danica; Vikartovska, Alica; Imrich, Richard; Kasak, Peter; Tkac, Jan

2018-06-01

In this study, one hundred serum samples from healthy people and patients with rheumatoid arthritis (RA) were analyzed. Standard immunoassays for detection of 10 different RA markers and analysis of glycan markers on antibodies in 10 different assay formats with several lectins were applied for each serum sample. A dataset containing 2000 data points was data mined using artificial neural networks (ANN). We identified key RA markers, which can discriminate between healthy people and seropositive RA patients (serum containing autoantibodies) with accuracy of 83.3%. Combination of RA markers with glycan analysis provided much better discrimination accuracy of 92.5%. Immunoassays completely failed to identify seronegative RA patients (serum not containing autoantibodies), while glycan analysis correctly identified 43.8% of these patients. Further, we revealed other critical parameters for successful glycan analysis such as type of a sample, format of analysis and orientation of captured antibodies for glycan analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pejchal, Robert; Doores, Katie J.; Walker, Laura M.

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment ofmore » the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.« less

All-atom ensemble modeling to analyze small angle X-ray scattering of glycosylated proteins

PubMed Central

Guttman, Miklos; Weinkam, Patrick; Sali, Andrej; Lee, Kelly K.

2013-01-01

Summary The flexible and heterogeneous nature of carbohydrate chains often renders glycoproteins refractory to traditional structure determination methods. Small Angle X-ray scattering (SAXS) can be a useful tool for obtaining structural information of these systems. All-atom modeling of glycoproteins with flexible glycan chains was applied to interpret the solution SAXS data for a set of glycoproteins. For simpler systems (single glycan, with a well defined protein structure), all-atom modeling generates models in excellent agreement with the scattering pattern, and reveals the approximate spatial occupancy of the glycan chain in solution. For more complex systems (several glycan chains, or unknown protein substructure), the approach can still provide insightful models, though the orientations of glycans become poorly determined. Ab initio shape reconstructions appear to capture the global morphology of glycoproteins, but in most cases offer little information about glycan spatial occupancy. The all-atom modeling methodology is available as a webserver at http://modbase.compbio.ucsf.edu/allosmod-foxs. PMID:23473666

Unique, polyfucosylated glycan-receptor interactions are essential for regeneration of Hydra magnipapillata.

PubMed

Sahadevan, Sonu; Antonopoulos, Aristotelis; Haslam, Stuart M; Dell, Anne; Ramaswamy, Subramanian; Babu, Ponnusamy

2014-01-17

Cell-cell communications, cell-matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan-receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan-receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan-receptor interactions in the regeneration of H. magnipapillata.

N-Glycan Modification in Aspergillus Species▿

PubMed Central

Kainz, Elke; Gallmetzer, Andreas; Hatzl, Christian; Nett, Juergen H.; Li, Huijuan; Schinko, Thorsten; Pachlinger, Robert; Berger, Harald; Reyes-Dominguez, Yazmid; Bernreiter, Andreas; Gerngross, Tillmann; Wildt, Stefan; Strauss, Joseph

2008-01-01

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi. PMID:18083888

The glycan structure of albumin Redhill, a glycosylated variant of human serum albumin.

PubMed

Kragh-Hansen, U; Donaldson, D; Jensen, P H

2001-11-26

Although human serum albumin is synthesized without carbohydrate, glycosylated variants of the protein can be found. We have determined the structure of the glycan bound to the double-mutant albumin Redhill (-1 Arg, 320 Ala-->Thr). The oligosaccharide was released from the protein using anhydrous hydrazine, and its structure was investigated using neuraminidase and a reagent array analysis method, which is based on the use of specific exoglycosidases. The glycan was shown to be a disialylated biantennary complex type oligosaccharide N-linked to 318 Asn. However, a minor part (11 mol%) of the glycan was without sialic acid. The structure is principally the same as that of glycans bound to two other types of glycosylated albumin variants. Glycosylation can affect, for example, the fatty acid binding properties of albumin. Taking the present information into account, it is apparent that different effects on binding are caused not by different glycan structures but by different locations of attachment, with the possible addition of local conformational changes in the protein molecule.

Silver nanorod structures for metal enhanced fluorescence

NASA Astrophysics Data System (ADS)

Badshah, Mohsin Ali; Lu, Xun; Ju, Jonghyun; Kim, Seok-min

2016-09-01

Fluorescence based detection is a commonly used methodology in biotechnology and medical diagnostics. Metalenhanced fluorescence (MEF) becomes a promising strategy to improve the sensitivity of fluorescence detection, where fluorophores coupling with surface plasmon on metallic structures results fluorescence enhancement. To apply the MEF methodology in real medical diagnostics, especially for protein or DNA microarray detection, a large area (e.g., slide glass, 75 × 25 mm2) with uniform metallic nanostructures is required. In this study, we fabricated a large area MEF substrates using oblique angle deposition (OAD), which is a single step, inexpensive large area fabrication method of nanostructures. To optimize the morphological effect, Ag-nanorods with various lengths were fabricated on the conventional slide glass substrates. Streptavidin-Cy5 dissolved in buffer solution with different concentration (100ng/ml 100μg/ml) were applied to MEF substrates using a pipette, and the fluorescence signals were measured. The enhancement factor increased with the increase in length of Ag-nanorods and maximum enhancement factor 91x was obtained from Ag-nanorods 750nm length compare to bare glass due to higher surface Plasmon effect.

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system

PubMed Central

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D.; Baba, Hiroko; Ikenaka, Kazuhiro

2017-01-01

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy. PMID:28186137

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system.

PubMed

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D; Baba, Hiroko; Ikenaka, Kazuhiro

2017-02-10

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P 0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy.

Glycosylation status of bone sialoprotein and its role in mineralization.

PubMed

Xu, Lan; Zhang, Zhenqing; Sun, Xue; Wang, Jingjing; Xu, Wei; Shi, Lv; Lu, Jiaojiao; Tang, Juan; Liu, Jingjing; Su, Xiong

2017-11-15

The highly glycosylated bone sialoprotein (BSP) is an abundant non-collagenous phosphoprotein in bone which enhances osteoblast differentiation and new bone deposition in vitro and in vivo. However, the structural details of its different glycosylation linkages have not been well studied and their functions in bone homeostasis are not clear. Previous studies suggested that the O-glycans, but not the N-glycans on BSP, are highly sialylated. Herein, we employed tandem mass spectrometry (MS/MS) to demonstrate that the N-glycanson the recombinant human integrin binding sialoprotein (rhiBSP) are also enriched in sialic acids (SAs) at their termini. We also identified multiple novel sites of N-glycan modification. Treatment of rhiBSP enhances osteoblast differentiation and mineralization of MC3T3-E1 cells and this effect could be partially reversed by efficient enzymatic removal of its N-glycans. Removal of all terminal SAs has a greater effect in reversing the effect of rhiBSP on osteogenesis, especially on mineralization, suggesting that sialylation at the termini of both N-glycans and O-glycans plays an important role in this regulation. Moreover, BSP-conjugated SAs may affect mineralization via ERK activation of VDR expression. Collectively, our results identified novel N-glycans enriched in SAs on the rhiBSP and demonstrated that SAs at both N- and O-glycans are important for BSP regulation of osteoblast differentiation and mineralization in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

Glycomics and glycoproteomics focused on aging and age-related diseases--Glycans as a potential biomarker for physiological alterations.

PubMed

Miura, Yuri; Endo, Tamao

2016-08-01

Since glycosylation depends on glycosyltransferases, glycosidases, and sugar nucleotide donors, it is susceptible to the changes associated with physiological and pathological conditions. Therefore, alterations in glycan structures may be good targets and biomarkers for monitoring health conditions. Since human aging and longevity are affected by genetic and environmental factors such as diseases, lifestyle, and social factors, a scale that reflects various environmental factors is required in the study of human aging and longevity. We herein focus on glycosylation changes elucidated by glycomic and glycoproteomic studies on aging, longevity, and age-related diseases including cognitive impairment, diabetes mellitus, and frailty. We also consider the potential of glycan structures as biomarkers and/or targets for monitoring physiological and pathophysiological changes. Glycan structures are altered in age-related diseases. These glycans and glycoproteins may be involved in the pathophysiology of these diseases and, thus, be useful diagnostic markers. Age-dependent changes in N-glycans have been reported previously in cohort studies, and characteristic N-glycans in extreme longevity have been proposed. These findings may lead to a deeper understanding of the mechanisms underlying aging as well as the factors influencing longevity. Alterations in glycosylation may be good targets and biomarkers for monitoring health conditions, and be applicable to studies on age-related diseases and healthy aging. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

Kinetics of N-Glycan Release from Human Immunoglobulin G (IgG) by PNGase F: All Glycans Are Not Created Equal.

PubMed

Huang, Yining; Orlando, Ron

2017-12-01

The biologic activity of IgG molecules is modulated by its crystallizable fragment N-glycosylation, and thus, the analysis of IgG glycosylation is critical. A standard approach to analyze glycosylation of IgGs involves the release of the N-glycans by the enzyme peptide N-glycosidase F, which cleaves the linkage between the asparagine residue and innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the reducing terminal GlcNAc residue. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this de-glycosylation reaction for IgG glycopeptides and to determine the effect of glycan structure and amino acid sequence on the rate of glycan release from glycopeptides of IgGs. This study revealed that the slight differences in amino acid sequences did not lead to a statistically different deglycosylation rate. However, statistically significant differences in the deglycosylation rate constants were observed between glycopeptides differing only in glycan structure ( i.e. , nonfucosylated, fucosylated, bisecting-GlcNAc, sialylated, etc .). For example, a single sialic acid residue was found to decrease the rate by a factor of 3. Similar reductions in rate were associated with the presence of a bisecting-GlcNAc. We predict the differences in release kinetics can lead to significant quantitative variations of the glycosylation study of IgGs.

Simultaneous Release and Labeling of O- and N-Glycans Allowing for Rapid Glycomic Analysis by Online LC-UV-ESI-MS/MS.

PubMed

Wang, Chengjian; Lu, Yu; Han, Jianli; Jin, Wanjun; Li, Lingmei; Zhang, Ying; Song, Xuezheng; Huang, Linjuan; Wang, Zhongfu

2018-05-24

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by β-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.

A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doores, Katie J.; Fulton, Zara; Hong, Vu

2011-08-24

Antibody 2G12 uniquely neutralizes a broad range of HIV-1 isolates by binding the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine. However, host-produced (self)-carbohydrate motifs have been unsuccessful so far at eliciting 2G12-like antibodies that cross-react with gp120. Based on the surprising observation that 2G12 binds nonproteinaceous monosaccharide D-fructose with higher affinity than D-mannose, we show here that a designed set of nonself, synthetic monosaccharides are potent antigens. When introduced to the terminus of the D1 arm of protein glycans recognized by 2G12,more » their antigenicity is significantly enhanced. Logical variation of these unnatural sugars pinpointed key modifications, and the molecular basis of this increased antigenicity was elucidated using high-resolution crystallographic analyses. Virus-like particle protein conjugates containing such nonself glycans are bound more tightly by 2G12. As immunogens they elicit higher titers of antibodies than those immunogenic conjugates containing the self D1 glycan motif. These antibodies generated from nonself immunogens also cross-react with this self motif, which is found in the glycan shield, when it is presented in a range of different conjugates and glycans. However, these antibodies did not bind this glycan motif when present on gp120.« less

Loss of intestinal core 1–derived O-glycans causes spontaneous colitis in mice

PubMed Central

Fu, Jianxin; Wei, Bo; Wen, Tao; Johansson, Malin E.V.; Liu, Xiaowei; Bradford, Emily; Thomsson, Kristina A.; McGee, Samuel; Mansour, Lilah; Tong, Maomeng; McDaniel, J. Michael; Sferra, Thomas J.; Turner, Jerrold R.; Chen, Hong; Hansson, Gunnar C.; Braun, Jonathan; Xia, Lijun

2011-01-01

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell–specific deficiency of core 1–derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1–derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1–derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 β1,3-galactosyltransferase–specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC. PMID:21383503

A label-free, fluorescence based assay for microarray

NASA Astrophysics Data System (ADS)

Niu, Sanjun

DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same intensity of excitation light. The fluorescence contrast is used to quantify the amount of probe-target hybridization. A mathematical model that considers multiple reflections and scattering is developed to explain the mechanism of the fluorescence contrast which depends on the thickness of the PS film. Scattering is the dominant factor that contributes to the contrast. The potential of this assay to detect single nucleotide polymorphism is also tested.

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

PubMed

Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

2015-01-01

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

PubMed Central

Busti, Elena; Bordoni, Roberta; Castiglioni, Bianca; Monciardini, Paolo; Sosio, Margherita; Donadio, Stefano; Consolandi, Clarissa; Rossi Bernardi, Luigi; Battaglia, Cristina; De Bellis, Gianluca

2002-01-01

Background PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Results Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. Conclusions The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria. PMID:12243651

Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides.

PubMed

Laassri, Majid; Chizhikov, Vladimir; Mikheev, Maxim; Shchelkunov, Sergei; Chumakov, Konstantin

2003-09-01

Variola virus (VARV), causing smallpox, is a potential biological weapon. Methods to detect VARV rapidly and to differentiate it from other viruses causing similar clinical syndromes are needed urgently. We have developed a new microarray-based method that detects simultaneously and discriminates four orthopoxvirus (OPV) species pathogenic for humans (variola, monkeypox, cowpox, and vaccinia viruses) and distinguishes them from chickenpox virus (varicella-zoster virus or VZV). The OPV gene C23L/B29R, encoding the CC-chemokine binding protein, was sequenced for 41 strains of seven species of orthopox viruses obtained from different geographical regions. Those C23L/B29R sequences and the ORF 62 sequences from 13 strains of VZV (selected from GenBank) were used to design oligonucleotide probes that were immobilized on an aldehyde-coated glass surface (a total of 57 probes). The microchip contained several unique 13-21 bases long oligonucleotide probes specific to each virus species to ensure redundancy and robustness of the assay. A region approximately 1100 bases long was amplified from samples of viral DNA and fluorescently labeled with Cy5-modified dNTPs, and single-stranded DNA was prepared by strand separation. Hybridization was carried out under plastic coverslips, resulting in a fluorescent pattern that was quantified using a confocal laser scanner. 49 known and blinded samples of OPV DNA, representing different OPV species, and two VZV strains were tested. The oligonucleotide microarray hybridization technique identified reliably and correctly all samples. This new procedure takes only 3 h, and it can be used for parallel testing of multiple samples.

Scanning microarray slides.

PubMed

Ares, Manuel

2014-02-01

Here we describe some practical concerns surrounding the scanning of microarray slides that have been hybridized with fluorescent dyes. We use a laser scanner that has two lasers, each set to excite a different fluor, and separate detectors to capture emission from each fluor. The laser passes over an address (position on the scanned surface) and the detectors capture photons emitted from each address. Two superimposed image files are written that carry intensities for each channel for each pixel of the image scan. These are the raw data. Image analysis software is used to identify and summarize the intensities of the pixels that make up each spot. After comparison to background pixels, the processed intensity levels representing the gene expression measurements are associated with the identity of each spot.

Databases and Associated Tools for Glycomics and Glycoproteomics.

PubMed

Lisacek, Frederique; Mariethoz, Julien; Alocci, Davide; Rudd, Pauline M; Abrahams, Jodie L; Campbell, Matthew P; Packer, Nicolle H; Ståhle, Jonas; Widmalm, Göran; Mullen, Elaine; Adamczyk, Barbara; Rojas-Macias, Miguel A; Jin, Chunsheng; Karlsson, Niclas G

2017-01-01

The access to biodatabases for glycomics and glycoproteomics has proven to be essential for current glycobiological research. This chapter presents available databases that are devoted to different aspects of glycobioinformatics. This includes oligosaccharide sequence databases, experimental databases, 3D structure databases (of both glycans and glycorelated proteins) and association of glycans with tissue, disease, and proteins. Specific search protocols are also provided using tools associated with experimental databases for converting primary glycoanalytical data to glycan structural information. In particular, researchers using glycoanalysis methods by U/HPLC (GlycoBase), MS (GlycoWorkbench, UniCarb-DB, GlycoDigest), and NMR (CASPER) will benefit from this chapter. In addition we also include information on how to utilize glycan structural information to query databases that associate glycans with proteins (UniCarbKB) and with interactions with pathogens (SugarBind).

Crystal structure of a fully glycosylated HIV-1 gp120 core reveals a stabilizing role for the glycan at Asn262

DOE PAGES

Kong, Leopold; Wilson, Ian A.; Kwong, Peter D.

2014-12-26

The crystal structure of a fully glycosylated HIV-1 gp120 core in complex with CD4 receptor and Fab 17b at 4.5-Å resolution reveals 9 of the 15 N-linked glycans of core gp120 to be partially ordered. The glycan at position Asn262 had the most extensive and well-ordered electron density, and a GlcNAc 2Man 7 was modeled. Lastly, the GlcNAc stem of this glycan is largely buried in a cleft in gp120, suggesting a role in gp120 folding and stability. Its arms interact with the stems of neighboring glycans from the oligomannose patch, which is a major target for broadly neutralizing antibodies.

Increased Bisecting N-Acetylglucosamine and Decreased Branched Chain Glycans of N-linked Glycoproteins in Expressed Prostatic Secretions Associated with Prostate Cancer Progression

PubMed Central

Nyalwidhe, Julius O.; Betesh, Lucy R.; Powers, Thomas W.; Jones, E. Ellen; White, Krista Y.; Burch, Tanya C.; Brooks, Jasmin; Watson, Megan T.; Lance, Raymond S.; Troyer, Dean A.; Semmes, O. John; Mehta, Anand; Drake, Richard R.

2013-01-01

Purpose Using prostatic fluids rich in glycoproteins like prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) , the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging mass spectrometry-based glycopeptide analysis platforms. Experimental Design A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides. Results Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data. Conclusion and clinical relevance Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis. PMID:23775902

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

PubMed

Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

2016-06-01

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of bisecting GlcNAc and core fucosylation on conformational properties of biantennary complex-type N-glycans in solution.

PubMed

Nishima, Wataru; Miyashita, Naoyuki; Yamaguchi, Yoshiki; Sugita, Yuji; Re, Suyong

2012-07-26

The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the conformational properties of N-glycans is examined at the atomic level by performing replica-exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex-type N-glycans, namely, unmodified, two single-substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Manα1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation, respectively. The population change is attributed to specific inter-residue hydrogen bonds, including water-mediated ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constants, are well reproduced taking the multiple conformers into account. Our structural model supports the concept of "conformer selection", which emphasizes the conformational flexibility of N-glycans in protein-glycan interactions.

Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

PubMed

Kato, Tatsuya; Kikuta, Kotaro; Kanematsu, Ayumi; Kondo, Sachiko; Yagi, Hirokazu; Kato, Koichi; Park, Enoch Y

2017-09-01

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man 3 GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

Separation of Two Distinct O-Glycoforms of Human IgA1 by Serial Lectin Chromatography Followed by Mass Spectrometry O-Glycan Analysis.

PubMed

Lehoux, S; Ju, T

2017-01-01

Human immunoglobulin A1 (IgA1), which carries four to six mucin-type O-glycans (O-glycans) on its hinge region (HR), is the most abundant O-glycoprotein in plasma or serum. While normal O-glycans from hematopoietic-originated cells are core 1-based complex structures, many reports showed that the IgA1 from patients with IgA nephropathy (IgAN) carries undergalactosylated or truncated O-glycans such as the Tn antigen and its sialylated version the SialylTn (STn) antigen on the HR. Yet, there is still a debate whether Tn/STn on the HR of IgA1 is specific to the IgA1 from patients with IgAN since these antigens have also been seen in serum IgA1 of healthy individuals. An additional question is whether the O-glycans at all sites on the two HRs of one IgA1 molecule are homogeneous (either all normal or all Tn/STn) or heterogeneous (both normal and Tn/STn O-glycans). To address these questions, we conducted a systematic study on the O-glycans of plasma IgA1 from both IgAN patients and healthy controls using serial HPA and PNA lectin chromatography followed by western blotting and further analysis of O-glycans from HPA-bound and PNA-bound IgA1 fractions by mass spectrometry. Unexpectedly, we found that a variable minor fraction of IgA1 from both IgAN patients and healthy controls had Tn/STn antigens, and that the O-glycoprotein IgA1 molecules from most samples had only two distinct O-glycoforms: one major glycoform with homogeneous normal core 1-based O-glycans and one minor glycoform with homogeneous Tn/STn antigens. These results raised a serious question about the role of Tn/STn antigens on IgA1 in pathogenesis of IgAN, and there is a demand for a practical methodology that any laboratory can utilize to analyze the O-glycans of IgA1. Herein, we describe the methodology we developed in more detail. The method could also be applied to the analysis of any other O-glycosylated proteins. © 2017 Elsevier Inc. All rights reserved.

VLSI Microsystem for Rapid Bioinformatic Pattern Recognition

NASA Technical Reports Server (NTRS)

Fang, Wai-Chi; Lue, Jaw-Chyng

2009-01-01

A system comprising very-large-scale integrated (VLSI) circuits is being developed as a means of bioinformatics-oriented analysis and recognition of patterns of fluorescence generated in a microarray in an advanced, highly miniaturized, portable genetic-expression-assay instrument. Such an instrument implements an on-chip combination of polymerase chain reactions and electrochemical transduction for amplification and detection of deoxyribonucleic acid (DNA).

Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates

PubMed Central

Yoshino, Timothy P.; Wu, Xiao-Jun; Gonzalez, Laura A.; Hokke, Cornelis H.

2013-01-01

Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval S. mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins, as well as the ability or hemocytes to acquire shared glycans by the selective binding of parasite-released LTP. Unraveling the functional significance of these naturally expressed and acquired shared glycans on specific hemocyte populations represents an important challenge for future investigations. PMID:23085445

Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.

PubMed

Newburg, D S

2009-04-01

This review discusses the role of human milk glycans in protecting infants, but the conclusion that the human milk glycans constitute an innate immune system whereby the mother protects her offspring may have general applicability in all mammals, including species of commercial importance. Infants that are not breastfed have a greater incidence of severe diarrhea and respiratory diseases than those who are breastfed. In the past, this had been attributed primarily to human milk secretory antibodies. However, the oligosaccharides are major components of human milk, and milk is also rich in other glycans, including glycoproteins, mucins, glycosaminoglycans, and glycolipids. These milk glycans, especially the oligosaccharides, are composed of thousands of components. The milk factor that promotes gut colonization by Bifidobacterium bifidum was found to be a glycan, and such prebiotic characteristics may contribute to protection against infectious agents. However, the ability of human milk glycans to protect the neonate seems primarily to be due to their inhibition of pathogen binding to their host cell target ligands. Many such examples include specific fucosylated oligosaccharides and glycans that inhibit specific pathogens. Most human milk oligosaccharides are fucosylated, and their production depends on fucosyltransferase enzymes; mutations in these fucosyltransferase genes are common and underlie the various Lewis blood types in humans. Variable expression of specific fucosylated oligosaccharides in milk, also a function of these genes (and maternal Lewis blood type), is significantly associated with the risk of infectious disease in breastfed infants. Human milk also contains major quantities and large numbers of sialylated oligosaccharides, many of which are also present in bovine colostrum. These could similarly inhibit several common viral pathogens. Moreover, human milk oligosaccharides strongly attenuate inflammatory processes in the intestinal mucosa. These results support the hypothesis that oligosaccharides and other glycans are the major constituents of an innate immune system of human milk whereby the mother protects her infant from enteric and other pathogens through breastfeeding. These protective glycans may prove useful as a basis for the development of novel prophylactic and therapeutic agents that inhibit disease by mucosal pathogens in many species.

Comparative Genomic Analysis of the Human Gut Microbiome Reveals a Broad Distribution of Metabolic Pathways for the Degradation of Host-Synthetized Mucin Glycans and Utilization of Mucin-Derived Monosaccharides

PubMed Central

Ravcheev, Dmitry A.; Thiele, Ines

2017-01-01

The colonic mucus layer is a dynamic and complex structure formed by secreted and transmembrane mucins, which are high-molecular-weight and heavily glycosylated proteins. Colonic mucus consists of a loose outer layer and a dense epithelium-attached layer. The outer layer is inhabited by various representatives of the human gut microbiota (HGM). Glycans of the colonic mucus can be used by the HGM as a source of carbon and energy when dietary fibers are not sufficiently available. Both commensals and pathogens can utilize mucin glycans. Commensals are mostly involved in the cleavage of glycans, while pathogens mostly utilize monosaccharides released by commensals. This HGM-derived degradation of the mucus layer increases pathogen susceptibility and causes many other health disorders. Here, we analyzed 397 individual HGM genomes to identify pathways for the cleavage of host-synthetized mucin glycans to monosaccharides as well as for the catabolism of the derived monosaccharides. Our key results are as follows: (i) Genes for the cleavage of mucin glycans were found in 86% of the analyzed genomes, which significantly higher than a previous estimation. (ii) Genes for the catabolism of derived monosaccharides were found in 89% of the analyzed genomes. (iii) Comparative genomic analysis identified four alternative forms of the monosaccharide-catabolizing enzymes and four alternative forms of monosaccharide transporters. (iv) Eighty-five percent of the analyzed genomes may be involved in potential feeding pathways for the monosaccharides derived from cleaved mucin glycans. (v) The analyzed genomes demonstrated different abilities to degrade known mucin glycans. Generally, the ability to degrade at least one type of mucin glycan was predicted for 81% of the analyzed genomes. (vi) Eighty-two percent of the analyzed genomes can form mutualistic pairs that are able to degrade mucin glycans and are not degradable by any of the paired organisms alone. Taken together, these findings provide further insight into the inter-microbial communications of the HGM as well as into host-HGM interactions. PMID:28912798

Comparative Genomic Analysis of the Human Gut Microbiome Reveals a Broad Distribution of Metabolic Pathways for the Degradation of Host-Synthetized Mucin Glycans and Utilization of Mucin-Derived Monosaccharides.

PubMed

Ravcheev, Dmitry A; Thiele, Ines

2017-01-01

The colonic mucus layer is a dynamic and complex structure formed by secreted and transmembrane mucins, which are high-molecular-weight and heavily glycosylated proteins. Colonic mucus consists of a loose outer layer and a dense epithelium-attached layer. The outer layer is inhabited by various representatives of the human gut microbiota (HGM). Glycans of the colonic mucus can be used by the HGM as a source of carbon and energy when dietary fibers are not sufficiently available. Both commensals and pathogens can utilize mucin glycans. Commensals are mostly involved in the cleavage of glycans, while pathogens mostly utilize monosaccharides released by commensals. This HGM-derived degradation of the mucus layer increases pathogen susceptibility and causes many other health disorders. Here, we analyzed 397 individual HGM genomes to identify pathways for the cleavage of host-synthetized mucin glycans to monosaccharides as well as for the catabolism of the derived monosaccharides. Our key results are as follows: (i) Genes for the cleavage of mucin glycans were found in 86% of the analyzed genomes, which significantly higher than a previous estimation. (ii) Genes for the catabolism of derived monosaccharides were found in 89% of the analyzed genomes. (iii) Comparative genomic analysis identified four alternative forms of the monosaccharide-catabolizing enzymes and four alternative forms of monosaccharide transporters. (iv) Eighty-five percent of the analyzed genomes may be involved in potential feeding pathways for the monosaccharides derived from cleaved mucin glycans. (v) The analyzed genomes demonstrated different abilities to degrade known mucin glycans. Generally, the ability to degrade at least one type of mucin glycan was predicted for 81% of the analyzed genomes. (vi) Eighty-two percent of the analyzed genomes can form mutualistic pairs that are able to degrade mucin glycans and are not degradable by any of the paired organisms alone. Taken together, these findings provide further insight into the inter-microbial communications of the HGM as well as into host-HGM interactions.

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

PubMed Central

Crooks, Ema T.; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S.; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O’Dell, Sijy; LaBranche, Celia; Robinson, James E.; Montefiori, David C.; McKee, Krisha; Du, Sean X.; Doria-Rose, Nicole; Kwong, Peter D.; Mascola, John R.; Zhu, Ping; Schief, William R.; Wyatt, Richard T.; Whalen, Robert G.; Binley, James M.

2015-01-01

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine. PMID:26023780

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

PubMed

Crooks, Ema T; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O'Dell, Sijy; LaBranche, Celia; Robinson, James E; Montefiori, David C; McKee, Krisha; Du, Sean X; Doria-Rose, Nicole; Kwong, Peter D; Mascola, John R; Zhu, Ping; Schief, William R; Wyatt, Richard T; Whalen, Robert G; Binley, James M

2015-05-01

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

PubMed

Kim, Chang Sup; Seo, Jeong Hyun; Cha, Hyung Joon

2012-08-07

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

PubMed

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

PubMed

Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

2016-02-01

Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates--both antibody-drug conjugates as well as constructs for in vivo imaging--has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities.

IL-15 regulates memory CD8+ T cell O-glycan synthesis and affects trafficking

PubMed Central

Nolz, Jeffrey C.; Harty, John T.

2014-01-01

Memory and naive CD8+ T cells exhibit distinct trafficking patterns. Specifically, memory but not naive CD8+ T cells are recruited to inflamed tissues in an antigen-independent manner. However, the molecular mechanisms that regulate memory CD8+ T cell trafficking are largely unknown. Here, using murine models of infection and T cell transfer, we found that memory but not naive CD8+ T cells dynamically regulate expression of core 2 O-glycans, which interact with P- and E-selectins to modulate trafficking to inflamed tissues. Following infection, antigen-specific effector CD8+ T cells strongly expressed core 2 O-glycans, but this glycosylation pattern was lost by most memory CD8+ T cells. After unrelated infection or inflammatory challenge, memory CD8+ T cells synthesized core 2 O-glycans independently of antigen restimulation. The presence of core 2 O-glycans subsequently directed these cells to inflamed tissue. Memory and naive CD8+ T cells exhibited the opposite pattern of epigenetic modifications at the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis. The open chromatin configuration in memory CD8+ T cells permitted de novo generation of core 2 O-glycans in a TCR-independent, but IL-15–dependent, manner. Thus, IL-15 stimulation promotes antigen-experienced memory CD8+ T cells to generate core 2 O-glycans, which subsequently localize them to inflamed tissues. These findings suggest that CD8+ memory T cell trafficking potentially can be manipulated to improve host defense and immunotherapy. PMID:24509081

Anti-microbial antibodies in celiac disease: Trick or treat?

PubMed Central

Papp, Maria; Foldi, Ildiko; Altorjay, Istvan; Palyu, Eszter; Udvardy, Miklos; Tumpek, Judit; Sipka, Sandor; Korponay-Szabo, Ilma Rita; Nemes, Eva; Veres, Gabor; Dinya, Tamas; Tordai, Attila; Andrikovics, Hajnalka; Norman, Gary L; Lakatos, Peter Laszlo

2009-01-01

AIM: To determine the prevalence of a new set of anti-glycan and anti-outer membrane protein (anti-OMP) antibodies in a Hungarian cohort of adult Celiac disease (CD) patients. METHODS: 190 consecutive CD patients [M/F: 71/119, age:39.9 (SD:14.1) years], 100 healthy, and 48 gastrointestinal controls were tested for glycan anti-Saccharomyces cerevisiae (gASCA), anti-laminaribioside (ALCA), anti-chitobioside, anti-mannobioside, anti-OMP antibodies and major NOD2/CARD15 mutations. Thirty out of 82 CD patients enrolled at the time of diagnosis were re-evaluated for the same antibodies after longstanding gluten-free diet (GFD). RESULTS: 65.9% of the CD patients were positive for at least one of the tested antibodies at the time of the diagnosis. Except anti-OMP and ALCA, anti-microbial antibodies were exclusively seen in untreated CD; however, the overall sensitivity was low. Any glycan positivity (LR+: 3.13; 95% CI: 2.08-4.73) was associated with an increased likelihood ratio for diagnosing CD. Significant correlation was found between the levels of anti-glycan and anti-endomysial or anti-transglutaminase antibodies. Anti-glycan positivity was lost after longstanding GFD. Anti-glycan antibody titers were associated with symptoms at presentation, but not the presence of NOD2/CARD15 mutations. Patients with severe malabsorption more frequently had multiple antibodies at diagnosis (P = 0.019). CONCLUSION: The presence of anti-glycan antibodies in CD seems to be secondary to the impaired small bowel mucosa which can lead to increased antigen presentation. Furthermore, anti-glycan positivity may be considered an additional marker of CD and dietary adherence. PMID:19701969

The Long Non-Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed-Batch Conditions.

PubMed

Amann, Thomas; Hansen, Anders Holmgaard; Kol, Stefan; Lee, Gyun Min; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup

2018-06-03

In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, we aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2 and-T3 with and without a disrupted B4Gal-T4 sequence resulted in only ∼1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. We revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ∼6% and ∼3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, we present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, we provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins. This article is protected by copyright. All rights reserved.

Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achyuthan, Komandoor E.; Wheeler, David R.

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of themore » YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.« less

Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

DOE PAGES

Achyuthan, Komandoor E.; Wheeler, David R.

2015-08-27

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of themore » YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.« less

Multiplexed protein profiling on microarrays by rolling-circle amplification

PubMed Central

Schweitzer, Barry; Roberts, Scott; Grimwade, Brian; Shao, Weiping; Wang, Minjuan; Fu, Qin; Shu, Quiping; Laroche, Isabelle; Zhou, Zhimin; Tchernev, Velizar T.; Christiansen, Jason; Velleca, Mark; Kingsmore, Stephen F.

2010-01-01

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys. PMID:11923841

Development of a cell microarray chip for detection of circulating tumor cells

NASA Astrophysics Data System (ADS)

Yamamura, S.; Yatsushiro, S.; Abe, K.; Baba, Y.; Kataoka, M.

2012-03-01

Detection of circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients has clinical significance in earlier diagnosis of metastases. In this study, a novel cell microarray chip for accurate and rapid detection of tumor cells from human leukocytes was developed. The chip with 20,944 microchambers (105 μm diameter and 50 μm depth) was made from polystyrene, and the surface was rendered to hydrophilic by means of reactive-ion etching, which led to the formation of mono-layers of leukocytes on the microchambers. As the model of CTCs detection, we spiked human bronchioalveolar carcinoma (H1650) cells into human T lymphoblastoid leukemia (CEM) cells suspension and detected H1650 cells using the chip. A CEM suspension contained with H1650 cells was dispersed on the chip surface, followed by 10 min standing to allow the cells to settle down into the microchambers. About 30 CEM cells were accommodated in each microchamber, over 600,000 CEM cells in total being on a chip. We could detect 1 H1650 cell per 106 CEM cells on the microarray by staining with fluorescence-conjugated antibody (Anti-Cytokeratin) and cell membrane marker (DiD). Thus, this cell microarray chip has highly potential to be a novel tool of accurate and rapid detection of CTCs.

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

One-pot enzymatic glycan remodeling of a therapeutic monoclonal antibody by endoglycosidase S (Endo-S) from Streptococcus pyogenes.

PubMed

Tong, Xin; Li, Tiezheng; Orwenyo, Jared; Toonstra, Christian; Wang, Lai-Xi

2018-04-01

A facile, one-pot enzymatic glycan remodeling of antibody rituximab to produce homogeneous high-mannose and hybrid type antibody glycoforms is described. This method was based on the unique substrate specificity of the endoglycosidase S (Endo-S) from Streptococcus pyogenes. While Endo-S efficiently hydrolyzes the bi-antennary complex type IgG Fc N-glycans, we found that Endo-S did not hydrolyze the "ground state" high-mannose or hybrid glycoforms, and only slowly hydrolyzed the highly activated high-mannose or hybrid N-glycan oxazolines. Moreover, we found that wild-type Endo-S could efficiently use high-mannose or hybrid glycan oxazolines for transglycosylation without product hydrolysis. The combination of the remarkable difference in substrate specificity of Endo-S allows the deglycosylation of heterogeneous rituximab and the transglycosylation with glycan oxazoline to take place in one-pot without the need of isolating the deglycosylated intermediate or changing the enzyme to afford the high-mannose type, hybrid type, and some selectively modified truncated form of antibody glycoforms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence that maturation of the N-linked glycans of the respiratory syncytial virus (RSV) glycoproteins is required for virus-mediated cell fusion: The effect of {alpha}-mannosidase inhibitors on RSV infectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDonald, Terence P.; Jeffree, Chris E.; Li, Ping

2006-07-05

Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the {alpha}-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affectedmore » by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity.« less

Notable Aspects of Glycan-Protein Interactions

PubMed Central

Cohen, Miriam

2015-01-01

This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). PMID:26340640

N-Glycosylation Profiling of Porcine Reproductive and Respiratory Syndrome Virus Envelope Glycoprotein 5

PubMed Central

Li, Juan; Tao, Shujuan; Orlando, Ron; Murtaugh, Michael P.

2015-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV. PMID:25726973

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

PubMed

Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

2004-12-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

PubMed Central

Panicker, Gitika; Call, Douglas R.; Krug, Melissa J.; Bej, Asim K.

2004-01-01

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. PMID:15574946

A Novel Method for Relative Quantitation of N-Glycans by Isotopic Labeling Using 18O-Water

PubMed Central

Tao, Shujuan; Orlando, Ron

2014-01-01

Quantitation is an essential aspect of comprehensive glycomics study. Here, a novel isotopic-labeling method is described for N-glycan quantitation using 18O-water. The incorporation of the 18O-labeling into the reducing end of N-glycans is simply and efficiently achieved during peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F release. This process provides a 2-Da mass difference compared with the N-glycans released in 16O-water. A mathematical calculation method was also developed to determine the 18O/16O ratios from isotopic peaks. Application of this method to several standard glycoprotein mixtures and human serum demonstrated that this method can facilitate the relative quantitation of N-glycans over a linear dynamic range of two orders, with high accuracy and reproducibility. PMID:25365792

Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein.

PubMed

Kim, Unyong; Oh, Myung Jin; Seo, Youngsuk; Jeon, Yinae; Eom, Joon-Ho; An, Hyun Joo

2017-09-01

Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.

The Emerging Importance of IgG Fab Glycosylation in Immunity.

PubMed

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

5-Nitrosalicylic Acid as a Novel Matrix for In-Source Decay in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

PubMed Central

Asakawa, Daiki

2013-01-01

The matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) of peptides and glycans was studied using an oxidizing chemical, 5-nitrosalicylic acid (5-NSA) as the matrix. The use of 5-NSA for the MALDI-ISD of peptides and glycans promoted fragmentation pathways involving “hydrogen-deficient” radical precursors. Hydrogen abstraction from peptides resulted in the production of a “hydrogen-deficient” peptide radical that contained a radical site on the amide nitrogen in the peptide backbone with subsequent radical-induced cleavage at the Cα–C bonds. Cleavage at the Cα–C bond leads to the production of an a•/x fragment pair and the radical a• ions then undergo further hydrogen abstraction to form a ions after Cα–C bond cleavage. Since the Pro residue does not contain a nitrogen-centered radical site, Cα–C bond cleavage does not occur at this site. Alternatively, the specific cleavage of CO−N bonds leads to a b•/y fragment pair at Xxx−Pro which occurs via hydrogen abstraction from the Cα−H in the Pro residue. In contrast, “hydrogen-deficient” glycan radicals were generated by hydrogen abstraction from hydroxyl groups in glycans. Both glycosidic and cross-ring cleavages occurred as the result of the degradation of “hydrogen-deficient” glycan radicals. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps in the characterization of glycans. Moreover, isobaric glycans could be distinguished by structure specific ISD ions, and the molar ratio of glycan isomers in a mixture can be estimated from their fragment ions abundance ratios. MALDI-ISD with 5-NSA could be a useful method for the sequencing of peptides including the location of post-translational modifications, identification and semi-quantitative analysis of mixtures of glycan isomers. PMID:24860709

Isomer-specific profiling of N-glycans derived from human serum for potential biomarker discovery in pancreatic cancer.

PubMed

Liu, Yufei; Wang, Chang; Wang, Ran; Wu, Yike; Zhang, Liang; Liu, Bi-Feng; Cheng, Liming; Liu, Xin

2018-06-15

Glycosylation is one of the most important post-translational modifications of protein. Recently, global profiling of human serum glycomics has become a noninvasive method for cancer-related biomarker discovery and many studies have focused on compositional glycan profiling. In contrast, structure-specific glycan profiling may provide more potential biomarkers with higher specificity than compositional profiling. In this work, N-glycans released from human serum were neutralized with methylamine and reduced by ammonia-borane complex prior to profiling using nanoLC-ESI-MS with porous graphitized carbon (PGC) and relative abundances of over 280 isomers were compared between pancreatic cancer (PC) cases (n = 32) and healthy controls (n = 32). Statistical analysis identified 25 specific-isomeric biomarkers with significant differences (p-value < 0.05). ROC and PCA analysis were performed to assess the potential biomarkers which were identified as being significantly altered in cancer. The AUCs of the significantly changed specific-isomers were ranging from 0.712 to 0.949. In addition, with the combination of all potential biomarkers, a higher AUC of 0.976 with sensitivity (93.5%) and specificity (90.6%) was obtained. Overall, the proposed strategy coupled to relative quantitative analysis of isomeric glycans make it possible to discover new biomarkers for the diagnosis of PC. Pancreatic cancer (PC) has a poor prognosis with a five-year survival rate <5%. Therefore, a strategy for accurate diagnosis of PC is indeed required. In this paper, a dual-derivatized strategy for structure-specific glycan profiling has been used and according to our best knowledge, this is the first application of this strategy for PC biomarker discovery, in which the separation, identification and relative quantification of isomeric glycans can be simultaneously obtained. In addition, by in-depth analysis of isomeric glycans, the full description of the stereo- and region- diversity of glycans can also be achieved, which might provide more potential information for PC biomarker discovery. Copyright © 2018 Elsevier B.V. All rights reserved.

Solution NMR Analyses of the C-type Carbohydrate Recognition Domain of DC-SIGNR Protein Reveal Different Binding Modes for HIV-derived Oligosaccharides and Smaller Glycan Fragments

PubMed Central

Probert, Fay; Whittaker, Sara B.-M.; Crispin, Max; Mitchell, Daniel A.; Dixon, Ann M.

2013-01-01

The C-type lectin DC-SIGNR (dendritic cell-specific ICAM-3-grabbing non-integrin-related; also known as L-SIGN or CD299) is a promising drug target due to its ability to promote infection and/or within-host survival of several dangerous pathogens (e.g. HIV and severe acute respiratory syndrome coronavirus (SARS)) via interactions with their surface glycans. Crystallography has provided excellent insight into the mechanism by which DC-SIGNR interacts with small glycans, such as (GlcNAc)2Man3; however, direct observation of complexes with larger, physiological oligosaccharides, such as Man9GlcNAc2, remains elusive. We have utilized solution-state nuclear magnetic resonance spectroscopy to investigate DC-SIGNR binding and herein report the first backbone assignment of its active, calcium-bound carbohydrate recognition domain. Direct interactions with the small sugar fragments Man3, Man5, and (GlcNAc)2Man3 were investigated alongside Man9GlcNAc derived from recombinant gp120 (present on the HIV viral envelope), providing the first structural data for DC-SIGNR in complex with a virus-associated ligand, and unique binding modes were observed for each glycan. In particular, our data show that DC-SIGNR has a different binding mode for glycans on the HIV viral envelope compared with the smaller glycans previously observed in the crystalline state. This suggests that using the binding mode of Man9GlcNAc, instead of those of small glycans, may provide a platform for the design of DC-SIGNR inhibitors selective for high mannose glycans (like those on HIV). 15N relaxation measurements provided the first information on the dynamics of the carbohydrate recognition domain, demonstrating that it is a highly flexible domain that undergoes ligand-induced conformational and dynamic changes that may explain the ability of DC-SIGNR to accommodate a range of glycans on viral surfaces. PMID:23788638

Changes to Serum Sample Tube and Processing Methodology Does Not Cause Inter-Individual Variation in Automated Whole Serum N-Glycan Profiling in Health and Disease

PubMed Central

Shubhakar, Archana; Kalla, Rahul; Nimmo, Elaine R.; Fernandes, Daryl L.; Satsangi, Jack; Spencer, Daniel I. R.

2015-01-01

Introduction Serum N-glycans have been identified as putative biomarkers for numerous diseases. The impact of different serum sample tubes and processing methods on N-glycan analysis has received relatively little attention. This study aimed to determine the effect of different sample tubes and processing methods on the whole serum N-glycan profile in both health and disease. A secondary objective was to describe a robot automated N-glycan release, labeling and cleanup process for use in a biomarker discovery system. Methods 25 patients with active and quiescent inflammatory bowel disease and controls had three different serum sample tubes taken at the same draw. Two different processing methods were used for three types of tube (with and without gel-separation medium). Samples were randomised and processed in a blinded fashion. Whole serum N-glycan release, 2-aminobenzamide labeling and cleanup was automated using a Hamilton Microlab STARlet Liquid Handling robot. Samples were analysed using a hydrophilic interaction liquid chromatography/ethylene bridged hybrid(BEH) column on an ultra-high performance liquid chromatography instrument. Data were analysed quantitatively by pairwise correlation and hierarchical clustering using the area under each chromatogram peak. Qualitatively, a blinded assessor attempted to match chromatograms to each individual. Results There was small intra-individual variation in serum N-glycan profiles from samples collected using different sample processing methods. Intra-individual correlation coefficients were between 0.99 and 1. Unsupervised hierarchical clustering and principal coordinate analyses accurately matched samples from the same individual. Qualitative analysis demonstrated good chromatogram overlay and a blinded assessor was able to accurately match individuals based on chromatogram profile, regardless of disease status. Conclusions The three different serum sample tubes processed using the described methods cause minimal inter-individual variation in serum whole N-glycan profile when processed using an automated workstream. This has important implications for N-glycan biomarker discovery studies using different serum processing standard operating procedures. PMID:25831126

Changes to serum sample tube and processing methodology does not cause Intra-Individual [corrected] variation in automated whole serum N-glycan profiling in health and disease.

PubMed

Ventham, Nicholas T; Gardner, Richard A; Kennedy, Nicholas A; Shubhakar, Archana; Kalla, Rahul; Nimmo, Elaine R; Fernandes, Daryl L; Satsangi, Jack; Spencer, Daniel I R

2015-01-01

Serum N-glycans have been identified as putative biomarkers for numerous diseases. The impact of different serum sample tubes and processing methods on N-glycan analysis has received relatively little attention. This study aimed to determine the effect of different sample tubes and processing methods on the whole serum N-glycan profile in both health and disease. A secondary objective was to describe a robot automated N-glycan release, labeling and cleanup process for use in a biomarker discovery system. 25 patients with active and quiescent inflammatory bowel disease and controls had three different serum sample tubes taken at the same draw. Two different processing methods were used for three types of tube (with and without gel-separation medium). Samples were randomised and processed in a blinded fashion. Whole serum N-glycan release, 2-aminobenzamide labeling and cleanup was automated using a Hamilton Microlab STARlet Liquid Handling robot. Samples were analysed using a hydrophilic interaction liquid chromatography/ethylene bridged hybrid(BEH) column on an ultra-high performance liquid chromatography instrument. Data were analysed quantitatively by pairwise correlation and hierarchical clustering using the area under each chromatogram peak. Qualitatively, a blinded assessor attempted to match chromatograms to each individual. There was small intra-individual variation in serum N-glycan profiles from samples collected using different sample processing methods. Intra-individual correlation coefficients were between 0.99 and 1. Unsupervised hierarchical clustering and principal coordinate analyses accurately matched samples from the same individual. Qualitative analysis demonstrated good chromatogram overlay and a blinded assessor was able to accurately match individuals based on chromatogram profile, regardless of disease status. The three different serum sample tubes processed using the described methods cause minimal inter-individual variation in serum whole N-glycan profile when processed using an automated workstream. This has important implications for N-glycan biomarker discovery studies using different serum processing standard operating procedures.

Trichuris suis-induced modulation of human dendritic cell function is glycan-mediated.

PubMed

Klaver, Elsenoor J; Kuijk, Loes M; Laan, Lisa C; Kringel, Helene; van Vliet, Sandra J; Bouma, Gerd; Cummings, Richard D; Kraal, Georg; van Die, Irma

2013-03-01

Human monocyte-derived dendritic cells (DCs) show remarkable phenotypic changes upon direct contact with soluble products (SPs) of Trichuris suis, a pig whipworm that is experimentally used in therapies to ameliorate inflammation in patients with Crohn's disease and multiple sclerosis. These changes may contribute to the observed induction of a T helper 2 (Th2) response and the suppression of Toll-like receptor (TLR)-induced Th1 and Th17 responses by human DCs primed with T. suis SPs. Here it is demonstrated that glycans of T. suis SPs contribute significantly to the suppression of the lipopolysaccharide (LPS)-induced expression in DCs of a broad variety of cytokines and chemokines, including important pro-inflammatory mediators such as TNF-α, IL-6, IL-12, lymphotoxin α (LTA), C-C Motif Ligand (CCL)2, C-X-C Motif Ligands (CXCL)9 and CXCL10. In addition, the data show that human DCs strongly bind T. suis SP-glycans via the C-type lectin receptors (CLRs) mannose receptor (MR) and DC-specific ICAM-3-grabbing non-integrin (DC-SIGN). The interaction of DCs with T. suis glycans likely involves mannose-type glycans, rather than fucosylated glycans, which differs from DC binding to soluble egg antigens of the human worm parasite, Schistosoma mansoni. In addition, macrophage galactose-type lectin (MGL) recognises T. suis SPs, which may contribute to the interaction with immature DCs or other MGL-expressing immune cells such as macrophages. The interaction of T. suis glycans with CLRs of human DCs may be essential for the ability of T. suis to suppress a pro-inflammatory phenotype of human DCs. The finding that the T. suis-induced modulation of human DC function is glycan-mediated is novel and indicates that helminth glycans contribute to the dampening of inflammation in a wide range of human inflammatory diseases. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes*

PubMed Central

Kaulfürst-Soboll, Heidi; Rips, Stephan; Koiwa, Hisashi; Kajiura, Hiroyuki; Fujiyama, Kazuhito; von Schaewen, Antje

2011-01-01

Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry β1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-β1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins. PMID:21478158

Revisiting the substrate specificity of mammalian α1,6-fucosyltransferase reveals that it catalyzes core fucosylation of N-glycans lacking α1,3-arm GlcNAc.

PubMed

Yang, Qiang; Zhang, Roushu; Cai, Hui; Wang, Lai-Xi

2017-09-08

The mammalian α1,6-fucosyltransferase (FUT8) catalyzes the core fucosylation of N -glycans in the biosynthesis of glycoproteins. Previously, intensive in vitro studies with crude extract or purified enzyme concluded that the attachment of a GlcNAc on the α1,3 mannose arm of N -glycan is essential for FUT8-catalyzed core fucosylation. In contrast, we have recently shown that expression of erythropoietin in a GnTI knock-out, FUT8-overexpressing cell line results in the production of fully core-fucosylated glycoforms of the oligomannose substrate Man 5 GlcNAc 2 , suggesting that FUT8 can catalyze core fucosylation of N -glycans lacking an α1,3-arm GlcNAc in cells. Here, we revisited the substrate specificity of FUT8 by examining its in vitro activity toward an array of selected N -glycans, glycopeptides, and glycoproteins. Consistent with previous studies, we found that free N -glycans lacking an unmasked α1,3-arm GlcNAc moiety are not FUT8 substrates. However, Man 5 GlcNAc 2 glycan could be efficiently core-fucosylated by FUT8 in an appropriate protein/peptide context, such as with the erythropoietin protein, a V3 polypeptide derived from HIV-1 gp120, or a simple 9-fluorenylmethyl chloroformate-protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature bi-antennary complex-type N -glycan also could be core-fucosylated by FUT8, albeit at much lower efficiency than the Man 5 GlcNAc 2 peptide. This study represents the first report of in vitro FUT8-catalyzed core fucosylation of N -glycans lacking the α1,3-arm GlcNAc moiety. Our results suggest that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Staged induction of HIV-1 glycan–dependent broadly neutralizing antibodies

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Bonsignori, Mattia; Kreider, Edward F.; Fera, Daniela; ...

2017-03-15

A preventive HIV-1 vaccine should induce HIV-1–specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3- glycan bnAb. Two autologousmore » neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3- glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.« less