Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation.

PubMed

Yang, Yu; Gu, Yuxin; Wan, Bin; Ren, Xiaomin; Guo, Liang-Hong

2017-09-15

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT. Copyright © 2017 Elsevier B.V. All rights reserved.

GWAS for serum galactose-deficient IgA1 implicates critical genes of the O-glycosylation pathway

PubMed Central

Kiryluk, Krzysztof; Moldoveanu, Zina; Suzuki, Hitoshi; Reily, Colin; Hou, Ping; Xie, Jingyuan; Mladkova, Nikol; Prakash, Sindhuri; Fischman, Clara; Shapiro, Samantha; Bradbury, Drew; Ionita-Laza, Iuliana; Eitner, Frank; Rauen, Thomas; Maillard, Nicolas; Floege, Jürgen; Chen, Nan; Zhang, Hong; Scolari, Francesco; Wyatt, Robert J.; Julian, Bruce A.; Gharavi, Ali G.; Novak, Jan

2017-01-01

Aberrant O-glycosylation of serum immunoglobulin A1 (IgA1) represents a heritable pathogenic defect in IgA nephropathy, the most common form of glomerulonephritis worldwide, but specific genetic factors involved in its determination are not known. We performed a quantitative GWAS for serum levels of galactose-deficient IgA1 (Gd-IgA1) in 2,633 subjects of European and East Asian ancestry and discovered two genome-wide significant loci, in C1GALT1 (rs13226913, P = 3.2 x 10−11) and C1GALT1C1 (rs5910940, P = 2.7 x 10−8). These genes encode molecular partners essential for enzymatic O-glycosylation of IgA1. We demonstrated that these two loci explain approximately 7% of variability in circulating Gd-IgA1 in Europeans, but only 2% in East Asians. Notably, the Gd-IgA1-increasing allele of rs13226913 is common in Europeans, but rare in East Asians. Moreover, rs13226913 represents a strong cis-eQTL for C1GALT1 that encodes the key enzyme responsible for the transfer of galactose to O-linked glycans on IgA1. By in vitro siRNA knock-down studies, we confirmed that mRNA levels of both C1GALT1 and C1GALT1C1 determine the rate of secretion of Gd-IgA1 in IgA1-producing cells. Our findings provide novel insights into the genetic regulation of O-glycosylation and are relevant not only to IgA nephropathy, but also to other complex traits associated with O-glycosylation defects, including inflammatory bowel disease, hematologic disease, and cancer. PMID:28187132

Dynamic Glycosylation Governs the Vertebrate COPII Protein Trafficking Pathway.

PubMed

Cox, Nathan J; Unlu, Gokhan; Bisnett, Brittany J; Meister, Thomas R; Condon, Brett M; Luo, Peter M; Smith, Timothy J; Hanna, Michael; Chhetri, Abhishek; Soderblom, Erik J; Audhya, Anjon; Knapik, Ela W; Boyce, Michael

2018-01-09

The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.

PubMed

Chigira, Yuko; Oka, Takuji; Okajima, Tetsuya; Jigami, Yoshifumi

2008-04-01

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

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

Jones, Meredith B., E-mail: mbauman7@jhu.edu; Tomiya, Noboru, E-mail: ntomiya1@jhu.edu; Betenbaugh, Michael J., E-mail: beten@jhu.edu

2010-04-23

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates inmore » the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.« less

Genotypic and phenotypic characterization of the O-linked protein glycosylation system reveals high glycan diversity in paired meningococcal carriage isolates.

PubMed

Børud, Bente; Bårnes, Guro K; Brynildsrud, Ola Brønstad; Fritzsønn, Elisabeth; Caugant, Dominique A

2018-03-19

Species within the genus Neisseria display significant glycan diversity associated with the O -linked protein glycosylation ( pgl ) systems due to phase variation, polymorphic genes and gene content. The aim of this study was to examine in detail the pgl genotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about two months apart, were analyzed with whole genome sequencing. The O -linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the PubMLST.org database. Immunoblotting with glycan specific antibodies were used to investigate the protein glycosylation phenotype. All major pgl locus polymorphisms identified in N. meningitidis to date were present in our isolate collection, with the variable presence of pglG-pglH, both in combination with either pglB or pglB2. We identified significant changes and diversity in the pgl genotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase variable expression of the involved glycosyltransferases and the O-acetyltransferase. To our knowledge, this is the first characterization of the pgl genotype and glycosylation phenotype in a larger strain collection. This study thus provides important insight into glycan diversity in N. meningitidis and phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage. Importance Bacterial meningitis is a serious global health problem and one of the major causative organisms is Neisseria meningitidis , which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

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

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylationmore » target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.« less

O-Glycosylation in Cell Wall Proteins in Scedosporium prolificans Is Critical for Phagocytosis and Inflammatory Cytokines Production by Macrophages

PubMed Central

Xisto, Mariana I. D. S.; Bittencourt, Vera C. B.; Liporagi-Lopes, Livia Cristina; Haido, Rosa M. T.; Mendonça, Morena S. A.; Sassaki, Guilherme; Figueiredo, Rodrigo T.; Romanos, Maria Teresa V.; Barreto-Bergter, Eliana

2015-01-01

In this study, we analyze the importance of O-linked oligosaccharides present in peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium prolificans for recognition and phagocytosis of conidia by macrophages. Adding PRM led to a dose-dependent inhibition of conidia phagocytosis, whereas de-O-glycosylated PRM did not show any effect. PRM induced the release of macrophage-derived antimicrobial compounds. However, O-linked oligosaccharides do not appear to be required for such induction. The effect of PRM on conidia-induced macrophage killing was examined using latex beads coated with PRM or de-O-glycosylated PRM. A decrease in macrophage viability similar to that caused by conidia was detected. However, macrophage killing was unaffected when beads coated with de-O-glycosylated PRM were used, indicating the toxic effect of O-linked oligosaccharides on macrophages. In addition, PRM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PRM abolished cytokine induction, suggesting that the O-linked oligosaccharidic chains are important moieties involved in inflammatory responses through the induction of TNF-α secretion. In summary, we show that O-glycosylation plays a role in the recognition and uptake of S. prolificans by macrophages, killing of macrophages and production of pro- inflammatory cytokines. PMID:25875427

Comparison of N- and O-linked glycosylation patterns of ebolavirus glycoproteins.

PubMed

Collar, Amanda L; Clarke, Elizabeth C; Anaya, Eduardo; Merrill, Denise; Yarborough, Sarah; Anthony, Scott M; Kuhn, Jens H; Merle, Christine; Theisen, Manfred; Bradfute, Steven B

2017-02-01

Ebolaviruses are emerging pathogens that cause severe and often fatal viral hemorrhagic fevers. Four distinct ebolaviruses are known to cause Ebola virus disease in humans. The ebolavirus envelope glycoprotein (GP 1,2 ) is heavily glycosylated, but the precise glycosylation patterns of ebolaviruses are largely unknown. Here we demonstrate that approximately 50 different N-glycan structures are present in GP 1,2 derived from the four pathogenic ebolaviruses, including high mannose, hybrid, and bi-, tri-, and tetra-antennary complex glycans with and without fucose and sialic acid. The overall N-glycan composition is similar between the different ebolavirus GP 1,2 s. In contrast, the amount and type of O-glycan structures varies widely between ebolavirus GP 1,2 s. Notably, this O-glycan dissimilarity is also present between two variants of Ebola virus, the original Yambuku variant and the Makona variant responsible for the most recent Western African epidemic. The data presented here should serve as the foundation for future ebolaviral entry and immunogenicity studies. Copyright © 2016 Elsevier Inc. All rights reserved.

A reagent-controlled SN2-glycosylation for the direct synthesis of β-linked 2-deoxy-sugars.

PubMed

Issa, John Paul; Bennett, Clay S

2014-04-16

The efficient and stereoselective construction of glycosidic linkages remains one of the most formidable challenges in organic chemistry. This is especially true in cases such as β-linked deoxy-sugars, where the outcome of the reaction cannot be controlled using the stereochemical information intrinsic to the glycosyl donor. Here we show that p-toluenesulfonic anhydride activates 2-deoxy-sugar hemiacetals in situ as electrophilic species, which react stereoselectively with nucleophilic acceptors to produce β-anomers exclusively. NMR studies confirm that, under these conditions, the hemiacetal is quantitatively converted into an α-glycosyl tosylate, which is presumably the reactive species in the reaction. This approach demonstrates that use of promoters that activate hemiacetals as well-defined intermediates can be used to permit stereoselective glycosylation through an SN2-pathway.

O-linked oligosaccharides on insulin receptor

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

Collier, E.; Gorden, P.

1991-02-01

The insulin receptor, an integral membrane glycoprotein, is synthesized as a single-chain precursor that is cleaved to produce two mature subunits, both of which contain N-linked oligosaccharide chains and covalently linked fatty acids. We report that the beta-subunit also contains O-linked oligosaccharides. The proreceptor, alpha-subunit, and beta-subunit were labeled with (3H)mannose and (3H)galactose in the presence or absence of an inhibitor of O-linked glycosylation. Tryptic peptides from each component were separated by reverse-phase high-performance liquid chromatography. N- and O-linked oligosaccharide chains were identified on these peptides by specific enzymatic digestions. The proreceptor and alpha-subunit contained only N-linked oligosaccharides, whereas themore » beta-subunit contained both N- and O-linked oligosaccharides. The O-linked oligosaccharide chains were attached to a single tryptic fraction of the beta-subunit, which also contained N-linked chains. This fraction was further localized to the NH2-terminal tryptic peptide of the beta-subunit by specific immunoprecipitation with an anti-peptide antibody with specificity for this region. Binding of insulin and autophosphorylation of the beta-subunit were not dependent on O-linked glycosylation, because cells grown in the presence of the inhibitor exhibited a normal dose response to insulin. Therefore, the insulin receptor contains O-linked oligosaccharides on the NH2-terminal tryptic peptide of the beta-subunit, and these O-linked oligosaccharides are not necessary to the binding or autophosphorylation function of the receptor.« less

Synthesis and evaluation of triazole linked glycosylated 18β-glycyrrhetinic acid derivatives as anticancer agents.

PubMed

Parida, Pravat Kumar; Sau, Abhijit; Ghosh, Tamashree; Jana, Kuladip; Biswas, Kaushik; Raha, Sanghamitra; Misra, Anup Kumar

2014-08-15

A series of glycosyl triazol linked 18β-glycyrrhetinic acid (GA) derivatives have been synthesized using 1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives (4a-h) with propargyl ester of 18β-glycyrrhetinic acid (GA) (2 and 3) following the concept of 'Click chemistry'. The synthesized triazole derivatives were de-O-acetylated to furnish compounds (7a-h and 8a-c) with free hydroxyl groups in the carbohydrate moieties, which were evaluated for their anticancer potential against human cervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA (1), compound 7d, compound 7g and compound 8c showed promising anticancer activities. Copyright © 2014 Elsevier Ltd. All rights reserved.

The impact of N- and O-glycosylation on the functions of Glut-1 transporter in human thyroid anaplastic cells.

PubMed

Samih, Nezha; Hovsepian, Sonia; Notel, Frédéric; Prorok, Maëlle; Zattara-Cannoni, Hélène; Mathieu, Sylvie; Lombardo, Dominique; Fayet, Guy; El-Battari, Assou

2003-04-07

It has been previously shown that glucose transporter Glut-1 expression was detectable by immunostaining in tissue sections from anaplastic carcinoma, but not in normal thyroid tissue. Using human thyroid anaplastic carcinoma cells, we studied the mechanism by which Glut-1 molecules are translocated from the endoplasmic reticulum to the cell surface. The contribution of N- and O-linked glycans for the translocation and activity of Glut-1 transporter is emphasized. The inhibition of N-glycosylation with tunicamycin (TM) led to a 50% decrease in glucose transport while glycosylated and unglycosylated forms of Glut-1 were found at the cell surface. However, the inhibition of N-linked oligosaccharide processing with deoxymannojirimycin (dMJ) and swainsonine (SW) influenced neither the intracellular trafficking nor the activity of the transporter. On the other hand, Glut-1 bound to the O-linked glycan-specific lectin jacalin and the O-glycosylation inhibitor benzyl-N-acetylgalactosamine dramatically inhibited glucose transport. These results show that O- and N-linked oligosaccharides arbored by Glut-1 are essential for glucose transport in anaplastic carcinoma cells. The quantitative and qualitative alterations of Glut-1 glycosylation and the increase in glucose transport are associated with the anaplastic phenotype of human thyroid cells.

N-/O-glycosylation analysis of human FVIIa produced in the milk of transgenic rabbits

PubMed Central

Chevreux, Guillaume; Faid, Valegh; Scohyers, Jean-Marc; Bihoreau, Nicolas

2013-01-01

Human coagulation factor VIIa is a glycoprotein that promotes haemostasis through activation of the coagulation cascade extrinsic pathway. Most haemophilia A/B patients with inhibitors are treated by injection of plasma-derived or recombinant FVIIa. The use of recombinant products raises questions about the ability of the host cell to produce efficiently post-translationally modified proteins. Glycosylation is especially critical considering that it can modulate protein safety and efficacy. The present paper reports the N-/O-glycosylation pattern of a new recombinant human factor VIIa expressed in the mammary glands of transgenic rabbits. Glycosylation was investigated by chromatography and advanced mass spectrometry techniques for glycan identification and quantitation. Mass spectrometry (MS)/MS analyses were performed to confirm the glycan structures as well as the position and branching of specific monosaccharides or substituents. The two N-glycosylation sites were found to be fully occupied mostly by mono- and bi-sialylated biantennary complex-type structures, the major form being A2G2S1. Some oligomannose/hybrid structures were retrieved in lower abundance, the major ones being GlcNAcα1,O-phosphorylated at the C6-position of a Man residue (Man-6-(GlcNAcα1,O-)phosphate motif) as commonly observed on lysosomal proteins. No immunogenic glycotopes such as Galili (Galα1,3Gal) and HD antigens (N-glycolylneuraminic acid (NeuGc)) were detected. Concerning O-glycosylation, the product exhibited O-fucose and O-glucose-(xylose)0, 1, 2 motifs as expected. The N-glycosylation consistency was also investigated by varying production parameters such as the period of lactation, the number of consecutive lactations and rabbit generations. Results show that the transgenesis technology is suitable for the long-term production of rhFVIIa with a reproducible glycosylation pattern. PMID:24092837

Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

PubMed Central

Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo

2014-01-01

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the

Deactivating Influence of 3-O-Glycosyl Substituent on Anomeric Reactivity of Thiomannoside Observed in Oligomannoside Synthesis.

PubMed

Zhou, Jun; Lv, Siying; Zhang, Dan; Xia, Fei; Hu, Wenhao

2017-03-03

It has been long recognized that, in chemical glycosylation, the anomeric reactivity of glycosyl donor can be influenced greatly by protecting groups. As opposed to the effects of protecting groups, we report herein a finding on how O-glycosyl substituent can affect the reactivity of oligosaccharyl donor, which in turn should have impact on convergent assembly of oligosaccharide. During our synthetic efforts toward Pichia holstii oligomannoside, a type of α-1,3-linked dimannosyl thioglycosides was found to exhibit unexpected low reactivity toward the activation of NIS/TMSOTf. This observation prompted us to perform a series of comparative reactivity studies, which attributed the donor deactivation to the presence of 3-O-glycosyl substituent, by comparison with O-acetyl group and O-glycosidic linkages at C-4/C-6 positions. To rationalize the unusual phenomenon, we hypothesize that O-glycosyl moiety at C-3 could destabilize the oxocarbenium ion intermediate by additionally increasing the O2-C2-C3-O3 torsional strain, which was further supported by DFT calculation of the hypothetical 4 H 3 -like oxocarbeniums. The observed deactivating influence provides a basis for estimation of donor reactivity and logical selection of synthetic strategy in oligosaccharide synthesis. Following this finding, we opted to use an iterative strategy for the synthesis of targeted pentamannoside 1 by using monomeric thiomannosides that ensured sufficient reactivity.

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

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

Matzuk, M.M.; Krieger, M.; Corless, C.L.

1987-09-01

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common ..cap alpha.. subunit but differ in their hormone-specific ..beta..-subunits. The ..beta.. subunit of hCG (hCG..beta..) is unique among the ..beta.. subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCG..beta.. gene alone or together with the hCG..cap alpha.. gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results revealmore » that hCG..beta.. can be secreted normally in the absence of its O-linked oligosaccharides. hCG..beta.. devoid of O-linked carbohydrate can also combine efficiently with hCG..cap alpha.. and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCG..beta.. O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG.« less

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins*

PubMed Central

Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

2016-01-01

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

Emerging facets of prokaryotic glycosylation

PubMed Central

Schäffer, Christina; Messner, Paul

2017-01-01

Glycosylation of proteins is one of the most prevalent post-translational modifications occurring in nature, with a wide repertoire of biological implications. Pathways for the main types of this modification, the N- and O-glycosylation, can be found in all three domains of life—the Eukarya, Bacteria and Archaea—thereby following common principles, which are valid also for lipopolysaccharides, lipooligosaccharides and glycopolymers. Thus, studies on any glycoconjugate can unravel novel facets of the still incompletely understood fundamentals of protein N- and O-glycosylation. While it is estimated that more than two-thirds of all eukaryotic proteins would be glycosylated, no such estimate is available for prokaryotic glycoproteins, whose understanding is lagging behind, mainly due to the enormous variability of their glycan structures and variations in the underlying glycosylation processes. Combining glycan structural information with bioinformatic, genetic, biochemical and enzymatic data has opened up an avenue for in-depth analyses of glycosylation processes as a basis for glycoengineering endeavours. Here, the common themes of glycosylation are conceptualised for the major classes of prokaryotic (i.e. bacterial and archaeal) glycoconjugates, with a special focus on glycosylated cell-surface proteins. We describe the current knowledge of biosynthesis and importance of these glycoconjugates in selected pathogenic and beneficial microbes. PMID:27566466

O-linked β-N-acetylglucosamine transferase directs cell proliferation in idiopathic pulmonary arterial hypertension.

PubMed

Barnes, Jarrod W; Tian, Liping; Heresi, Gustavo A; Farver, Carol F; Asosingh, Kewal; Comhair, Suzy A A; Aulak, Kulwant S; Dweik, Raed A

2015-04-07

Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway. We measured the levels of O-linked β-N-acetylglucosamine modification, O-linked β-N-acetylglucosamine transferase (OGT), and O-linked β-N-acetylglucosamine hydrolase in control and IPAH cells and tissues. Our data suggest that the activation of the hexosamine biosynthetic pathway directly increased OGT levels and activity, triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-linked β-N-acetylglucosamine modification levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1. Our data demonstrate that hexosamine biosynthetic pathway flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of host cell factor-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH. © 2015 American Heart Association, Inc.

Taming the Reactivity of Glycosyl Iodides To Achieve Stereoselective Glycosidation.

PubMed

Gervay-Hague, Jacquelyn

2016-01-19

that even highly functionalized aglycon acceptors add. Following the coupling event, the TMS ethers are readily removed by methanolysis, and since all of the byproducts are volatile, multiple reactions can be performed in a single reaction vessel without isolation of intermediates. In this fashion, per-O-TMS monosaccharides can be converted to biologically relevant α-linked glycolipids in one pot. The stereochemical outcome of these reactions can also be switched to β-glycoside formation by addition of silver to chelate the iodide, thus favoring SN2 displacement of the α-iodide. While iodides derived from benzyl and silyl ether-protected oligosaccharides are susceptible to interglycosidic bond cleavage when treated with TMSI, the introduction of a single acetate protecting group prevents this unwanted side reaction. Partial acetylation of armed glycosyl iodides also attenuates HI elimination side reactions. Conversely, fully acetylated glycosyl iodides are deactivated and require metal catalysis in order for glycosidation to occur. Recent findings indicate that I2 activation of per-O-acetylated mono-, di-, and trisaccharides promotes glycosidation of cyclic ethers to give β-linked iodoalkyl glycoconjugates in one step. Products of these reactions have been converted into multivalent carbohydrate displays. With these synthetic pathways elucidated, chemical reactivity can be exquisitely controlled by the judicious selection of protecting groups to achieve high stereocontrol in step-economical processes.

Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins.

PubMed

Stepper, Judith; Shastri, Shilpa; Loo, Trevor S; Preston, Joanne C; Novak, Petr; Man, Petr; Moore, Christopher H; Havlíček, Vladimír; Patchett, Mark L; Norris, Gillian E

2011-02-18

O-Glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine β-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC(50) 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein.

PubMed

Liu, Li; Bastien, Nathalie; Li, Yan

2007-12-01

The biosynthesis and posttranslational processing of human metapneumovirus attachment G glycoprotein were investigated. After pulse-labeling, the G protein accumulated as three species with molecular weights of 45,000, 50,000, and 53,000 (45K, 50K, and 53K, respectively). N-Glycosidase digestion indicated that these forms represent the unglycosylated precursor and N-glycosylated intermediate products, respectively. After an appropriate chase, these three naive forms were further processed to a mature 97K form. The presence of O-linked sugars in mature G protein was confirmed by O-glycanase digestion and lectin-binding assay using Arachis hypogaea (peanut agglutinin), an O-glycan-specific lectin. In addition, in the O-glycosylation-deficient cell line (CHO ldlD cell), the G protein could not be processed to the mature form unless the exogenous Gal and GalNAc were supplemented, which provided added evidence supporting the O-linked glycosylation of G protein. The maturation of G was completely blocked by monensin but was partially sensitive to brefeldin A (BFA), suggesting the O-linked glycosylation of G initiated in the trans-Golgi compartment and terminated in the trans-Golgi network. Enzymatic deglycosylation analysis confirmed that the BFA-G was a partial mature form containing N-linked oligosaccharides and various amounts of O-linked carbohydrate side chains. The expression of G protein at the cell surface could be detected by indirect immunofluorescence staining assay. Furthermore, cell surface immunoprecipitation displayed an efficient intracellular transport of G protein.

Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation.

PubMed

Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol; Yoon, Sung-il

2015-03-20

Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer.

PubMed

Peiris, Diluka; Ossondo, Marlène; Fry, Simon; Loizidou, Marilena; Smith-Ravin, Juliette; Dwek, Miriam V

2015-01-01

Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

PubMed Central

Peiris, Diluka; Ossondo, Marlène; Fry, Simon; Loizidou, Marilena; Smith-Ravin, Juliette; Dwek, Miriam V.

2015-01-01

Background Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. Methodology In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Results Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Conclusion Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer. PMID:26495974

Mass spectrometric analysis of O-linked oligosaccharides from various recombinant expression systems.

PubMed

Kenny, Diarmuid T; Gaunitz, Stefan; Hayes, Catherine A; Gustafsson, Anki; Sjöblom, Magnus; Holgersson, Jan; Karlsson, Niclas G

2013-01-01

Analysis of O-linked glycosylation is one of the main challenges during structural validation of recombinant glycoproteins. With methods available for N-linked glycosylation in regard to oligosaccharide analysis as well as glycopeptide mapping, there are still challenges for O-linked glycan analysis. Here, we present mass spectrometric methodology for O-linked oligosaccharides released by reductive β-elimination. Using LC-MS and LC-MS(2) with graphitized carbon columns, oligosaccharides are analyzed without derivatization. This approach provides a high-throughput method for screening during clonal selection, as well as product structure verification, without impairing sequencing ability. The protocols are exemplified by analysis of glycoproteins from mammalian cell cultures (CHO cells) as well as insect cells and yeast. The data shows that the method can be successfully applied to both neutral and acidic O-linked oligosaccharides, where sialic acid, hexuronic acid, and sulfate are common substituents. Further characterization of O-glycans can be achieved using permethylation. Permethylation of O-linked oligosaccharides followed by direct infusion into the mass spectrometer provide information about oligosaccharide composition, and subsequent MS (n) experiments can be carried out to elucidate oligosaccharide structure including linkage information and sequence.

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.

PubMed

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Joshi, Hiren Jitendra; Siukstaite, Lina; Harrison, Oliver J; Brasch, Julia; Goodman, Kerry M; Hansen, Lars; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y; Clausen, Henrik; Halim, Adnan

2017-10-17

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1-4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3.

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins

PubMed Central

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Siukstaite, Lina; Harrison, Oliver J.; Brasch, Julia; Goodman, Kerry M.; Hansen, Lars; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y.; Clausen, Henrik

2017-01-01

The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1–4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3. PMID:28973932

Advances in the biotechnological glycosylation of valuable flavonoids.

PubMed

Xiao, Jianbo; Muzashvili, Tamar S; Georgiev, Milen I

2014-11-01

The natural flavonoids, especially their glycosides, are the most abundant polyphenols in foods and have diverse bioactivities. The biotransformation of flavonoid aglycones into their glycosides is vital in flavonoid biosynthesis. The main biological strategies that have been used to achieve flavonoid glycosylation in the laboratory involve metabolic pathway engineering and microbial biotransformation. In this review, we summarize the existing knowledge on the production and biotransformation of flavonoid glycosides using biotechnology, as well as the impact of glycosylation on flavonoid bioactivity. Uridine diphosphate glycosyltransferases play key roles in decorating flavonoids with sugars. Modern metabolic engineering and proteomic tools have been used in an integrated fashion to generate numerous structurally diverse flavonoid glycosides. In vitro, enzymatic glycosylation tends to preferentially generate flavonoid 3- and 7-O-glucosides; microorganisms typically convert flavonoids into their 7-O-glycosides and will produce 3-O-glycosides if supplied with flavonoid substrates having a hydroxyl group at the C-3 position. In general, O-glycosylation reduces flavonoid bioactivity. However, C-glycosylation can enhance some of the benefits of flavonoids on human health, including their antioxidant and anti-diabetic potential. Copyright © 2014 Elsevier Inc. All rights reserved.

Glycosylation Is a Major Regulator of Phenylpropanoid Availability and Biological Activity in Plants

PubMed Central

Le Roy, Julien; Huss, Brigitte; Creach, Anne; Hawkins, Simon; Neutelings, Godfrey

2016-01-01

The phenylpropanoid pathway in plants is responsible for the biosynthesis of a huge amount of secondary metabolites derived from phenylalanine and tyrosine. Both flavonoids and lignins are synthesized at the end of this very diverse metabolic pathway, as well as many intermediate molecules whose precise biological functions remain largely unknown. The diversity of these molecules can be further increased under the action of UDP-glycosyltransferases (UGTs) leading to the production of glycosylated hydroxycinnamates and related aldehydes, alcohols and esters. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. (De)-glycosylation therefore represents an extremely important regulation point in phenylpropanoid homeostasis. In this article we review recent knowledge on the enzymes involved in regulating phenylpropanoid glycosylation status and availability in different subcellular compartments. We also examine the potential link between monolignol glycosylation and lignification by exploring co-expression of lignin biosynthesis genes and phenolic (de)glycosylation genes. Of the different biological roles linked with their particular chemical properties, phenylpropanoids are often correlated with the plant's stress management strategies that are also regulated by glycosylation. UGTs can for instance influence the resistance of plants during infection by microorganisms and be involved in the mechanisms related to environmental changes. The impact of flavonoid glycosylation on the color of flowers, leaves, seeds and fruits will also be discussed. Altogether this paper underlies the fact that glycosylation and deglycosylation are powerful mechanisms allowing plants to regulate phenylpropanoid localisation, availability and biological activity. PMID:27303427

The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering

PubMed Central

Bossé, Janine T.; Abouelhadid, Sherif; Li, Yanwen; Lin, Chia-Wei; Vohra, Prerna; Tucker, Alexander W.; Rycroft, Andrew N.; Maskell, Duncan J.; Aebi, Markus; Langford, Paul R.

2017-01-01

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering. PMID:28077594

The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering.

PubMed

Cuccui, Jon; Terra, Vanessa S; Bossé, Janine T; Naegeli, Andreas; Abouelhadid, Sherif; Li, Yanwen; Lin, Chia-Wei; Vohra, Prerna; Tucker, Alexander W; Rycroft, Andrew N; Maskell, Duncan J; Aebi, Markus; Langford, Paul R; Wren, Brendan W

2017-01-01

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering. © 2017 The Authors.

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch*

PubMed Central

Harvey, Beth M.; Rana, Nadia A.; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S.

2016-01-01

Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity. PMID:27268051

An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

PubMed Central

Lee, Yujean; Kim, Hyori; Chung, Junho

2014-01-01

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding. PMID:25236766

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

PubMed Central

Jones, Meredith B.; Rosenberg, Julian N.; Betenbaugh, Michael J.; Krag, Sharon S.

2009-01-01

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide. PMID:19348869

Resveratrol triggers ER stress-mediated apoptosis by disrupting N-linked glycosylation of proteins in ovarian cancer cells.

PubMed

Gwak, HyeRan; Kim, Soochi; Dhanasekaran, Danny N; Song, Yong Sang

2016-02-28

Malignant tumors have a high glucose demand and alter cellular metabolism to survive. Herein, focusing on the utility of glucose metabolism as a therapeutic target, we found that resveratrol induced endoplasmic reticulum (ER) stress-mediated apoptosis by interrupting protein glycosylation in a cancer-specific manner. Our results indicated that resveratrol suppressed the hexosamine biosynthetic pathway and interrupted protein glycosylation through GSK3β activation. Application of either biochemical intermediates of the hexosamine pathway or small molecular inhibitors of GSK3β reversed the effects of resveratrol on the disruption of protein glycosylation. Additionally, an ER UDPase, ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), modulated protein glycosylation by Akt attenuation in response to resveratrol. By inhibition or overexpression of Akt functions, we confirmed that the glycosylation activities were dependent on ENTPD5 expression and regulated by the action of Akt in ovarian cancer cells. Resveratrol-mediated disruption of protein glycosylation induced cellular apoptosis as indicated by the up-regulation of GADD153, followed by the activation of ER-stress sensors (PERK and ATF6α). Thus, our results provide novel insight into cancer cell metabolism and protein glycosylation as a therapeutic target for cancers. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages.

PubMed

Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Autosomal recessive PGM3 mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment

PubMed Central

Zhang, Yu; Yu, Xiaomin; Ichikawa, Mie; Lyons, Jonathan J.; Datta, Shrimati; Lamborn, Ian T.; Jing, Huie; Kim, Emily S.; Biancalana, Matthew; Wolfe, Lynne A.; DiMaggio, Thomas; Matthews, Helen F.; Kranick, Sarah M.; Stone, Kelly D.; Holland, Steven M.; Reich, Daniel S.; Hughes, Jason D.; Mehmet, Huseyin; McElwee, Joshua; Freeman, Alexandra F.; Freeze, Hudson H.; Su, Helen C.; Milner, Joshua D.

2014-01-01

Background Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. Objective To define a genetic syndrome of severe atopy, elevated serum IgE, immune deficiency, autoimmunity, and motor and neurocognitive impairment. Methods Eight patients from two families who had similar syndromic features were studied. Thorough clinical evaluations, including brain MRI and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T cell cytokine production were measured. Whole exome sequencing was performed to identify disease-causing mutations. Immunoblotting, qRT-PCR, enzymatic assays, nucleotide sugar and sugar phosphate analyses along with MALDI-TOF mass spectrometry of glycans were used to determine the molecular consequences of the mutations. Results Marked atopy and autoimmunity were associated with increased TH2 and TH17 cytokine production by CD4+ T cells. Bacterial and viral infection susceptibility were noted along with T cell lymphopenia, particularly of CD8+ T cells, and reduced memory B cells. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurological abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced UDP-GlcNAc, along with decreased O- and N-linked protein glycosylation in patients’ cells. These results define a new Congenital Disorder of Glycosylation. Conclusions Autosomal recessive, hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability and hypomyelination. PMID:24589341

A Point Mutation in the Gene for Asparagine-Linked Glycosylation 10B (Alg10b) Causes Nonsyndromic Hearing Impairment in Mice (Mus musculus)

PubMed Central

Probst, Frank J.; Corrigan, Rebecca R.; del Gaudio, Daniela; Salinger, Andrew P.; Lorenzo, Isabel; Gao, Simon S.; Chiu, Ilene; Xia, Anping

2013-01-01

The study of mouse hearing impairment mutants has led to the identification of a number of human hearing impairment genes and has greatly furthered our understanding of the physiology of hearing. The novel mouse mutant neurological/sensory 5 (nse5) demonstrates a significantly reduced or absent startle response to sound and is therefore a potential murine model of human hearing impairment. Genetic analysis of 500 intercross progeny localized the mutant locus to a 524 kilobase (kb) interval on mouse chromosome 15. A missense mutation in a highly-conserved amino acid was found in the asparagine-linked glycosylation 10B gene (Alg10b), which is within the critical interval for the nse5 mutation. A 20.4 kb transgene containing a wildtype copy of the Alg10b gene rescued the mutant phenotype in nse5/nse5 homozygous animals, confirming that the mutation in Alg10b is responsible for the nse5/nse5 mutant phenotype. Homozygous nse5/nse5 mutants had abnormal auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), and cochlear microphonics (CMs). Endocochlear potentials (EPs), on the other hand, were normal. ABRs and DPOAEs also confirmed the rescue of the mutant nse5/nse5 phenotype by the wildtype Alg10b transgene. These results suggested a defect in the outer hair cells of mutant animals, which was confirmed by histologic analysis. This is the first report of mutation in a gene involved in the asparagine (N)-linked glycosylation pathway causing nonsyndromic hearing impairment, and it suggests that the hearing apparatus, and the outer hair cells in particular, are exquisitely sensitive to perturbations of the N-linked glycosylation pathway. PMID:24303013

Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

PubMed

Karst, Daniel J; Scibona, Ernesto; Serra, Elisa; Bielser, Jean-Marc; Souquet, Jonathan; Stettler, Matthieu; Broly, Hervé; Soos, Miroslav; Morbidelli, Massimo; Villiger, Thomas K

2017-09-01

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can

Predicting the Retention Behavior of Specific O-Linked Glycopeptides.

PubMed

Badgett, Majors J; Boyes, Barry; Orlando, Ron

2017-09-01

O -Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O -glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O - N -acetylgalactosamine ( O -GalNAc), O - N -acetylglucosamine ( O -GlcNAc), and O -fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications.

Predicting the Retention Behavior of Specific O-Linked Glycopeptides

PubMed Central

Badgett, Majors J.; Boyes, Barry; Orlando, Ron

2017-01-01

O-Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O-glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O-N-acetylgalactosamine (O-GalNAc), O-N-acetylglucosamine (O-GlcNAc), and O-fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications. PMID:28785176

Functional Roles of N-Linked Glycosylation of Human Matrix Metalloproteinase 9.

PubMed

Duellman, Tyler; Burnett, John; Yang, Jay

2015-10-01

Matrix metalloproteinase-9 (MMP-9) is a secreted endoproteinase with a critical role in the regulation of the extracellular matrix and proteolytic activation of signaling molecules. Human (h)MMP-9 has two well-defined N-glycosylation sites at residues N38 and N120; however, their role has remained mostly unexplored partly because expression of the N-glycosylation-deficient N38S has been difficult due to a recently discovered single nucleotide polymorphism-dependent miRNA-mediated inhibitory mechanism. hMMP-9 cDNA encoding amino acid substitutions at residues 38 (modified-S38, mS38) or 120 (N120S) were created in the background of a miRNA-binding site disrupted template and expressed by transient transfection. hMMP-9 harboring a single mS38 replacement secreted well, whereas N120S, or a double mS38/N120S hMMP-9 demonstrated much reduced secretion. Imaging indicated endoplasmic reticulum (ER) retention of the non-secreted variants and co-immunoprecipitation confirmed an enhanced strong interaction between the non-secreted hMMP-9 and the ER-resident protein calreticulin (CALR). Removal of N-glycosylation at residue 38 revealed an amino acid-dependent strong interaction with CALR likely preventing unloading of the misfolded protein from the ER chaperone down the normal secretory pathway. As with other glycoproteins, N-glycosylation strongly regulates hMMP-9 secretion. This is mediated, however, through a novel mechanism of cloaking an N-glycosylation-independent strong interaction with the ER-resident CALR. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

PubMed

Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Functional Relevance of Protein Glycosylation to the Pro-Inflammatory Effects of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) on Monocytes/Macrophages

PubMed Central

Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Background and Objective Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. Methods The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. Results 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Conclusions Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages. PMID:25658763

Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy.

PubMed

Ravidà, Alessandra; Musante, Luca; Kreivi, Marjut; Miinalainen, Ilkka; Byrne, Barry; Saraswat, Mayank; Henry, Michael; Meleady, Paula; Clynes, Martin; Holthofer, Harry

2015-05-01

Diabetic nephropathy often progresses to end-stage kidney disease and, ultimately, to renal replacement therapy. Hyperglycemia per se is expected to have a direct impact on the biosynthesis of N- and O-linked glycoproteins. This study aims to establish the link between protein glycosylation and progression of experimental diabetic kidney disease using orthogonal methods. Kidneys of streptozotocin-diabetic and control rats were harvested at three different time points post streptozotocin injection. A panel of 12 plant lectins was used in the screening of lectin blots. The lectins UEAI, PHA-E, GSI, PNA, and RCA identified remarkable disease-associated differences in glycoprotein expression. Lectin affinity chromatography followed by mass spectrometric analyses led to the identification of several glycoproteins involved in salt-handling, angiogenesis, and extracellular matrix degradation. Our data confirm a substantial link between glycosylation signature and diabetes progression. Furthermore, as suggested by our findings on dipeptidyl peptidase-IV, altered protein glycosylation may reflect changes in biochemical properties such as enzymatic activity. Thus, our study demonstrates the unexplored potential of protein glycosylation analysis in the discovery of molecules linked to diabetic kidney disease.

A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri CIDCA 83111 is O- and N-glycosylated.

PubMed

Cavallero, Gustavo J; Malamud, Mariano; Casabuono, Adriana C; Serradell, M de Los Ángeles; Couto, Alicia S

2017-06-06

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471-476, with a Glc 5-8 GalA 2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

PubMed

Eklund, Erik A; Merbouh, Nabyl; Ichikawa, Mie; Nishikawa, Atsushi; Clima, Jessica M; Dorman, James A; Norberg, Thomas; Freeze, Hudson H

2005-11-01

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

NASA Astrophysics Data System (ADS)

Wadzinski, Tyler J.; Steinauer, Angela; Hie, Liana; Pelletier, Guillaume; Schepartz, Alanna; Miller, Scott J.

2018-06-01

Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

PubMed Central

Kapuria, Vaibhav; Röhrig, Ute F.; Bhuiyan, Tanja; Borodkin, Vladimir S.; van Aalten, Daan M.F.; Zoete, Vincent; Herr, Winship

2016-01-01

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc), O-linked-GlcNAc transferase (OGT) catalyzes Ser/Thr O-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase–protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase–protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT. PMID:27056667

Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation

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

Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol

2015-03-20

Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helicesmore » with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis. - Highlights: • cjPseH adopts a single-domain structure of a central β-sheet decorated by α-helices. • cjPseH features two continuously connected grooves on the protein surface. • Acetyl coenzyme A (AcCoA) binds into a deep groove of cjPseH in an ‘L’ shape. • The acetyl end of AcCoA points to a wide groove, a potential substrate-binding site.« less

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

PubMed Central

King, Sarah L.; Joshi, Hiren J.; Schjoldager, Katrine T.; Halim, Adnan; Madsen, Thomas D.; Dziegiel, Morten H.; Woetmann, Anders; Vakhrushev, Sergey Y.

2017-01-01

The hemostatic system comprises platelet aggregation, coagulation, and fibrinolysis, and is critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play important roles in the hemostatic system; however, most investigations have focused on N-glycans because of the complexity of O-glycan analysis. Here we performed the first systematic analysis of native-O-glycosylation using lectin affinity chromatography coupled to liquid chromatography mass spectrometry (LC-MS)/MS to determine the precise location of O-glycans in human plasma, platelets, and endothelial cells, which coordinately regulate hemostasis. We identified the hitherto largest O-glycoproteome from native tissue with a total of 649 glycoproteins and 1123 nonambiguous O-glycosites, demonstrating that O-glycosylation is a ubiquitous modification of extracellular proteins. Investigation of the general properties of O-glycosylation established that it is a heterogeneous modification, frequently occurring at low density within disordered regions in a cell-dependent manner. Using an unbiased screen to identify associations between O-glycosites and protein annotations we found that O-glycans were over-represented close (± 15 amino acids) to tandem repeat regions, protease cleavage sites, within propeptides, and located on a select group of protein domains. The importance of O-glycosites in proximity to proteolytic cleavage sites was further supported by in vitro peptide assays demonstrating that proteolysis of key hemostatic proteins can be inhibited by the presence of O-glycans. Collectively, these data illustrate the global properties of native O-glycosylation and provide the requisite roadmap for future biomarker and structure-function studies. PMID:29296958

The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

PubMed Central

Peyfoon, Elham; Meyer, Benjamin; Hitchen, Paul G.; Panico, Maria; Morris, Howard R.; Haslam, Stuart M.; Albers, Sonja-Verena; Dell, Anne

2010-01-01

Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS), consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya. PMID:20936123

Glycosylation: a hallmark of cancer?

PubMed

Vajaria, Bhairavi N; Patel, Prabhudas S

2017-04-01

The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during the multistep tumorigenesis. Cancer being a cellular disease, changes in cellular glycoproteins play an important role in malignant transformation and cancer progression. The present review summarizes various studies that depicted correlation of glycosylation with tumor initiation, progression and metastasis, which are helpful in early diagnosis, disease monitoring and prognosis. The results are further strengthened by our reports, which depicted alterations in sialylation and fucosylation in different cancers. Alterations in glycosyltransferases are also involved in formation of various tumor antigens (e.g. Sialyl Lewis x) which serves as ligand for the cell adhesion molecule, selectin which is involved in adhesion of cancer cells to vascular endothelium and thus contributes to hematogenous metastasis. Increased glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC)s are also involved in loss of E-cadherin, a key molecule implicated in metastatic dissemination of cells. The present review also summarizes the correlation of glycosylation with all the hallmarks of cancer. The enormous progress in the design of novel inhibitors of pathway intermediates of sialylation and fucosylation can prove wonders in combating the dreadful disease. The results provide the evidence that altered glycosylation is linked to tumor initiation, progression and metastasis. Hence, it can be considered as a new hallmark of cancer development and strategies to develop novel glycosylation targeted molecules should be strengthened.

Molecular-Scale Features that Govern the Effects of O-Glycosylation on a Carbohydrate-Binding Module

DOE PAGES

Guan, Xiaoyang; Chaffey, Patrick K.; Zeng, Chen; ...

2015-09-21

The protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. This study suggests the possibility ofmore » designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.« less

N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma.

PubMed

Li, Jiang-Hua; Huang, Wan; Lin, Peng; Wu, Bo; Fu, Zhi-Guang; Shen, Hao-Miao; Jing, Lin; Liu, Zhen-Yu; Zhou, Yang; Meng, Yao; Xu, Bao-Qing; Chen, Zhi-Nan; Jiang, Jian-Li

2016-11-21

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.

N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma

PubMed Central

Li, Jiang-Hua; Huang, Wan; Lin, Peng; Wu, Bo; Fu, Zhi-Guang; Shen, Hao-Miao; Jing, Lin; Liu, Zhen-Yu; Zhou, Yang; Meng, Yao; Xu, Bao-Qing; Chen, Zhi-Nan; Jiang, Jian-Li

2016-01-01

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147. PMID:27869218

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules.

PubMed

Kersten, Roland D; Ziemert, Nadine; Gonzalez, David J; Duggan, Brendan M; Nizet, Victor; Dorrestein, Pieter C; Moore, Bradley S

2013-11-19

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

Structure of a Novel O-Linked N-Acetyl-d-glucosamine (O-GlcNAc) Transferase, GtfA, Reveals Insights into the Glycosylation of Pneumococcal Serine-rich Repeat Adhesins*

PubMed Central

Shi, Wei-Wei; Jiang, Yong-Liang; Zhu, Fan; Yang, Yi-Hu; Shao, Qiu-Yan; Yang, Hong-Bo; Ren, Yan-Min; Wu, Hui; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a β-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides. PMID:24936067

Post-translational Modification of Extremophilic Proteins: N-glycosylation in Archaea

DTIC Science & Technology

2014-12-02

Kaminski, Z. Guan, S. Yurist-Doutsch, J. Eichler. Two Distinct N-Glycosylation Pathways Process the Haloferax volcanii S-Layer Glycoprotein upon Changes...Promiscuity: AglB, the Archaeal Oligosaccharyltransferase, Can Process a Variety of Lipid-Linked Glycans, Applied and Environmental Microbiology, (11 2013...Archaea,  N-­‐linked   oligosaccharides  are   assembled  on  dolichol  phosphate  prior  to  transfer  of  the  glycan

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.

A Knowledge-Based System for Display and Prediction of O-Glycosylation Network Behaviour in Response to Enzyme Knockouts

PubMed Central

McDonald, Andrew G.; Tipton, Keith F.; Davey, Gavin P.

2016-01-01

O-linked glycosylation is an important post-translational modification of mucin-type protein, changes to which are important biomarkers of cancer. For this study of the enzymes of O-glycosylation, we developed a shorthand notation for representing GalNAc-linked oligosaccharides, a method for their graphical interpretation, and a pattern-matching algorithm that generates networks of enzyme-catalysed reactions. Software for generating glycans from the enzyme activities is presented, and is also available online. The degree distributions of the resulting enzyme-reaction networks were found to be Poisson in nature. Simple graph-theoretic measures were used to characterise the resulting reaction networks. From a study of in-silico single-enzyme knockouts of each of 25 enzymes known to be involved in mucin O-glycan biosynthesis, six of them, β-1,4-galactosyltransferase (β4Gal-T4), four glycosyltransferases and one sulfotransferase, play the dominant role in determining O-glycan heterogeneity. In the absence of β4Gal-T4, all Lewis X, sialyl-Lewis X, Lewis Y and Sda/Cad glycoforms were eliminated, in contrast to knockouts of the N-acetylglucosaminyltransferases, which did not affect the relative abundances of O-glycans expressing these epitopes. A set of 244 experimentally determined mucin-type O-glycans obtained from the literature was used to validate the method, which was able to predict up to 98% of the most common structures obtained from human and engineered CHO cell glycoforms. PMID:27054587

Golgi Glycosylation

PubMed Central

Stanley, Pamela

2011-01-01

Glycosylation is a very common modification of protein and lipid, and most glycosylation reactions occur in the Golgi. Although the transfer of initial sugar(s) to glycoproteins or glycolipids occurs in the ER or on the ER membrane, the subsequent addition of the many different sugars that make up a mature glycan is accomplished in the Golgi. Golgi membranes are studded with glycosyltransferases, glycosidases, and nucleotide sugar transporters arrayed in a generally ordered manner from the cis-Golgi to the trans-Golgi network (TGN), such that each activity is able to act on specific substrate(s) generated earlier in the pathway. The spectrum of glycosyltransferases and other activities that effect glycosylation may vary with cell type, and thus the final complement of glycans on glycoconjugates is variable. In addition, glycan synthesis is affected by Golgi pH, the integrity of Golgi peripheral membrane proteins, growth factor signaling, Golgi membrane dynamics, and cellular stress. Knowledge of Golgi glycosylation has fostered the development of assays to identify mechanisms of intracellular vesicular trafficking and facilitated glycosylation engineering of recombinant glycoproteins. PMID:21441588

N-linked glycosylation of cortical N-methyl-D-aspartate and kainate receptor subunits in schizophrenia.

PubMed

Tucholski, Janusz; Simmons, Micah S; Pinner, Anita L; McMillan, Laurence D; Haroutunian, Vahram; Meador-Woodruff, James H

2013-08-21

Dysfunctional glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. Abnormal expressions in schizophrenia of ionotropic glutamate receptors (iGluRs) and the proteins that regulate their trafficking have been found to be region and subunit specific in brain, suggesting that abnormal trafficking of iGluRs may contribute toward altered glutamatergic neurotransmission. The post-translational modification N-glycosylation of iGluR subunits can be used as a proxy for their intracellular localization. Receptor complexes assemble in the lumen of the endoplasmic reticulum, where N-glycosylation begins with the addition of N-linked oligomannose glycans, and is subsequently trimmed and replaced by more elaborate glycans while trafficking through the Golgi apparatus. Previously, we found abnormalities in N-glycosylation of the GluR2 AMPA receptor subunit in schizophrenia. Here, we investigated N-glycosylation of N-methyl-D-aspartate and kainate (KA) receptor subunits in the dorsolateral prefrontal cortex from patients with schizophrenia and a comparison group. We used enzymatic deglycosylation with two glycosidases: endoglycosidase H (Endo H), which removes immature high mannose-containing sugars, and peptide-N-glycosidase F (PNGase F), which removes all N-linked sugars. The NR1, NR2A, NR2B, GluR6, and KA2 subunits were all sensitive to treatment with Endo H and PNGase F. The GluR6 KA receptor subunit was significantly more sensitive to Endo H-mediated deglycosylation in schizophrenia, suggesting a larger molecular mass of N-linked high mannose and/or hybrid sugars on GluR6. This finding, taken with our previous work, suggests that a cellular mechanism underlying abnormal glutamate neurotransmission in schizophrenia may involve abnormal trafficking of both AMPA and KA receptors.

Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M.

PubMed

Oostra, M; de Haan, C A M; de Groot, R J; Rottier, P J M

2006-03-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N

Sioxanthin, a novel glycosylated carotenoid, reveals an unusual subclustered biosynthetic pathway.

PubMed

Richter, Taylor K S; Hughes, Chambers C; Moore, Bradley S

2015-06-01

Members of the marine actinomycete genus Salinispora constitutively produce a characteristic orange pigment during vegetative growth. Contrary to the understanding of widespread carotenoid biosynthesis pathways in bacteria, Salinispora carotenoid biosynthesis genes are not confined to a single cluster. Instead, bioinformatic and genetic investigations confirm that four regions of the Salinispora tropica CNB-440 genome, consisting of two gene clusters and two independent genes, contribute to the in vivo production of a single carotenoid. This compound, namely (2'S)-1'-(β-D-glucopyranosyloxy)-3',4'-didehydro-1',2'-dihydro-φ,ψ-caroten-2'-ol, is novel and has been given the trivial name 'sioxanthin'. Sioxanthin is a C40 -carotenoid, glycosylated on one end of the molecule and containing an aryl moiety on the opposite end. Glycosylation is unusual among actinomycete carotenoids, and sioxanthin joins a rare group of carotenoids with polar and non-polar head groups. Gene sequence homology predicts that the sioxanthin biosynthetic pathway is present in all of the Salinispora as well as other members of the family Micromonosporaceae. Additionally, this study's investigations of clustering of carotenoid biosynthetic genes in heterotrophic bacteria show that a non-clustered genome arrangement is more common than previously suggested, with nearly half of the investigated genomes showing a non-clustered architecture. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin

PubMed Central

Maky, Mohamed Abdelfattah; Ishibashi, Naoki; Zendo, Takeshi; Perez, Rodney Honrada; Doud, Jehan Ragab; Karmi, Mohamed

2015-01-01

Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat. PMID:25956765

Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin.

PubMed

Maky, Mohamed Abdelfattah; Ishibashi, Naoki; Zendo, Takeshi; Perez, Rodney Honrada; Doud, Jehan Ragab; Karmi, Mohamed; Sonomoto, Kenji

2015-07-01

Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Total synthesis of agalloside, isolated from Aquilaria agallocha, by the 5-O-glycosylation of flavan.

PubMed

Arai, Midori A; Yamaguchi, Yumi; Ishibashi, Masami

2017-06-14

Agalloside (1) is a neural stem cell differentiation activator isolated from Aquilaria agallocha by our group using Hes1 immobilized beads. We conducted the first total synthesis of agalloside (1) via the 5-O-glycosylation of flavan 25 using glycosyl fluoride 20 in the presence of BF 3 ·Et 2 O. Subsequent oxidation with DDQ to flavanone 2 and deprotection successively provided agalloside (1). This synthetic strategy holds promise for use in the synthesis of 5-O-glycosylated flavonoids. The synthesized agalloside (1) accelerated neural stem cell differentiation, which is a result comparable to that for the naturally occurring compound 1.

Changes in isovitexin-O-glycosylation during the development of young barley plants.

PubMed

Brauch, Dominic; Porzel, Andrea; Schumann, Erika; Pillen, Klaus; Mock, Hans-Peter

2018-04-01

Phenylpropanoids are a class of plant natural products that have many biological functions, including stress defence. In barley, phenylpropanoids have been described as having protective properties against excess UV-B radiation and have been linked to resistance to pathogens. Although the phenylpropanoid composition of barley has recently been addressed in more detail, the biosynthesis and regulation of this pathway have not been fully established. Barley introgression lines, such as the S42IL-population offer a set of genetically diverse plants that enable the correlation of metabolic data to distinct genetic regions on the barley genome and, subsequently, identification of relevant genes. The phenylpropanoid profiles of the first and third leaf of barley seedlings in Scarlett and four members of the S42IL-population were obtained by LC-MS. Comparison of the leaf profiles revealed a change in the glycosylation pattern of the flavone-6-C-glucoside isovitexin in the elite cultivar Scarlett. The change was characterized by the stepwise decrease in isovitexin-7-O-glucoside (saponarin) and an increase in isovitexin-2″-O-β-D-glucoside content. The lines S42IL-101-, -177 and -178 were completely devoid of isovitexin-2″-O-β-D-glucoside. Parallel glucosyltransferase assays were consistent with the observed metabolic patterns. The genetic region responsible for this metabolic effect was located on chromosome 1H between 0.21 and 15.08 cM, encompassing 505 gene candidates in the genome of the sequenced cultivar Morex. Only one of these genes displayed sequence similarity with glucosyltransferases of plant secondary metabolism that possessed the characteristic PSPG motif. Copyright © 2018 Elsevier Ltd. All rights reserved.

Links between CD147 function, glycosylation, and caveolin-1.

PubMed

Tang, Wei; Chang, Sharon B; Hemler, Martin E

2004-09-01

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-catalyzed, beta1,6-branched, polylactosamine-type sugars, which account for its excess size. Therefore, CD147, which is itself elevated on invasive tumor cells, may make a major contribution to the abundance of beta1,6-branched polylactosamine sugars that appear on invasive tumor cells. It was shown previously that caveolin-1 associates with CD147, thus inhibiting CD147 self-aggregation and MMP induction; now we show that caveolin-1 associates with LG-CD147 and restricts the biosynthetic conversion of LG-CD147 to HG-CD147. In addition, HG-CD147 (but not LG-CD147) was preferentially captured as a multimer after treatment of cells with a homobifunctional cross-linking agent and was exclusively recognized by monoclonal antibody AAA6, a reagent that selectively recognizes self-associated CD147 and inhibits CD147-mediated MMP induction. In conclusion, we have 1) determined the biochemical basis for the unusual size variation in CD147, 2) established that CD147 is a major carrier of beta1,6-branched polylactosamine sugars on tumor cells, and 3) determined that caveolin-1 can inhibit the conversion of LG-CD147 to HG-CD147. Because it is HG-CD147 that self-aggregates and stimulates MMP induction, we now have a mechanism to explain how caveolin-1 inhibits these processes. These results help explain the previously established tumor suppressor functions of caveolin-1.

Protein glycosylation in gastric and colorectal cancers: Toward cancer detection and targeted therapeutics.

PubMed

Ferreira, José Alexandre; Magalhães, Ana; Gomes, Joana; Peixoto, Andreia; Gaiteiro, Cristiana; Fernandes, Elisabete; Santos, Lúcio Lara; Reis, Celso A

2017-02-28

Glycosylation is the most frequent and structurally complex posttranslational modification in cell-surface and secreted proteins. Glycans are major orchestrators of biological processes, namely, by controlling protein folding and key biological functions such as cell adhesion, migration, signaling and immune recognition. Altered glycosylation is considered a hallmark of malignant transformations that decisively contributes to disease outcome. This review comprehensively summarizes the main findings related with gastrointestinal cancers and the decisive impact of aberrant glycosylation on tumor biology toward more aggressive phenotypes. Particular emphasis is given to alterations in O-glycosylation, namely, the overexpression of immature O-glycans, and the sialylated Lewis antigens sialyl-LeA and sialyl-LeX, frequently implicated in lymphohematogenous metastasis. We further discuss how recent contributions from glycoproteomics and glycoengineering fields have broadened our understanding of the human O-glycoproteome and its implications for cancer research. Finally, we address the tremendous potential of glycans in the context of targeted therapeutics (selective inhibition of glycosylation pathways, immunotherapy) and discuss the need to include glycomics/glycoproteomics in holistic panomics models toward true precision medicine settings. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry.

PubMed

Zhang, Yong; Xie, Xinfang; Zhao, Xinyuan; Tian, Fang; Lv, Jicheng; Ying, Wantao; Qian, Xiaohong

2018-01-06

Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took

Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase

Treesearch

Franz St. Johns; Jason C. Hurlbert; John D. Rice; James F. Preston; Edwin Pozharski

2011-01-01

Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGXn by...

Antisense RNA to the first N-glycosylation gene, ALG7, inhibits protein N-glycosylation and secretion by Xenopus oocytes.

PubMed

Kukuruzinska, M A; Apekin, V; Lamkin, M S; Hiltz, A; Rodriguez, A; Lin, C C; Paz, M A; Oppenheim, F G

1994-02-15

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

Recent structural and mechanistic insights into post-translational enzymatic glycosylation.

PubMed

Hurtado-Guerrero, Ramon; Davies, Gideon J

2012-12-01

Enzymatic glycosylation of proteins, a post-transitional modification of great significance, is carried out by diverse glycosyltransferases (GTs) that harness activated sugar donors, typically nucleotide or lipid-phosphate linked species. Recent work has seen a major increase in the study of the 3D structure and reaction mechanism of these enzymes. Key advances include the dissection of the classical O-glycosylating and N-glycosylating apparatus, revealing unusual folds and hitherto unconsidered chemical mechanisms for acceptor activation. There has been considerable success in the application of kinetic isotope effects and quantum simulations to address the controversial issue of the reaction mechanism of retaining GTs. New roles for old modifications, exemplified by potential epigenetic roles for glycosylation, have been discovered and there has also been a plethora of studies into important mammalian glycosylations that play key roles in cellular biology, opening up new targets for chemical intervention approaches. Copyright © 2012 Elsevier Ltd. All rights reserved.

Deciphering the functions of O-GlcNAc glycosylation in the brain: The role of site-specific quantitative O-GlcNAcomics.

PubMed

Thompson, John W; Sorum, Alexander W; Hsieh-Wilson, Linda C

2018-06-23

The dynamic posttranslational modification O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) is present on thousands of intracellular proteins in the brain. Like phosphorylation, O-GlcNAcylation is inducible and plays important functional roles in both physiology and disease. Recent advances in mass spectrometry (MS) and bioconjugation methods are now enabling the mapping of O-GlcNAcylation events to individual sites in proteins. However, our understanding of which glycosylation events are necessary for regulating protein function and controlling specific processes, phenotypes, or diseases remains in its infancy. Given the sheer number of O-GlcNAc sites, methods are greatly needed to identify promising sites and prioritize them for time- and resource-intensive functional studies. Revealing sites that are dynamically altered by different stimuli or disease states will likely to go a long way in this regard. Here, we describe advanced methods for identifying O-GlcNAc sites on individual proteins and across the proteome, and for determining their stoichiometry in vivo. We also highlight emerging technologies for quantitative, site-specific MS-based O-GlcNAc proteomics (O-GlcNAcomics), which allow proteome-wide tracking of O-GlcNAcylation dynamics at individual sites. These cutting-edge technologies are beginning to bridge the gap between the high-throughput cataloging of O-GlcNAcylated proteins and the relatively low-throughput study of individual proteins. By uncovering the O-GlcNAcylation events that change in specific physiological and disease contexts, these new approaches are providing key insights into the regulatory functions of O-GlcNAc in the brain, including their roles in neuroprotection, neuronal signaling, learning and memory, and neurodegenerative diseases.

Plasma Glycoproteomics Reveals Sepsis Outcomes Linked to Distinct Proteins in Common Pathways

PubMed Central

DeLeon-Pennell, Kristine Y.; Nguyen, Nguyen T.; de Castro Brás, Lisandra E.; Flynn, Elizabeth R.; Cannon, Presley L.; Griswold, Michael E.; Jin, Yu-Fang; Puskarich, Michael A.; Jones, Alan E.; Lindsey, Merry L.

2015-01-01

Objective Sepsis remains a predominant cause of mortality in the ICU, yet strategies to increase survival have proved largely unsuccessful. This study aimed to identify proteins linked to sepsis outcomes using a glycoproteomic approach to target extracellular proteins that trigger downstream pathways and direct patient outcomes. Design Plasma was obtained from the LacTATEs cohort. N-linked plasma glycopeptides were quantified by solid-phase extraction coupled with mass spectrometry. Glycopeptides were assigned to proteins using RefSeq and visualized in a heat map. Protein differences were validated by immunoblotting, and proteins were mapped for biological processes using Database for Annotation, Visualization and Integrated Discovery and for functional pathways using Kyoto Encyclopedia of Genes and Genomes databases. Setting Hospitalized care. Measurements and Main Results A total of 501 glycopeptides corresponding to 234 proteins were identified. Of these, 66 glycopeptides were unique to the survivor group and corresponded to 54 proteins, 60 were unique to the nonsurvivor group and corresponded to 43 proteins, and 375 were common responses between groups and corresponded to 137 proteins. Immunoblotting showed that nonsurvivors had increased total kininogen; decreased total cathepsin-L1, vascular cell adhesion molecule, periostin, and neutrophil gelatinase–associated lipocalin; and a two-fold decrease in glycosylated clusterin (all p < 0.05). Kyoto Encyclopedia of Genes and Genomes analysis identified six enriched pathways. Interestingly, survivors relied on the extrinsic pathway of the complement and coagulation cascade, whereas nonsurvivors relied on the intrinsic pathway. Conclusion This study identifies proteins linked to patient outcomes and provides insight into unexplored mechanisms that can be investigated for the identification of novel therapeutic targets. (Crit Care Med 2015; XX:00–00) PMID:26086942

The use of O-trifluoroacetyl protection and profound influence of the nature of glycosyl acceptor in benzyl-free arabinofuranosylation.

PubMed

Abronina, Polina I; Fedina, Ksenia G; Podvalnyy, Nikita M; Zinin, Alexander I; Chizhov, Alexander O; Kondakov, Nikolay N; Torgov, Vladimir I; Kononov, Leonid O

2014-09-19

The influence of O-trifluoroacetyl (TFA) groups at different positions of thioglycoside glycosyl donors on stereoselectivity of α-arabinofuranosylation leading to corresponding disaccharides was studied. It was shown that TFA group in thioglycoside glycosyl donors, when combined with 2-O-(triisopropylsilyl) (TIPS) non-participating group, may be regarded as an electron-withdrawing protecting group that may enhance 1,2-cis-selectivity in arabinofuranosylation, the results strongly depending on the nature of glycosyl acceptor. The reactivities of the glycosyl donors were compared with those of a similar thioglycoside with O-pentafluoropropionyl groups and the known phenyl 3,5-O-(di-tert-butylsilylene)-1-thio-α-d-arabinofuranosides with 2-O-TIPS and 2-O-benzyl groups. The 'matching' in the donor-acceptor combination was found to be critical for achieving both high reactivity of glycosyl donor and β-stereoselectivity of arabinofuranosylation. The use of glycosyl donors with TFA and silyl protection may be useful in the realization of the benzyl-free approach to oligoarabinofuranosides with azido group in aglycon-convenient building blocks for the preparation of neoglycoconjugates. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

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

Li, Qiang; Kamemura, Kazuo, E-mail: k_kamemura@nagahama-i-bio.ac.jp

2014-07-18

Highlights: • The majority of EWS localizes stably in the cytosol in 3T3-L1 preadipocytes. • Adipogenic stimuli induce the nuclear localization of EWS. • Adipogenesis promotes O-GlcNAcylation of EWS. • O-GlcNAcylation stimulates the recruitment of EWS to the nuclear periphery. - Abstract: Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughoutmore » adipogenesis. Instead, an adipogenesis-dependent increase in O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.« less

O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

PubMed

Tzeng, Sheue-Fen; Tsai, Chin-Hsien; Chao, Tai-Kuang; Chou, Yu-Ching; Yang, Yu-Chih; Tsai, Mong-Hsun; Cha, Tai-Lung; Hsiao, Pei-Wen

2018-06-15

Disseminated castration-resistant prostate cancer (CRPC) is a common disease in men that is characterized by limited survival and resistance to androgen-deprivation therapy. The increase in human epidermal growth factor receptor 2 (HER2) signaling contributes to androgen receptor activity in a subset of patients with CRPC; however, enigmatically, HER2-targeted therapies have demonstrated a lack of efficacy in patients with CRPC. Aberrant glycosylation is a hallmark of cancer and involves key processes that support cancer progression. Using transcriptomic analysis of prostate cancer data sets, histopathologic examination of clinical specimens, and in vivo experiments of xenograft models, we reveal in this study a coordinated increase in glycan-binding protein, galectin-4, specific glycosyltransferases of core 1 synthase, glycoprotein- N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) and ST3 beta-galactoside α-2,3-sialyltransferase 1 (ST3GAL1), and resulting mucin-type O-glycans during the progression of CRPC. Furthermore, galectin-4 engaged with C1GALT1-dependent O-glycans to promote castration resistance and metastasis by activating receptor tyrosine kinase signaling and cancer cell stemness properties mediated by SRY-box 9 (SOX9). This galectin-glycan interaction up-regulated the MYC-dependent expression of C1GALT1 and ST3GAL1, which altered cellular mucin-type O-glycosylation to allow for galectin-4 binding. In clinical prostate cancer, high-level expression of C1GALT1 and galectin-4 together predict poor overall survival compared with low-level expression of C1GALT1 and galectin-4. In summary, MYC regulates abnormal O-glycosylation, thus priming cells for binding to galectin-4 and downstream signaling, which promotes castration resistance and metastasis.-Tzeng, S.-F., Tsai, C.-H., Chao, T.-K., Chou, Y.-C., Yang, Y.-C., Tsai, M.-H., Cha, T.-L., Hsiao, P.-W. O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate

5-thiomannosides block the biosynthesis of dolichol-linked oligosaccharides and mimic class I congenital disorders of glycosylation.

PubMed

Zandberg, Wesley F; Gao, Ningguo; Kumarasamy, Jayakanthan; Lehrman, Mark A; Seidah, Nabil G; Pinto, B Mario

2012-02-13

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

PubMed

Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K; Lira-Navarrete, Erandi; Pedersen, Nis B; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G; Nielsen, Rikke; Christensen, Erik I; Hansen, Lars; Bennett, Eric P; Vakhrushev, Sergey Y; Schjoldager, Katrine T; Clausen, Henrik

2018-05-11

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O -glycan sites. Moreover, we found that O -glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O -glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O -glycosylation of LDLR-related proteins and identified conserved O -glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O -glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O -glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Flagellar glycosylation in Clostridium botulinum.

PubMed

Twine, Susan M; Paul, Catherine J; Vinogradov, Evgeny; McNally, David J; Brisson, Jean-Robert; Mullen, James A; McMullin, David R; Jarrell, Harold C; Austin, John W; Kelly, John F; Logan, Susan M

2008-09-01

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Glycosylation enables aesculin to activate Nrf2.

PubMed

Kim, Kyun Ha; Park, Hyunsu; Park, Hee Jin; Choi, Kyoung-Hwa; Sadikot, Ruxana T; Cha, Jaeho; Joo, Myungsoo

2016-07-15

Since aesculin, 6,7-dihydroxycoumarin-6-O-β-glucopyranoside, suppresses inflammation, we asked whether its anti-inflammatory activity is associated with the activation of nuclear factor-E2-related factor 2 (Nrf2), a key anti-inflammatory factor. Our results, however, show that aesculin marginally activated Nrf2. Since glycosylation can enhance the function of a compound, we then asked whether adding a glucose makes aesculin activate Nrf2. Our results show that the glycosylated aesculin, 3-O-β-d-glycosyl aesculin, robustly activated Nrf2, inducing the expression of Nrf2-dependent genes, such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinone oxidoreductase 1 in macrophages. Mechanistically, 3-O-β-d-glycosyl aesculin suppressed ubiquitination of Nrf2, retarding degradation of Nrf2. Unlike aesculin, 3-O-β-d-glycosyl aesculin significantly suppressed neutrophilic lung inflammation, a hallmark of acute lung injury (ALI), in mice, which was not recapitulated in Nrf2 knockout mice, suggesting that the anti-inflammatory function of the compound largely acts through Nrf2. In a mouse model of sepsis, a major cause of ALI, 3-O-β-d-glycosyl aesculin significantly enhanced the survival of mice, compared with aesculin. Together, these results show that glycosylation could confer the ability to activate Nrf2 on aesculin, enhancing the anti-inflammatory function of aesculin. These results suggest that glycosylation can be a way to improve or alter the function of aesculin.

Concurrent Automated Sequencing of the Glycan and Peptide Portions of O-Linked Glycopeptide Anions by Ultraviolet Photodissociation Mass Spectrometry

PubMed Central

Madsen, James A.; Ko, Byoung Joon; Xu, Hua; Iwashkiw, Jeremy A.; Robotham, Scott A.; Shaw, Jared B.; Feldman, Mario F.; Brodbelt, Jennifer S.

2013-01-01

O -glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly- and multiply-deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products, which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. Based on the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and non-glycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the “superbug” A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis. PMID:24006841

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus*

PubMed Central

Bagdonaite, Ieva; Nordén, Rickard; Joshi, Hiren J.; King, Sarah L.; Vakhrushev, Sergey Y.; Olofsson, Sigvard; Wandall, Hans H.

2016-01-01

Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a “bottom up” mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation. PMID:27129252

Different glycosylation in acetylcholinesterases from mammalian brain and erythrocytes.

PubMed

Liao, J; Heider, H; Sun, M C; Brodbeck, U

1992-04-01

Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine-linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate-polyacrylamide gel before and after deglycosylation with N-glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent-soluble AChE from electric organs of Torpedo nacline timilei as well as Elec-39 raised against AChE from Electrophorus electricus cross-reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N-glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N-linked carbohydrates. Analysis of N-acetylglucosamine sugars revealed the presence of N-acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N-linked glycosylation. On the other hand, N-acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O-linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.

Protein Glycosylation in Helicobacter pylori: Beyond the Flagellins?

PubMed Central

Hopf, Patrick S.; Ford, Rachel S.; Zebian, Najwa; Merkx-Jacques, Alexandra; Vijayakumar, Somalinga; Ratnayake, Dinath; Hayworth, Jacqueline; Creuzenet, Carole

2011-01-01

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori. PMID:21984942

CBP70, a glycosylated nuclear lectin.

PubMed

Rousseau, C; Felin, M; Doyennette-Moyne, M A; Sève, A P

1997-09-01

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.

Glucosamine Modulates T Cell Differentiation through Down-regulating N-Linked Glycosylation of CD25*

PubMed Central

Chien, Ming-Wei; Lin, Ming-Hong; Huang, Shing-Hwa; Fu, Shin-Huei; Hsu, Chao-Yuan; Yen, B. Lin-Ju; Chen, Jiann-Torng; Chang, Deh-Ming; Sytwu, Huey-Kang

2015-01-01

Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through down-regulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both down-regulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis. PMID:26468284

Hijacking bacterial glycosylation for the production of glycoconjugates, from vaccines to humanised glycoproteins.

PubMed

Cuccui, Jon; Wren, Brendan

2015-03-01

Glycosylation or the modification of a cellular component with a carbohydrate moiety has been demonstrated in all three domains of life as a basic post-translational process important in a range of biological processes. This review will focus on the latest studies attempting to exploit bacterial N-linked protein glycosylation for glycobiotechnological applications including glycoconjugate vaccine and humanised glycoprotein production. The challenges that remain for these approaches to reach full biotechnological maturity will be discussed. Oligosaccharyltransferase-dependent N-linked glycosylation can be exploited to make glycoconjugate vaccines against bacterial pathogens. Few technical limitations remain, but it is likely that the technologies developed will soon be considered a cost-effective and flexible alternative to current chemical-based methods of vaccine production. Some highlights from current glycoconjugate vaccines developed using this in-vivo production system include a vaccine against Shigella dysenteriae O1 that has passed phase 1 clinical trials, a vaccine against the tier 1 pathogen Francisella tularensis that has shown efficacy in mice and a vaccine against Staphylococcus aureus serotypes 5 and 8. Generation of humanised glycoproteins within bacteria was considered impossible due to the distinct nature of glycan modification in eukaryotes and prokaryotes. We describe the method used to overcome this conundrum to allow engineering of a eukaryotic pentasaccharide core sugar modification within Escherichia coli. This core was assembled by combining the function of the initiating transferase WecA, several Alg genes from Saccharomyces cerevisiae and the oligosaccharyltransferase function of the Campylobacter jejuni PglB. Further exploitation of a cytoplasmic N-linked glycosylation system found in Actinobacillus pleuropneumoniae where the central enzyme is known as N-linking glycosyltransferase has overcome some of the limitations demonstrated by the

The O-Glycosylated Linker from the Trichoderma reesei Family 7 Cellulase Is a Flexible, Disordered Protein

PubMed Central

Beckham, Gregg T.; Bomble, Yannick J.; Matthews, James F.; Taylor, Courtney B.; Resch, Michael G.; Yarbrough, John M.; Decker, Steve R.; Bu, Lintao; Zhao, Xiongce; McCabe, Clare; Wohlert, Jakob; Bergenstråhle, Malin; Brady, John W.; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.

2010-01-01

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study. PMID:21112302

First evidence of C- and O-glycosyl flavone in blood orange (Citrus sinensis (L.) Osbeck) juice and their influence on antioxidant properties.

PubMed

Barreca, Davide; Bellocco, Ersilia; Leuzzi, Ugo; Gattuso, Giuseppe

2014-04-15

RP-LC-DAD-ESI-MS-MS separation/identification protocol has been employed for the identification and characterisation of nine C- and O-glycosyl flavonoids in Moro (Citrus sinensis (L.) Osbeck) juice grown in Southern Italy. For the first time we reported the presence of five C-glycosyl flavones (lucenin-2, vicenin-2, stellarin-2, lucenin-2 4'-methyl ether and scoparin), a 3-hydroxy-3-methylglutaryl glycosyl flavonol (3-hydroxy-3-methylglutaryl glycosyl quercetin) and a flavone O-glycosides (chrysoeriol 7-O-neoesperidoside). Moreover, the influence of the identified C- and O-glycosyl flavonoids on the total antioxidant activity of crude juice has been evaluated on the basis of its ability to scavenge DPPH•, OH• and ABTS•+ radicals and to reduce iron. Copyright © 2013 Elsevier Ltd. All rights reserved.

O-Linked β-N-acetylglucosamine (O-GlcNAc) modification: a new pathway to decode pathogenesis of diabetic retinopathy.

PubMed

Gurel, Zafer; Sheibani, Nader

2018-01-31

The incidence of diabetes continues to rise among all ages and ethnic groups worldwide. Diabetic retinopathy (DR) is a complication of diabetes that affects the retinal neurovasculature causing serious vision problems, including blindness. Its pathogenesis and severity is directly linked to the chronic exposure to high glucose conditions. No treatments are currently available to stop the development and progression of DR. To develop new and effective therapeutic approaches, it is critical to better understand how hyperglycemia contributes to the pathogenesis of DR at the cellular and molecular levels. We propose alterations in O-GlcNAc modification of target proteins during diabetes contribute to the development and progression of DR. The O-GlcNAc modification is regulated through hexosamine biosynthetic pathway. We showed this pathway is differentially activated in various retinal vascular cells under high glucose conditions perhaps due to their selective metabolic activity. O-GlcNAc modification can alter protein stability, activity, interactions, and localization. By targeting the same amino acid residues (serine and threonine) as phosphorylation, O-GlcNAc modification can either compete or cooperate with phosphorylation. Here we will summarize the effects of hyperglycemia-induced O-GlcNAc modification on the retinal neurovasculature in a cell-specific manner, providing new insight into the role of O-GlcNAc modification in early loss of retinal pericytes and the pathogenesis of DR. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Glycosylation potential of human prostate cancer cell lines

PubMed Central

Gao, Yin; Chachadi, Vishwanath B.; Cheng, Pi-Wan

2014-01-01

Altered glycosylation is a universal feature of cancer cells and altered glycans can help cancer cells escape immune surveillance, facilitate tumor invasion, and increase malignancy. The goal of this study was to identify specific glycoenzymes, which could distinguish prostate cancer cells from normal prostatic cells. We investigated enzymatic activities and gene expression levels of key glycosyl- and sulfotransferases responsible for the assembly of O- and N-glycans in several prostatic cells. These cells included immortalized RWPE-1 cells derived from normal prostatic tissues, and prostate cancer cells derived from metastasis in bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP). We found that all cells were capable of synthesizing complex N-glycans and O-glycans with the core 1 structure, and each cell line had characteristic bio-synthetic pathways to modify these structures. The in vitro measured activities corresponded well to the mRNA levels of glycosyltransferases and sulfotransferases. Lectin and antibody binding to whole cells supported these results, which form the basis for the development of tumor cell-specific targeting strategies. PMID:22843320

Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.

PubMed

Harada, Yoichiro; Buser, Reto; Ngwa, Elsy M; Hirayama, Hiroto; Aebi, Markus; Suzuki, Tadashi

2013-11-08

Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.

Serum under-O-glycosylated IgA1 level is not correlated with glomerular IgA deposition based upon heterogeneity in the composition of immune complexes in IgA nephropathy.

PubMed

Satake, Kenji; Shimizu, Yoshio; Sasaki, Yohei; Yanagawa, Hiroyuki; Suzuki, Hitoshi; Suzuki, Yusuke; Horikoshi, Satoshi; Honda, Shinichiro; Shibuya, Kazuko; Shibuya, Akira; Tomino, Yasuhiko

2014-06-13

Although serum under-O-glycosylated IgA1 in IgA nephropathy (IgAN) patients may deposit more preferentially in glomeruli than heavily-O-glycosylated IgA1, the relationship between the glomerular IgA deposition level and the O-glycan profiles of serum IgA1 remains obscure. Serum total under-O-glycosylated IgA1 levels were quantified in 32 IgAN patients by an enzyme-linked immunosorbent assay (ELISA) with Helix aspersa (HAA) lectin. Serum under-O-glycosylated polymeric IgA1 (pIgA1) was selectively measured by an original method using mouse Fcα/μ receptor (mFcα/μR) transfectant and flow cytometry (pIgA1 trap). The percentage area of IgA deposition in the whole glomeruli (Area-IgA) was quantified by image analysis on the immunofluorescence of biopsy specimens. Correlations were assessed between the Area-IgA and data from HAA-ELISA or pIgA1 trap. The relationships between clinical parameters and data from HAA-ELISA or pIgA1 trap were analyzed by data mining approach. While the under-O-glycosylated IgA1 levels in IgAN patients were significantly higher than those in healthy controls when measured (p<0.05), there was no significant difference in under-O-glycosylated pIgA1. There was neither a correlation observed between the data from HAA-ELISA and pIgA1 trap (r2=0.09) in the IgAN patients (r2=0.005) nor was there a linear correlation between Area-IgA and data from HAA-ELISA or the pIgA1 trap (r2=0.005, 0.03, respectively). Contour plots of clinical parameters versus data from HAA-ELISA and the pIgA1 trap revealed that patients with a high score in each clinical parameter concentrated in specific areas, showing that patients with specific O-glycan profiles of IgA1 have similar clinical parameters. A decision tree analysis suggested that dominant immune complexes in glomeruli were consisted of: 1) IgA1-IgG and complements, 2) pIgA1 and complements, and 3) monomeric IgA1-IgA or aggregated monomeric IgA1. Serum under-O-glycosylated IgA1 levels are not correlated with

Metabolically programmed quality control system for dolichol-linked oligosaccharides

PubMed Central

Harada, Yoichiro; Nakajima, Kazuki; Masahara-Negishi, Yuki; Freeze, Hudson H.; Angata, Takashi; Taniguchi, Naoyuki; Suzuki, Tadashi

2013-01-01

The glycolipid Glc3Man9GlcNAc2-pyrophosphate-dolichol serves as the precursor for asparagine (N)-linked protein glycosylation in mammals. The biosynthesis of dolichol-linked oligosaccharides (DLOs) is arrested in low-glucose environments via unknown mechanisms, resulting in abnormal N-glycosylation. Here, we show that under glucose deprivation, DLOs are prematurely degraded during the early stages of DLO biosynthesis by pyrophosphatase, leading to the release of singly phosphorylated oligosaccharides into the cytosol. We identified that the level of GDP-mannose (Man), which serves as a donor substrate for DLO biosynthesis, is substantially reduced under glucose deprivation. We provide evidence that the selective shutdown of the GDP-Man biosynthetic pathway is sufficient to induce the release of phosphorylated oligosaccharides. These results indicate that glucose-regulated metabolic changes in the GDP-Man biosynthetic pathway cause the biosynthetic arrest of DLOs and facilitate their premature degradation by pyrophosphatase. We propose that this degradation system may avoid abnormal N-glycosylation with premature oligosaccharides under conditions that impair efficient DLO biosynthesis. PMID:24218558

Laminin alterations after in vitro nonenzymatic glycosylation.

PubMed

Charonis, A S; Reger, L A; Dege, J E; Kouzi-Koliakos, K; Furcht, L T; Wohlhueter, R M; Tsilibary, E C

1990-07-01

Laminin, a basement membrane protein derived from the matrix of the Engelbreth-Holm-Swarm murine tumor, was nonenzymatically glycosylated in vitro in the presence of increasing glucose concentrations. The amount of glucose incorporated per laminin molecule was shown to be proportional to the molarity of glucose used. Nonenzymatic glycosylation resulted in formation of cross-links and alterations of the cruciform shape of laminin molecules; these alterations were dramatic when high concentrations of glucose were used. One of the functions of laminin, the process of self-assembly, was shown to be impaired after in vitro nonenzymatic glycosylation. Glucose incorporation resulted in a dramatic decrease of long-to-long laminin dimers, which normally form during the initial steps of assembly. Furthermore, nonenzymatic glycosylation of laminin reduced its ability to self-associate into complexes larger than dimers, as judged by turbidimetry. The observed decrease of maximal turbidity was proportional to the degree of nonenzymatic glycosylation. Aminoguanidine, which has been suggested to inhibit cross-link formation, was shown to restore to a large extent the shape of laminin, the percentage of long-to-long arm dimers, and the maximal turbidity when included in the mixtures of laminin and glucose. These data suggest that structural and functional alterations of laminin may be primarily due to formation of cross-links. Such modifications of laminin (along with our basement membrane components) may contribute to the morphological and physiological changes observed in basement membranes under diabetic conditions.

O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome*

PubMed Central

Yehezkel, Galit; Cohen, Liz; Kliger, Adi; Manor, Esther; Khalaila, Isam

2012-01-01

O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer. PMID:22730328

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

PubMed Central

Chang, Xiu-bao; Mengos, April; Hou, Yue-xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei; Riordan, John R.; Gentzsch, Martina

2009-01-01

Summary The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway. PMID:18682497

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

PubMed

Chang, Xiu-Bao; Mengos, April; Hou, Yue-Xian; Cui, Liying; Jensen, Timothy J; Aleksandrov, Andrei; Riordan, John R; Gentzsch, Martina

2008-09-01

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

PubMed Central

Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y.; Strahl, Sabine; Clausen, Henrik

2015-01-01

Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575

Recombinant Human Lysyl Oxidase-like 2 Secreted from Human Embryonic Kidney Cells Displays Complex and Acidic Glycans at All Three N-Linked Glycosylation Sites.

PubMed

Go, Eden P; Moon, Hee-Jung; Mure, Minae; Desaire, Heather

2018-05-04

Human lysyl oxidase-like 2 (hLOXL2), a glycoprotein implicated in tumor progression and organ fibrosis, is a molecular target for anticancer and antifibrosis treatment. This glycoprotein contains three predicted N-linked glycosylation sites; one is near the protein's active site, and at least one more is known to facilitate the protein's secretion. Because the glycosylation impacts the protein's biology, we sought to characterize the native, mammalian glycosylation profile and to determine how closely this profile is recapitulated when the protein is expressed in insect cells. All three glycosylation sites on the protein, expressed in human embryonic kidney (HEK) cells, were characterized individually using a mass spectrometry-based glycopeptide analysis workflow. These data were compared to the glycosylation profile of the same protein expressed in insect cells. We found that the producer cell type imparts a substantial influence on the glycosylation of this important protein. The more-relevant version, expressed in HEK cells, contains large, acidic glycoforms; these glycans are not generated in insect cells. The glycosylation differences likely have structural and functional consequences, and these data should be considered when generating protein for functional studies or for high-throughput screening campaigns.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells*

PubMed Central

de Queiroz, Rafaela Muniz; Madan, Rashna; Chien, Jeremy; Dias, Wagner Barbosa; Slawson, Chad

2016-01-01

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma. PMID:27402830

Rate-dependent inverse-addition beta-selective mannosylation and contiguous sequential glycosylation involving beta-mannosidic bond formation.

PubMed

Chang, Shih-Sheng; Shih, Che-Hao; Lai, Kwun-Cheng; Mong, Kwok-Kong Tony

2010-05-03

The beta-selectivity of mannosylation has been found to be dependent on the addition rate of the mannosyl trichloroacetimidate donor in an inverse-addition (I-A) procedure. This rate dependent I-A procedure can improve the selectivity of direct beta-mannosylation and is applicable to orthogonal glycosylations of thioglycoside acceptors. Further elaboration of this novel procedure enables the development of the contiguous sequential glycosylation strategy, which streamlines the preparation of oligosaccharides invoking beta-mannosidic bond formation. The synthetic utility of the contiguous glycosylation strategy was demonstrated by the preparation of the trisaccharide core of human N-linked glycoproteins and the trisaccharide repeating unit of the O-specific polysaccharide found in the cellular capsule of Salmonelle bacteria.

[Synthesis and properties of O- and S-glycosylated derivatives of pyropheophorbide a].

PubMed

Aksenova, A A; Sebiakin, Iu L; Mironov, A F

2001-01-01

New O- and S-glycosylated derivatives of pyropheophorbide a were synthesized in the context of the design of photosensitizers for photodynamic cancer therapy. The resulting amphiphilic conjugates were found to be sufficiently water-soluble and suitable for the study of the photosensitizer penetration and accumulation in tumors.

UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation.

PubMed

Wen, Chao; Huang, Wei; Zhu, Xue-Lin; Li, Xiao-San; Zhang, Fan; Jiang, Ren-Wang

2018-02-02

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C 21 steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

Mining gene link information for survival pathway hunting.

PubMed

Jing, Gao-Jian; Zhang, Zirui; Wang, Hong-Qiang; Zheng, Hong-Mei

2015-08-01

This study proposes a gene link-based method for survival time-related pathway hunting. In this method, the authors incorporate gene link information to estimate how a pathway is associated with cancer patient's survival time. Specifically, a gene link-based Cox proportional hazard model (Link-Cox) is established, in which two linked genes are considered together to represent a link variable and the association of the link with survival time is assessed using Cox proportional hazard model. On the basis of the Link-Cox model, the authors formulate a new statistic for measuring the association of a pathway with survival time of cancer patients, referred to as pathway survival score (PSS), by summarising survival significance over all the gene links in the pathway, and devise a permutation test to test the significance of an observed PSS. To evaluate the proposed method, the authors applied it to simulation data and two publicly available real-world gene expression data sets. Extensive comparisons with previous methods show the effectiveness and efficiency of the proposed method for survival pathway hunting.

Stress disrupts intestinal mucus barrier in rats via mucin O-glycosylation shift: prevention by a probiotic treatment.

PubMed

Da Silva, Stéphanie; Robbe-Masselot, Catherine; Ait-Belgnaoui, Afifa; Mancuso, Alessandro; Mercade-Loubière, Myriam; Salvador-Cartier, Christel; Gillet, Marion; Ferrier, Laurent; Loubière, Pascal; Dague, Etienne; Theodorou, Vassilia; Mercier-Bonin, Muriel

2014-08-15

Despite well-known intestinal epithelial barrier impairment and visceral hypersensitivity in irritable bowel syndrome (IBS) patients and IBS-like models, structural and physical changes in the mucus layer remain poorly understood. Using a water avoidance stress (WAS) model, we aimed at evaluating whether 1) WAS modified gut permeability, visceral sensitivity, mucin expression, biochemical structure of O-glycans, and related mucus physical properties, and 2) whether Lactobacillus farciminis treatment prevented these alterations. Wistar rats received orally L. farciminis or vehicle for 14 days; at day 10, they were submitted to either sham or 4-day WAS. Intestinal paracellular permeability and visceral sensitivity were measured in vivo. The number of goblet cells and Muc2 expression were evaluated by histology and immunohistochemistry, respectively. Mucosal adhesion of L. farciminis was determined ex situ. The mucin O-glycosylation profile was obtained by mass spectrometry. Surface imaging of intestinal mucus was performed at nanoscale by atomic force microscopy. WAS induced gut hyperpermeability and visceral hypersensitivity but did not modify either the number of intestinal goblet cells or Muc2 expression. In contrast, O-glycosylation of mucins was strongly affected, with the appearance of elongated polylactosaminic chain containing O-glycan structures, associated with flattening and loss of the mucus layer cohesive properties. L. farciminis bound to intestinal Muc2 and prevented WAS-induced functional alterations and changes in mucin O-glycosylation and mucus physical properties. WAS-induced functional changes were associated with mucus alterations resulting from a shift in O-glycosylation rather than from changes in mucin expression. L. farciminis treatment prevented these alterations, conferring epithelial and mucus barrier strengthening. Copyright © 2014 the American Physiological Society.

Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae *

PubMed Central

Neubert, Patrick; Halim, Adnan; Zauser, Martin; Essig, Andreas; Joshi, Hiren J.; Zatorska, Ewa; Larsen, Ida Signe Bohse; Loibl, Martin; Castells-Ballester, Joan; Aebi, Markus; Clausen, Henrik; Strahl, Sabine

2016-01-01

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammals O-mannosylation is the only type of O-glycosylation. In an essential step toward the full understanding of protein O-mannosylation we mapped the O-mannose glycoproteome in baker's yeast. Taking advantage of an O-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500 O-glycoproteins from all subcellular compartments for which over 2300 O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293 O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized protein O-mannosyltransferases. We find that O-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed that O-mannosylation is favored in unstructured regions and β-strands. Furthermore, O-mannosylation is impeded in the proximity of N-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and their O-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types of O-glycosylation from yeast to mammals. PMID:26764011

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

PubMed

Anastasia, Luigi; Rota, Paola; Anastasia, Mario; Allevi, Pietro

2013-09-21

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

PubMed

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Synthesis of S-linked trisaccharide glycal of derhodinosylurdamycin A: Discovery of alkyl thiocyanate as an efficient electrophile for stereoselective sulfenylation of 2-deoxy glycosyl lithium.

PubMed

Acharya, Padam P; Baryal, Kedar N; Reno, Cristin E; Zhu, Jianglong

2017-08-07

Stereoselective synthesis of S-linked trisaccharide glycal of angucycline antitumor antibiotic derhodinosylurdamycin A is described. The synthesis has been accomplished employing our previously reported umpolung S-glycosylation strategy - stereoselective sulfenylation of 2-deoxy glycosyl lithium. It was found that sugar-derived thiocyanate was a better electrophile than corresponding asymmetric disulfide in this type of stereoselective sulfenylation. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Nutrient-Sensing Hexosamine Biosynthetic Pathway as the Hub of Cancer Metabolic Rewiring.

PubMed

Chiaradonna, Ferdinando; Ricciardiello, Francesca; Palorini, Roberta

2018-06-02

Alterations in glucose and glutamine utilizing pathways and in fatty acid metabolism are currently considered the most significant and prevalent metabolic changes observed in almost all types of tumors. Glucose, glutamine and fatty acids are the substrates for the hexosamine biosynthetic pathway (HBP). This metabolic pathway generates the "sensing molecule" UDP- N -Acetylglucosamine (UDP-Glc N Ac). UDP-Glc N Ac is the substrate for the enzymes involved in protein N - and O -glycosylation, two important post-translational modifications (PTMs) identified in several proteins localized in the extracellular space, on the cell membrane and in the cytoplasm, nucleus and mitochondria. Since protein glycosylation controls several key aspects of cell physiology, aberrant protein glycosylation has been associated with different human diseases, including cancer. Here we review recent evidence indicating the tight association between the HBP flux and cell metabolism, with particular emphasis on the post-transcriptional and transcriptional mechanisms regulated by the HBP that may cause the metabolic rewiring observed in cancer. We describe the implications of both protein O - and N -glycosylation in cancer cell metabolism and bioenergetics; focusing our attention on the effect of these PTMs on nutrient transport and on the transcriptional regulation and function of cancer-specific metabolic pathways.

Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation.

PubMed

Thompson, John W; Griffin, Matthew E; Hsieh-Wilson, Linda C

2018-01-01

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition "click" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation. © 2018 Elsevier Inc. All rights reserved.

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

Quantitative Glycoproteomics Analysis Reveals Changes in N-Glycosylation Level Associated with Pancreatic Ductal Adenocarcinoma

PubMed Central

2015-01-01

Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-β, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events. PMID:24471499

Quantitative glycoproteomics analysis reveals changes in N-glycosylation level associated with pancreatic ductal adenocarcinoma.

PubMed

Pan, Sheng; Chen, Ru; Tamura, Yasuko; Crispin, David A; Lai, Lisa A; May, Damon H; McIntosh, Martin W; Goodlett, David R; Brentnall, Teresa A

2014-03-07

Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-β, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events.

Synthesis of 4-O-glycosylated 1,5-anhydro-D-fructose and of 1,5-anhydro-D-tagatose from a common intermediate 2,3-O-isopropylidene-D-fructose.

PubMed

Agoston, Károly; Dékány, Gyula; Lundt, Inge

2009-05-26

Four novel disaccharides of glycosylated 1,5-anhydro-D-ketoses have been prepared: 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-tagatose, and 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-beta-D-fructopyranose, was prepared from D-fructose and was converted into the D-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.

Glycosylation status of vitamin D binding protein in cancer patients

PubMed Central

Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

2009-01-01

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages. PMID:19642159

Glycosylation status of vitamin D binding protein in cancer patients.

PubMed

Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

2009-10-01

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

Impact of O-glycosylation on the molecular and cellular adhesion properties of the Escherichia coli autotransporter protein Ag43.

PubMed

Reidl, Sebastian; Lehmann, Annika; Schiller, Roswitha; Salam Khan, A; Dobrindt, Ulrich

2009-08-01

Antigen 43 (Ag43) represents an entire family of closely related autotransporter proteins in Escherichia coli and has been described to confer aggregation and fluffing of cells, to promote biofilm formation, uptake and survival in macrophages as well as long-term persistence of uropathogenic E. coli in the murine urinary tract. Furthermore, it has been reported that glycosylation of the Ag43 passenger domain (alpha(43)) stabilizes its conformation and increases adhesion to Hep-2 cells. We characterized the role of Ag43 as an adhesin and the impact of O-glycosylation on the function of Ag43. To analyze whether structural variations in the alpha(43) domain correlate with different functional properties, we cloned 5 different agn43 alleles from different E. coli subtypes and tested them for autoaggregation, biofilm formation, adhesion to different eukaryotic cell lines as well as to purified components of the extracellular matrix. These experiments were performed with nonglycosylated and O-glycosylated Ag43 variants. We show for the first time that Ag43 mediates bacterial adhesion in a cell line-specific manner and that structural variations of the alpha(43) domain correlate with increased adhesive properties to proteins of the extracellular matrix such as collagen and laminin. Whereas O-glycosylation of many alpha(43) domains led to impaired autoaggregation and a significantly reduced adhesion to eukaryotic cell lines, their interaction with collagen was significantly increased. These data demonstrate that O-glycosylation is not a prerequisite for Ag43 function and that the different traits mediated by Ag43, i.e., biofilm formation, autoaggregation, adhesion to eukaryotic cells and extracellular matrix proteins, rely on distinct mechanisms.

Pharmacological Inhibition of O-GlcNAcase Enhances Autophagy in Brain through an mTOR-Independent Pathway.

PubMed

Zhu, Yanping; Shan, Xiaoyang; Safarpour, Farzaneh; Erro Go, Nancy; Li, Nancy; Shan, Alice; Huang, Mina C; Deen, Matthew; Holicek, Viktor; Ashmus, Roger; Madden, Zarina; Gorski, Sharon; Silverman, Michael A; Vocadlo, David J

2018-03-05

The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer's disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic.

Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

PubMed

Hauptmann, Peter; Lehle, Ludwig

2008-07-04

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

Effects of cell culture conditions on antibody N-linked glycosylation--what affects high mannose 5 glycoform.

PubMed

Pacis, Efren; Yu, Marcella; Autsen, Jennifer; Bayer, Robert; Li, Feng

2011-10-01

The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process. Copyright © 2011 Wiley Periodicals, Inc.

Advanced glycosylation end product promotes forkhead box O1 and inhibits Wnt pathway to suppress capacities of epidermal stem cells.

PubMed

Zhu, Jie; Wang, Peng; Yu, Zhimin; Lai, Wei; Cao, Yi; Huang, Pinbo; Xu, Qiaodong; Yu, Menglei; Xu, Junyao; Huang, Zitong; Zeng, Bing

2016-01-01

Diabetes mellitus is frequently accompanied by chronic complications like delayed wound healing, which is consider to be attributed to the accumulation of advanced glycosylation end product (AGE). However, the impacts of AGE on epidermal stem cells (ESCs) are largely unknown. This study aims to address the influence and mechanism of AGE on ESCs. ESCs isolated from rats were cultured in AGE-modified bovine serum albumin and transfected with small interfering RNA to knock down AGE-specific receptor (AGER). Expression of stem cell markers integrin β1 (ITGB1) and keratin 19 (KRT19), cell viability, apoptosis and reactive oxygen species (ROS) were examined. Wnt pathway-related factors Wnt family member 1 (WNT1), WNT3A, β-catenin, v-myc avian myelocytomatosis viral oncogene homolog (MYC), cyclin D1 (CCND1) and matrix metallopeptidase 7 (MMP7) were quantified. The interaction between forkhead box O1 (FOXO1) and β-catenin was assessed by co-immunoprecipitation. Results indicated that AGE down-regulated ITGB1 and KRT19 expression, suppressed ESC viability and promoted apoptosis, and ROS level ( P < 0.01), implying decreased capacities of ESCs. AGE also promoted AGER and FOXO1, while AGER knockdown had the opposite effects. Moreover, AGER knockdown elevated the level of WNT1, WNT3A, MYC, CCND1 and MMP7 that were suppressed by AGE ( P < 0.01). Immunoprecipitation analysis showed that FOXO1 could compete with lymphoid enhancer binding factor 1 to interact with β-catenin, which might help to elucidate the mechanism of AGE repressing ESCs. This study helps to understand the mechanism of accumulated AGE in affecting ESC capacities, and provides potential therapeutic targets to meliorate diabetic wound healing.

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

PubMed

Lackman, Jarkko J; Goth, Christoffer K; Halim, Adnan; Vakhrushev, Sergey Y; Clausen, Henrik; Petäjä-Repo, Ulla E

2018-01-01

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors

Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products.

PubMed

Kuglstatter, A; Stihle, M; Neumann, C; Müller, C; Schaefer, W; Klein, C; Benz, J

2017-09-01

An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation. Both homodimer structures show parallel Fc fragment architectures, in contrast to recently reported crystal structures of the corresponding aglycosylated Fc fragments which in the absence of disulfide mutations show an unexpected antiparallel arrangement. The glycosylated Knob-Knob Fc fragment is destabilized as indicated by variability in the relative orientation of its CH3 domains. The glycosylated Hole-Hole Fc fragment shows an unexpected intermolecular disulfide bond via the introduced Y349C Hole mutation which results in a large CH3 domain shift and a new CH3-CH3 interface. The crystal structures of glycosylated, disulfide-linked KiH Fc fragment and its Knob-Knob and Hole-Hole side products reported here will facilitate further design of highly efficient antibody heterodimerization strategies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Arginine insertion and loss of N-linked glycosylation site in HIV-1 envelope V3 region confer CXCR4-tropism

PubMed Central

Tsuchiya, Kiyoto; Ode, Hirotaka; Hayashida, Tsunefusa; Kakizawa, Junko; Sato, Hironori; Oka, Shinichi; Gatanaga, Hiroyuki

2013-01-01

The third variable region (V3) of HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism). However, which combinations of mutations in V3 confer a tropism shift is still unclear. A unique pattern of mutations in antiretroviral therapy-naive HIV-1 patient was observed associated with the HIV-1 tropism shift CCR5 to CXCR4. The insertion of arginine at position 11 and the loss of the N-linked glycosylation site were indispensable for acquiring pure CXCR4-tropism, which were confirmed by cell-cell fusion assay and phenotype analysis of recombinant HIV-1 variants. The same pattern of mutations in V3 and the associated tropism shift were identified in two of 53 other patients (3.8%) with CD4+ cell count <200/mm3. The combination of arginine insertion and loss of N-linked glycosylation site usually confers CXCR4-tropism. Awareness of this rule will help to confirm the tropism prediction from V3 sequences by conventional rules. PMID:23925152

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.

PubMed

Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun; Lackman, Jarkko J; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H; Overall, Christopher M; Clausen, Henrik; Schjoldager, Katrine T; Petäjä-Repo, Ulla E

2017-03-17

The β 1 -adrenergic receptor (β 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O -glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O -glycosylates β 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O -glycosylation and proteolytic cleavage assays, a cell line deficient in O -glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β 1 AR. Furthermore, we demonstrate that impaired O -glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O -glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage*

PubMed Central

Goth, Christoffer K.; Tuhkanen, Hanna E.; Khan, Hamayun; Lackman, Jarkko J.; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H.; Overall, Christopher M.; Clausen, Henrik; Schjoldager, Katrine T.; Petäjä-Repo, Ulla E.

2017-01-01

The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. PMID:28167537

Mapping N-linked glycosylation of carbohydrate-active enzymes in the secretome of Aspergillus nidulans grown on lignocellulose.

PubMed

Rubio, Marcelo Ventura; Zubieta, Mariane Paludetti; Franco Cairo, João Paulo Lourenço; Calzado, Felipe; Paes Leme, Adriana Franco; Squina, Fabio Marcio; Prade, Rolf Alexander; de Lima Damásio, André Ricardo

2016-01-01

The genus Aspergillus includes microorganisms that naturally degrade lignocellulosic biomass, secreting large amounts of carbohydrate-active enzymes (CAZymes) that characterize their saprophyte lifestyle. Aspergillus has the capacity to perform post-translational modifications (PTM), which provides an additional advantage for the use of these organisms as a host for the production of heterologous proteins. In this study, the N-linked glycosylation of CAZymes identified in the secretome of Aspergillus nidulans grown on lignocellulose was mapped. Aspergillus nidulans was grown in glucose, xylan and pretreated sugarcane bagasse (SCB) for 96 h, after which glycoproteomics and glycomics were carried out on the extracellular proteins (secretome). A total of 265 proteins were identified, with 153, 210 and 182 proteins in the glucose, xylan and SCB substrates, respectively. CAZymes corresponded to more than 50 % of the total secretome in xylan and SCB. A total of 182 N-glycosylation sites were identified, of which 121 were detected in 67 CAZymes. A prevalence of the N-glyc sequon N-X-T (72.2 %) was observed in N-glyc sites compared with N-X-S (27.8 %). The amino acids flanking the validated N-glyc sites were mainly composed of hydrophobic and polar uncharged amino acids. Selected proteins were evaluated for conservation of the N-glyc sites in Aspergilli homologous proteins, but a pattern of conservation was not observed. A global analysis of N-glycans released from the proteins secreted by A. nidulans was also performed. While the proportion of N-glycans with Hex5 to Hex9 was similar in the xylan condition, a prevalence of Hex5 was observed in the SCB and glucose conditions. The most common and frequent N-glycosylated motifs, an overview of the N-glycosylation of the CAZymes and the number of mannoses found in N-glycans were analyzed. There are many bottlenecks in protein production by filamentous fungi, such as folding, transport by vesicles and secretion, but N-glycosylation

Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

PubMed Central

Chen, Liqun; Drake, Matthew R.; Resch, Michael G.; Greene, Eric R.; Himmel, Michael E.; Chaffey, Patrick K.; Beckham, Gregg T.; Tan, Zhongping

2014-01-01

The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. PMID:24821760

Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins.

PubMed

Brooks, Susan A

2006-06-01

A major challenge for the biotechnology industry is to engineer the glycosylation pathways of expression systems to synthesize recombinant proteins with human glycosylation. Inappropriate glycosylation can result in reduced activity, limited half-life in circulation and unwanted immunogenicity. In this review, the complexities of glycosylation in human cells are explained and compared with glycosylation in bacteria, yeasts, fungi, insects, plants and nonhuman mammalian species. Key advances in the engineering of the glycosylation of expression systems are highlighted. Advances in the challenging and technically complex field of glycan analysis are also described. The emergence of a new generation of expression systems with sophisticated engineering for humanized glycosylation of glycoproteins appears to be on the horizon.

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

PubMed

Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

2011-02-03

Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

PubMed Central

2011-01-01

Background Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr

Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

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

Xiang, Yong; Karaveg, Khanita; Moremen, Kelley W.

2016-11-17

Asn-linked glycosylation of newly synthesized polypeptides occurs in the endoplasmic reticulum of eukaryotic cells. Glycan structures are trimmed and remodeled as they transit the secretory pathway, and processing intermediates play various roles as ligands for folding chaperones and signals for quality control and intracellular transport. Key steps for the generation of these trimmed intermediates are catalyzed by glycoside hydrolase family 47 (GH47) α-mannosidases that selectively cleave α1,2-linked mannose residues. Despite the sequence and structural similarities among the GH47 enzymes, the molecular basis for residue-specific cleavage remains obscure. The present studies reveal enzyme–substrate complex structures for two related GH47 α-mannosidases andmore » provide insights into how these enzymes recognize the same substrates differently and catalyze the complementary glycan trimming reactions necessary for glycan maturation.« less

KT5823 Differentially Modulates Sodium Iodide Symporter Expression, Activity, and Glycosylation between Thyroid and Breast Cancer Cells

PubMed Central

Beyer, Sasha; Lakshmanan, Aparna; Liu, Yu-Yu; Zhang, Xiaoli; Wapnir, Irene; Smolenski, Albert

2011-01-01

Na+/I− symporter (NIS)-mediated iodide uptake into thyroid follicular cells serves as the basis of radioiodine therapy for thyroid cancer. NIS protein is also expressed in the majority of breast tumors, raising potential for radionuclide therapy of breast cancer. KT5823, a staurosporine-related protein kinase inhibitor, has been shown to increase thyroid-stimulating hormone-induced NIS expression, and thus iodide uptake, in thyroid cells. In this study, we found that KT5823 does not increase but decreases iodide uptake within 0.5 h of treatment in trans-retinoic acid and hydrocortisone-treated MCF-7 breast cancer cells. Moreover, KT5823 accumulates hypoglycosylated NIS, and this effect is much more evident in breast cancer cells than thyroid cells. The hypoglycosylated NIS is core glycosylated, has not been processed through the Golgi apparatus, but is capable of trafficking to the cell surface. KT5823 impedes complex NIS glycosylation at a regulatory point similar to brefeldin A along the N-linked glycosylation pathway, rather than targeting a specific N-glycosylated site of NIS. KT5823-mediated effects on NIS activity and glycosylation are also observed in other breast cancer cells as well as human embryonic kidney cells expressing exogenous NIS. Taken together, KT5823 will serve as a valuable pharmacological reagent to uncover mechanisms underlying differential NIS regulation between thyroid and breast cancer cells at multiple levels. PMID:21209020

Glycosylation of Cblns attenuates their receptor binding.

PubMed

Rong, Yongqi; Bansal, Parmil K; Wei, Peng; Guo, Hong; Correia, Kristen; Parris, Jennifer; Morgan, James I

2018-05-18

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Reconstruction and visualization of carbohydrate, N-glycosylation pathways in Pichia pastoris CBS7435 using computational and system biology approaches.

PubMed

Srivastava, Akriti; Somvanshi, Pallavi; Mishra, Bhartendu Nath

2013-06-01

Pichia pastoris is an efficient expression system for production of recombinant proteins. To understand its physiology for building novel applications it is important to understand and reconstruct its metabolic network. The metabolic reconstruction approach connects genotype with phenotype. Here, we have attempted to reconstruct carbohydrate metabolism pathways responsible for high biomass density and N-glycosylation pathways involved in the post translational modification of proteins of P. pastoris CBS7435. Both these metabolic pathways play a crucial role in heterologous protein production. We report novel, missing and unannotated enzymes involved in the target metabolic pathways. A strong possibility of cellulose and xylose metabolic processes in P. pastoris CBS7435 suggests its use in the area of biofuels. The reconstructed metabolic networks can be used for increased yields and improved product quality, for designing appropriate growth medium, for production of recombinant therapeutics and for making biofuels.

Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids.

PubMed

Dutta, Devawati; Mandal, Chhabinath; Mandal, Chitra

2017-12-01

Glycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc). Protein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the +2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions. Therefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers. Copyright © 2017 Elsevier B.V. All rights reserved.

GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

PubMed Central

Lee, I-Chiao; van Swam, Iris I.; Tomita, Satoru; Morsomme, Pierre; Rolain, Thomas; Hols, Pascal; Bron, Peter A.

2014-01-01

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively. PMID:24532775

[Pathogenicity factors of bacteria with glycosylating activity].

PubMed

Tartakovskaia, D I; Araslanova, V A; Belyĭ, Iu F

2011-01-01

A and B toxins of Clostridium difficile, a-toxin of C. novyi, lehal toxin of C. sordellii, and TpeL toxin of C. perfringens belong to the group of the so-called large Clostridium toxins. These toxins modify low-molecular weight guanosine triphosphate-binding proteins of the Rho/Ras family by their glycosylation that results in inactivation of major signal pathways in eukaryotic cells. Lgt glycosyltransferases, a new group of pathogenicity factors also capable of inactivating eukaryotic substrates via glycosylation, have recently been identified in Legionella. They are transported into cytoplasm of eukaryotic target cells by type 4 secretory system of Legionella. After translocation, the enzyme inhibits protein synthesis by attaching glucose residue to Ser53 of 1A elongation factor. The available data suggest an important role of bacterial glycosylating factors in the action of pathogens causing infectious diseases.

Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development.

PubMed

Dehennaut, Vanessa; Lefebvre, Tony; Leroy, Yves; Vilain, Jean-Pierre; Michalski, Jean-Claude; Bodart, Jean-François

2009-04-01

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I-V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease.

PubMed Central

Wang, Guirong; Christensen, Neil D; Wigdahl, Brian; Guttentag, Susan H; Floros, Joanna

2003-01-01

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions. PMID:12356334

Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro-B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure.

PubMed

Halfinger, Bernhard; Hammerer-Lercher, Angelika; Amplatz, Benno; Sarg, Bettina; Kremser, Leopold; Lindner, Herbert H

2017-01-01

Currently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms. The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry. We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable. Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms. © 2016 American Association for Clinical Chemistry.

C- and O-glycosyl flavonoids in Sanguinello and Tarocco blood orange (Citrus sinensis (L.) Osbeck) juice: Identification and influence on antioxidant properties and acetylcholinesterase activity.

PubMed

Barreca, Davide; Gattuso, Giuseppe; Laganà, Giuseppina; Leuzzi, Ugo; Bellocco, Ersilia

2016-04-01

Sanguinello and Tarocco are the blood orange (Citrus sinensis (L.) Osbeck) cultivars most diffused worldwide. Reversed phase liquid chromatography coupled with MS-MS analysis showed that these two varieties have a similar chromatographic pattern, characterised by the presence of C- and O-glycosyl flavonoids. Of the two, Sanguinello was found to be far richer in flavonoids than Tarocco. In the juices, twelve individual components were identified for the first time, namely, four C-glycosyl flavones (lucenin-2, vicenin-2, stellarin-2, lucenin-2 4'-methyl ether and scoparin), three flavonol derivatives (quercetin-3-O-(2-rhamnosyl)-rutinoside, quercetin-3-O-hexoside, quercetin 3-hydroxy-3-methylglutaryl-glycoside), an O-triglycosyl flavanone (narirutin 4'-O-glucoside) and a flavone O-glycosides (chrysoeriol 7-O-neoesperidoside). Moreover, the influence of the identified C- and O-glycosyl flavonoids on the antioxidant and acetylcholinesterase activity of these juices has been evaluated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)*

PubMed Central

van der Post, Sjoerd; Subramani, Durai B.; Bäckström, Malin; Johansson, Malin E. V.; Vester-Christensen, Malene B.; Mandel, Ulla; Bennett, Eric P.; Clausen, Henrik; Dahlén, Gunnar; Sroka, Aneta; Potempa, Jan; Hansson, Gunnar C.

2013-01-01

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation. PMID:23546879

Trivalent chromium inhibits TSP-1 expression, proliferation, and O-GlcNAc signaling in vascular smooth muscle cells in response to high glucose in vitro.

PubMed

Ganguly, Rituparna; Sahu, Soumyadip; Chavez, Ronaldo J; Raman, Priya

2015-01-15

Trivalent chromium (Cr(3+)) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. In vitro and in vivo studies suggest that Cr(3+) supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr(3+) in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study we show that a therapeutically relevant concentration of Cr(3+) (100 nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr(3+) occurs at the level of transcription. The inhibitory effects of Cr(3+) on TSP-1 were accompanied by significant reductions in O-glycosylation of cytoplasmic and nuclear proteins. Using Western blotting and immunofluorescence studies, we demonstrate that reduced protein O-glycosylation by Cr(3+) is mediated via inhibition of glutamine: fructose 6-phosphate amidotransferase, a rate-limiting enzyme of the hexosamine pathway, and O-linked N-acetylglucosamine (O-GlcNAc) transferase, a distal enzyme in the pathway that controls intracellular protein O-glycosylation. Additionally, we found that Cr(3+) attenuates reactive oxygen species formation in glucose-stimulated HASMC, suggesting an antioxidant effect. Finally, we report an antiproliferative effect of Cr(3+) that is specific for high glucose and conditions triggering elevated protein O-glycosylation. Taken together, these findings provide the first cellular evidence for a novel role of Cr(3+) to modulate aberrant vascular smooth muscle cell function associated with hyperglycemia-induced vascular complications. Copyright © 2015 the American Physiological Society.

Evidence for an asialoglycoprotein receptor on nonparenchymal cells for O-linked glycoproteins.

PubMed

Stefanich, Eric G; Ren, Song; Danilenko, Dimitry M; Lim, Amy; Song, An; Iyer, Suhasini; Fielder, Paul J

2008-11-01

B cell-activating factor receptor 3 (BR3)-Fc is an IgG1-receptor dimeric fusion protein that has multiple O-linked glycosylation sites and sialylation levels that can vary in the manufacturing process. Increased sialic acid levels resulted from increased site occupancy with the O-linked N-acetylgalactosamine (GalNAc-Gal), but because the ratio of sialic acid per mole of oligosaccharide remained approximately 1, this led to increased asialo terminal GalNAc. Previous studies have demonstrated an effect of terminal asialo Gal or GalNAc on the clearance of glycoproteins due to uptake and degradation by lectin receptors in the liver. However, the previous studies examined N-linked oligosaccharides, and there are less data regarding O-linked oligosaccharides. The objective of these studies was to determine the effects on the pharmacokinetics and distribution of the asialo terminal GalNAc and varying amounts of sialic acid residues on BR3-Fc. The results of the data presented here suggest that exposed Gal on the desialylated BR3-Fc led to rapid clearance due to uptake and degradation in the liver that was associated with nonparenchymal cells. It is interesting to note that the data indicated a decreased clearance and increased exposure of BR3-Fc as the sialic acid levels increased, even though increased sialic acid was associated with increased asialo GalNAc. Therefore, the exposed GalNAc did not seem to play a role in the clearance of BR3-Fc; although the Gal linked to the hydroxyl group at position 3 may have prevented an interaction. Because we did not see uptake of desialylated BR3-Fc in hepatocytes where the asialoglycoprotein receptor is localized, this nonparenchymal cell lectin may have preference for O-linked glycoproteins.

Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

PubMed Central

Larsen, Ida Signe Bohse; Narimatsu, Yoshiki; Joshi, Hiren Jitendra; Yang, Zhang; Harrison, Oliver J.; Brasch, Julia; Shapiro, Lawrence; Honig, Barry; Vakhrushev, Sergey Y.; Clausen, Henrik; Halim, Adnan

2017-01-01

Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated α-dystroglycanopathies, because deficient O-Man glycosylation of α-dystroglycan disrupts laminin interaction with α-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose β-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to α-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to α-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins. PMID:28512129

Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

PubMed

Mahan, Alison E; Jennewein, Madeleine F; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D; Baden, Lindsey; Barouch, Dan H; Alter, Galit

2016-03-01

Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

Appropriate polarization following pharmacological rescue of V2 vasopressin receptors encoded by X-linked nephrogenic diabetes insipidus alleles involves a conformation of the receptor that also attains mature glycosylation.

PubMed

Tan, Christopher M; Nickols, Hilary Highfield; Limbird, Lee E

2003-09-12

To understand the mechanisms of G protein-coupled receptor delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2Rs) in polarized Madin-Darby canine kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates arginine vasopressin (AVP)-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, Delta V278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of Delta V278 V2R, parallels acquisition of AVP-stimulated cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized epithelial cells.

Activation of liver alcohol dehydrogenase by glycosylation.

PubMed Central

Tsai, C S; White, J H

1983-01-01

D-Fructose and D-glucose activate alcohol dehydrogenase from horse liver to oxidize ethanol. One mol of D-[U-14C]fructose or D-[U-14C]glucose is covalently incorporated per mol of the maximally activated enzyme. Amino acid and N-terminal analyses of the 14C-labelled glycopeptide isolated from a proteolytic digest of the [14C]glycosylated enzyme implicate lysine-315 as the site of the glycosylation. 13C-n.m.r.-spectroscopic studies indicate that D-[13C]glucose is covalently linked in N-glucosidic and Amadori-rearranged structures in the [13C]glucosylated alcohol dehydrogenase. Experimental results are consistent with the formation of the N-glycosylic linkage between glycose and lysine-315 of liver alcohol dehydrogenase in the initial step that results in an enhanced catalytic efficiency to oxidize ethanol. PMID:6342612

O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

PubMed

Sheikh, M Osman; Thieker, David; Chalmers, Gordon; Schafer, Christopher M; Ishihara, Mayumi; Azadi, Parastoo; Woods, Robert J; Glushka, John N; Bendiak, Brad; Prestegard, James H; West, Christopher M

2017-11-17

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF ( S kp1/ C ullin-1/ F -box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box-binding domain. Prolyl hydroxylation of Skp1 contributes to O 2 -dependent Dictyostelium development, but full glycosylation at that position is required for optimal O 2 sensing. Previous studies have shown that the glycan promotes organization of the F-box-binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box-binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box-combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 Linked Disaccharides Via Tandem Mass Spectrometry and 18O-labeling

NASA Astrophysics Data System (ADS)

Konda, Chiharu; Bendiak, Brad; Xia, Yu

2012-02-01

Collision-induced dissociation (CID) of deprotonated hexose-containing disaccharides ( m/z 341) with 1-2, 1-4, and 1-6 linkages yields product ions at m/z 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. However, only trace quantities of m/z 221 ions can be generated for 1-3 linkages in Paul or linear ion traps, preventing further CID analysis. Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3) from beam-type CID of a series of 1-3 linked disaccharides conducted on a triple quadrupole/linear ion trap mass spectrometer. 18O-labeling at the carbonyl position of the reducing sugar allowed mass-discrimination of the "sidedness" of dissociation events to either side of the glycosidic linkage. Under relatively low energy beam-type CID and ion trap CID, an m/z 223 product ion containing 18O predominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Under higher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have the glycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns were observed for each m/z 221 ion from higher energy beam-type CID of 1-3 linked disaccharides and the stereochemistry of the non-reducing sugar, together with the anomeric configuration, were successfully identified both with and without 18O-labeling of the reducing sugar carbonyl group.

Mapping the Relationship between Glycosyl Acceptor Reactivity and Glycosylation Stereoselectivity.

PubMed

van der Vorm, Stefan; van Hengst, Jacob M A; Bakker, Marloes; Overkleeft, Herman S; van der Marel, Gijsbert A; Codée, Jeroen D C

2018-03-30

The reactivity of both coupling partners-the glycosyl donor and acceptor-is decisive for the outcome of a glycosylation reaction, in terms of both yield and stereoselectivity. Where the reactivity of glycosyl donors is well understood and can be controlled through manipulation of the functional/protecting-group pattern, the reactivity of glycosyl acceptor alcohols is poorly understood. We here present an operationally simple system to gauge glycosyl acceptor reactivity, which employs two conformationally locked donors with stereoselectivity that critically depends on the reactivity of the nucleophile. A wide array of acceptors was screened and their structure-reactivity/stereoselectivity relationships established. By systematically varying the protecting groups, the reactivity of glycosyl acceptors can be adjusted to attain stereoselective cis-glucosylations. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins--towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation.

PubMed

Daskalova, Sasha M; Radder, Josiah E; Cichacz, Zbigniew A; Olsen, Sam H; Tsaprailis, George; Mason, Hugh; Lopez, Linda C

2010-08-24

Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform. Plant

Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans

PubMed Central

De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R.; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G.; Van Etten, James L.

2013-01-01

The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms. PMID:23918378

Versatile On-Resin Synthesis of High Mannose Glycosylated Asparagine with Functional Handles

PubMed Central

Chen, Rui; Pawlicki, Mark A.; Tolbert, Thomas J.

2013-01-01

Here we present a synthetic route for solid phase synthesis of N-linked glycoconjugates containing high mannose oligosaccharides which allows the incorporation of useful functional handles on the N-terminus of asparagine. In this strategy, the C-terminus of an Fmoc protected aspartic acid residue is first attached to a solid phase support. The side chain of aspartic acid is protected by a 2-phenylisopropyl protecting group, which allows selective deprotection for the introduction of glycosylation. By using a convergent on-resin glycosylamine coupling strategy, an N-glycosidic linkage is successfully formed on the free side chain of the resin bound aspartic acid with a large high mannose oligosaccharide, Man8GlcNAc2, to yield N-linked high mannose glycosylated asparagine. The use of on-resin glycosylamine coupling provides excellent glycosylation yield, can be applied to couple other types of oligosaccharides, and also makes it possible to recover excess oligosaccharides conveniently after the on-resin coupling reaction. Useful functional handles including an alkene (p-vinylbenzoic acid), an alkyne (4-pentynoic acid), biotin, and 5-carboxyfluorescein are then conjugated onto the N-terminal amine of asparagine on-resin after the removal of the Fmoc protecting group. In this way, useful functional handles are introduced onto the glycosylated asparagine while maintaining the structural integrity of the reducing end of the oligosaccharide. The asparagine side chain also serves as a linker between the glycan and the functional group and preserves the native presentation of N-linked glycan which may aid in biochemical and structural studies. As an example of a biochemical study using functionalized high mannose glycosylated asparagine, a fluorescence polarization assay has been utilized to study the binding of the lectin Concanavalin A (ConA) using 5-carboxyfluorescein labeled high mannose glycosylated asparagine. PMID:24326091

Mutation of NgBR, a subunit of cis-prenyltransferase, causes a congenial disorder of glycosylation

PubMed Central

Park, Eon Joo; Grabińska, Kariona A.; Guan, Ziqiang; Stránecký, Viktor; Hartmannová, Hana; Hodaňová, Kateřina; Barešová, Veronika; Sovová, Jana; Jozsef, Levente; Ondrušková, Nina; Hansíková, Hana; Honzík, Tomáš; Zeman, Jiří; Hůlková, Helena; Wen, Rong; Kmoch, Stanislav; Sessa, William C.

2014-01-01

Summary Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. Cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provides a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation. PMID:25066056

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.

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors

PubMed Central

Vitiazeva, Varvara; Kattla, Jayesh J.; Flowers, Sarah A.; Lindén, Sara K.; Premaratne, Pushpa; Weijdegård, Birgitta; Sundfeldt, Karin; Karlsson, Niclas G.

2015-01-01

Background Mucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer. Methods In this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant) and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC) coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSn). The LC-ESI-MSn of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes. Results The obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline) and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC). Conclusion Mucinous benign and LMPs along with mucinous low-grade carcinomas

Regulation of TNF-Related Apoptosis-Inducing Ligand Signaling by Glycosylation

PubMed Central

2018-01-01

Tumor necrosis-factor related apoptosis-inducing ligand, also known as TRAIL or APO2L (Apo-2 ligand), is a cytokine of the TNF superfamily acknowledged for its ability to trigger selective apoptosis in tumor cells while being relatively safe towards normal cells. Its binding to its cognate agonist receptors, namely death receptor 4 (DR4) and/or DR5, can induce the formation of a membrane-bound macromolecular complex, coined DISC (death-signaling inducing complex), necessary and sufficient to engage the apoptotic machinery. At the very proximal level, TRAIL DISC formation and activation of apoptosis is regulated both by antagonist receptors and by glycosylation. Remarkably, though, despite the fact that all membrane-bound TRAIL receptors harbor putative glycosylation sites, only pro-apoptotic signaling through DR4 and DR5 has, so far, been found to be regulated by N- and O-glycosylation, respectively. Because putative N-glycosylation sequons and O-glycosylation sites are also found and conserved in all these receptors throughout all animal species (in which these receptors have been identified), glycosylation is likely to play a more prominent role than anticipated in regulating receptor/receptor interactions or trafficking, ultimately defining cell fate through TRAIL stimulation. This review aims to present and discuss these emerging concepts, the comprehension of which is likely to lead to innovative anticancer therapies. PMID:29498673

Expression of cancer-associated simple mucin-type O-glycosylated antigens in parasites.

PubMed

Osinaga, Eduardo

2007-01-01

Simple mucin-type O-glycan structures, such as Tn, TF, sialyl-Tn and Tk antigens, are among of the most specific human cancer-associated structures. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. In the last few years several simple mucin-type O-glycan antigens were identified in different species belonging to the main two helminth parasite phyla, and sialyl-Tn bearing glycoproteins were detected in Trypanosoma cruzi. These results are of interest to understand new aspects in parasite glycoimmunology and may help identify new biological characteristics of parasites as well of the host-parasite relationship. Considering that different groups reported a negative correlation between certain parasite infections and cancer development, we could hypothesize that simple mucin-type O-glycosylated antigens obtained from parasites could be good potential targets for cancer immunotherapy.

Bufo arenarum egg jelly coat: purification and characterization of two highly glycosylated proteins.

PubMed Central

Arranz, S E; Albertali, I E; Cabada, M O

1997-01-01

Egg jelly coats from Bufo arenarum are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they transit along the different oviductal portions. In this study, we have isolated two highly glycosylated proteins of the jelly coat, which are secreted almost all the way along the oviduct. Both glycoproteins [designated as highly glycosylated protein (HGP) and low-molecular-mass highly glycosylated protein (L-HGP)] were purified to homogeneity, from the secretion of the caudal oviduct portion, by CsCl density gradient ultracentrifugation. HGP is a high-molecular-mass protein with mucin-like characteristics: high viscosity, a high content of serine and threonine, about 70% carbohydrate by weight, and a protease-resistant domain. Cleavage of disulphide bridges with reducing agents resulted in the release of a single subunit (300000 Da). L-HGP is also a disulphide-cross-linked protein with lower apparent monomeric molecular mass, in the range 100-120 kDa and containing 50% carbohydrate by weight. HGP contains galactose, fucose, N-acetylgalactosamine and sialic acid, but no mannose, suggesting the presence of O-linked oligosaccharides exclusively. The secretion ratio of HGP increases from cephalic (16% of total protein in pars preconvoluta) to caudal (40% of total protein in pars convoluta) oviductal portions. It appears to be the major structural component of the jelly coat. Our purification data suggest that HGP is non-covalently linked to the other egg jelly proteins. Polyclonal antiserum to each purified glycoprotein from secretion was raised in rabbits and used to localize both glycoproteins in the different oviductal portions, total egg jelly and the aqueous medium where oocyte strings were incubated. HGP forms a stable fibre matrix around the oocyte. L-HGP is present in the jelly coat and is released into the incubation medium. PMID:9173897

Building blocks for the synthesis of glycosyl-myo-inositols involved in the insulin intracellular signalling process.

PubMed

Zapata, A; Martín-Lomas, M

1992-10-09

Glycosylation of (+/- )-1-O-benzyl-2,3:5,6-di-O-isopropylidene-myo-inositol (4) with 6-O-acetyl-4-O-allyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranosyl trichloroacetimidate (6) gave the 4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)- myo-inositol derivative (9) as a mixture of diastereoisomers which could be resolved by chromatography. Likewise alpha-glycosylation of 4 with 6-O-acetyl-2-azido-3-O-benzoyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta- D- galactopyranosyl)-D-glucopyranosyl trichloroacetimidate (10) gave the corresponding pseudotrisaccharide derivative 16 as a mixture of diastereomers which could be resolved partially by chromatography. alpha-Glycosylation of enantiomerically pure 2,3:5,6- (18) and 2,3:4,5-di-O-isopropylidene-1-O-menthoxycarbonyl-myo-inositol (19) with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate (20) gave the pseudodisaccharide derivatives 21 and 22, respectively. Likewise, alpha-glycosylation of 18 with 10 afforded a pseudotrisaccharide derivative (23).

Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction.

PubMed

Ding, Ning; Yang, Chunguang; Sun, Shenxia; Han, Lichi; Ruan, Yao; Guo, Longhua; Hu, Xuejun; Zhang, Jianing

2017-03-25

Escherichia coli cells have been considered as promising hosts for producing N-glycosylated proteins since the successful production of N-glycosylated protein in E. coli with the pgl (N-linked protein glycosylation) locus from Campylobacter jejuni. However, one hurdle in producing N-glycosylated proteins in large scale using E. coli is inefficient glycan glycosylation. In this study, we developed a strategy for the production of N-glycosylated proteins with high efficiency via an optimized auto-induction method. The 10th human fibronectin type III domain (FN3) was engineered with native glycosylation sequon DFNRSK and optimized DQNAT sequon in C-terminus with flexible linker as acceptor protein models. The resulting glycosylation efficiencies were confirmed by Western blots with anti-FLAG M1 antibody. Increased efficiency of glycosylation was obtained by changing the conventional IPTG induction to auto-induction method, which increased the glycosylation efficiencies from 60% and 75% up to 90% and 100% respectively. Moreover, in the condition of inserting the glycosylation sequon in the loop of FN3 (the acceptor sequon with local structural conformation), the glycosylation efficiency was increased from 35% to 80% by our optimized auto-induction procedures. To justify the potential for general application of the optimized auto-induction method, the reconstituted lsg locus from Haemophilus influenzae and PglB from C. jejuni were utilized, and this led to 100% glycosylation efficiency. Our studies provided quantitative evidence that the optimized auto-induction method will facilitate the large-scale production of pure exogenous N-glycosylation proteins in E. coli cells. Copyright © 2017 Elsevier Inc. All rights reserved.

A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein.

PubMed

Crimmins, Dan L; Kao, Jeffrey L-F

2008-07-01

The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles' heel of immunoassays in general. A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays.

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

PubMed

Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

2013-02-01

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts. Copyright © 2012 Elsevier B.V. All rights reserved.

Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions.

PubMed

Park, Yongho; Harper, Kaid C; Kuhl, Nadine; Kwan, Eugene E; Liu, Richard Y; Jacobsen, Eric N

2017-01-13

Carbohydrates are involved in nearly all aspects of biochemistry, but their complex chemical structures present long-standing practical challenges to their synthesis. In particular, stereochemical outcomes in glycosylation reactions are highly dependent on the steric and electronic properties of coupling partners; thus, carbohydrate synthesis is not easily predictable. Here we report the discovery of a macrocyclic bis-thiourea derivative that catalyzes stereospecific invertive substitution pathways of glycosyl chlorides. The utility of the catalyst is demonstrated in the synthesis of trans-1,2-, cis-1,2-, and 2-deoxy-β-glycosides. Mechanistic studies are consistent with a cooperative mechanism in which an electrophile and a nucleophile are simultaneously activated to effect a stereospecific substitution reaction. Copyright © 2017, American Association for the Advancement of Science.

Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, causes a congenital disorder of glycosylation.

PubMed

Park, Eon Joo; Grabińska, Kariona A; Guan, Ziqiang; Stránecký, Viktor; Hartmannová, Hana; Hodaňová, Kateřina; Barešová, Veronika; Sovová, Jana; Jozsef, Levente; Ondrušková, Nina; Hansíková, Hana; Honzík, Tomáš; Zeman, Jiří; Hůlková, Helena; Wen, Rong; Kmoch, Stanislav; Sessa, William C

2014-09-02

Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice, and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provide a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection of site specific glycosylation in proteins using flow cytometry†

PubMed Central

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation.

PubMed

Peng, Changmin; Zhu, Yue; Zhang, Wanjun; Liao, Qinchao; Chen, Yali; Zhao, Xinyuan; Guo, Qiang; Shen, Pan; Zhen, Bei; Qian, Xiaohong; Yang, Dong; Zhang, Jin-San; Xiao, Dongguang; Qin, Weijie; Pei, Huadong

2017-11-02

The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

PubMed

Gurung, Rit Bahadur; Gong, So Youn; Dhakal, Dipesh; Le, Tuoi Thi; Jung, Na Rae; Jung, Hye Jin; Oh, Tae Jin; Sohng, Jae Kyung

2017-09-28

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, antiinflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP- α-D-glucose or UDP-α-D-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'- O -β- glucoside, curcumin 4',4''-di- O -β-glucoside, curcumin 4'- O -β-2-deoxyglucoside, and curcumin 4',4''-di- O -β-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'- O -β-glucoside and curcumin 4'- O -β-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

Hypoxia-elicited impairment of cell wall integrity, glycosylation precursor synthesis, and growth in scaled-up high-cell density fed-batch cultures of Saccharomyces cerevisiae.

PubMed

Aon, Juan C; Sun, Jianxin; Leighton, Julie M; Appelbaum, Edward R

2016-08-15

In this study we examine the integrity of the cell wall during scale up of a yeast fermentation process from laboratory scale (10 L) to industrial scale (10,000 L). In a previous study we observed a clear difference in the volume fraction occupied by yeast cells as revealed by wet cell weight (WCW) measurements between these scales. That study also included metabolite analysis which suggested hypoxia during scale up. Here we hypothesize that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance, which in turn may lead to the observed difference in WCW. We tested the cell wall integrity by probing the cell wall sensitivity to Zymolyase. Also exometabolomics data showed changes in supply of precursors for the glycosylation pathway. The results show a more sensitive cell wall later in the production process at industrial scale, while the sensitivity at early time points was similar at both scales. We also report exometabolomics data, in particular a link with the protein glycosylation pathway. Significantly lower levels of Man6P and progressively higher GDP-mannose indicated partially impaired incorporation of this sugar nucleotide during co- or post-translational protein glycosylation pathways at the 10,000 L compared to the 10 L scale. This impairment in glycosylation would be expected to affect cell wall integrity. Although cell viability from samples obtained at both scales were similar, cells harvested from 10 L bioreactors were able to re-initiate growth faster in fresh shake flask media than those harvested from the industrial scale. The results obtained help explain the WCW differences observed at both scales by hypoxia-triggered weakening of the yeast cell wall during the scale up.

Diversity and functions of protein glycosylation in insects.

PubMed

Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

NASA Astrophysics Data System (ADS)

Zhu, Zhikai; Go, Eden P.; Desaire, Heather

2014-06-01

N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.

PubMed

Biswas, Himadri; Chattopadhyaya, Rajagopal

2016-04-01

Curcuma longa rhizome lectin, a mannose-binding protein of non-seed portions of turmeric, is known to have antifungal, antibacterial and α-glucosidase inhibitory activities. We studied the role of complex-type glycans attached to asparagine (Asn) 66 and Asn 110 to elucidate the role of carbohydrates in lectin activity and stability. Apart from the native lectin, the characteristics of a deglycosylated Escherichia coli expressed lectin, high-mannose oligosaccharides at both asparagines and its glycosylation mutants N66Q and N110Q expressed in Pichia pastoris, were compared to understand the relationship between glycosylation and activity. Far UV circular dichroism (CD) spectra, fluorescence emission maximum, hemagglutination assay show no change in secondary or tertiary structures or sugar-binding properties between wild-type and aforementioned recombinant lectins under physiological pH. But reduced agglutination activity and loss of tertiary structure are observed in the acidic pH range for the deglycosylated and the N110Q protein. In thermal and guanidine hydrochloride (GdnCl)-induced unfolding, the wild-type and high-mannose lectins possess higher stability compared with the deglycosylated recombinant lectin and both mutants, as measured by a higher Tm of denaturation or a greater free energy change, respectively. Reversibility experiments after thermal denaturation reveal that deglycosylated proteins tend to aggregate during thermal inactivation but the wild type shows a much greater recovery to the native state upon refolding. These results suggest that N-glycosylation in turmeric lectin is important for the maintenance of its proper folding upon changes in pH, and that the oligosaccharides help in maintaining the active conformation and prevent aggregation in unfolded or partially folded molecules. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

NASA Astrophysics Data System (ADS)

Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

2016-07-01

Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

Comprehensive identification of novel proteins and N-glycosylation sites in royal jelly

PubMed Central

2014-01-01

Background Royal jelly (RJ) is a proteinaceous secretion produced from the hypopharyngeal and mandibular glands of nurse bees. It plays vital roles in honeybee biology and in the improvement of human health. However, some proteins remain unknown in RJ, and mapping N-glycosylation modification sites on RJ proteins demands further investigation. We used two different liquid chromatography-tandem mass spectrometry techniques, complementary N-glycopeptide enrichment strategies, and bioinformatic approaches to gain a better understanding of novel and glycosylated proteins in RJ. Results A total of 25 N-glycosylated proteins, carrying 53 N-glycosylation sites, were identified in RJ proteins, of which 42 N-linked glycosylation sites were mapped as novel on RJ proteins. Most of the glycosylated proteins were related to metabolic activities and health improvement. The 13 newly identified proteins were also mainly associated with metabolic processes and health improvement activities. Conclusion Our in-depth, large-scale mapping of novel glycosylation sites represents a crucial step toward systematically revealing the functionality of N-glycosylated RJ proteins, and is potentially useful for producing a protein with desirable pharmacokinetic and biological activity using a genetic engineering approach. The newly-identified proteins significantly extend the proteome coverage of RJ. These findings contribute vital and new knowledge to our understanding of the innate biochemical nature of RJ at both the proteome and glycoproteome levels. PMID:24529077

Analysis of urinary PSA glycosylation is not indicative of high-risk prostate cancer.

PubMed

Barrabés, Sílvia; Llop, Esther; Ferrer-Batallé, Montserrat; Ramírez, Manel; Aleixandre, Rosa N; Perry, Antoinette S; de Llorens, Rafael; Peracaula, Rosa

2017-07-01

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA. Copyright © 2017 Elsevier B.V. All rights reserved.

Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.

PubMed Central

Bäckström, Malin; Link, Thomas; Olson, Fredrik J; Karlsson, Hasse; Graham, Rosalind; Picco, Gianfranco; Burchell, Joy; Taylor-Papadimitriou, Joyce; Noll, Thomas; Hansson, Gunnar C

2003-01-01

We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer. PMID:12950230

Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics.

PubMed

Costa, Céu; Pereira, Sofia; Lima, Luís; Peixoto, Andreia; Fernandes, Elisabete; Neves, Diogo; Neves, Manuel; Gaiteiro, Cristiana; Tavares, Ana; Gil da Costa, Rui M; Cruz, Ricardo; Amaro, Teresina; Oliveira, Paula A; Ferreira, José Alexandre; Santos, Lúcio L

2015-01-01

Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing.

PubMed

Brown, P H; Hickman, S

1986-02-25

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.

Pathways linking parental divorce with adolescent depression.

PubMed

Aseltine, R H

1996-06-01

This article examines the intervening pathways linking parental divorce with adolescent depression, using both cross-sectional and prospective data from a study of high school students in the Boston metropolitan area. Overall, findings reveal that parental divorce is linked with adolescent depression in two ways: (1) it is a source of numerous secondary problems and stresses that are causally related to depression, and (2) it alters youths' reactivity to these stresses, in some cases enhancing, but in other cases mitigating, their depressive effects. Analyses demonstrated the central role of economic hardship in linking family status with depression, with the strength of this indirect pathway partly attributable to the greater vulnerability of youths in single-parent families to financial stresses. In contrast, family conflict did not account for the distress of youths in single-parent families, largely because of their immunity to the effects of such conflict. Finally, prospective data failed to support the hypothesis that differences between youths in single-parent and intact families predate the divorce.

Glycosylated Metal Phthalocyanines.

PubMed

Hanack, Michael

2015-11-10

In the first part; the syntheses of mono-; di-; and tetra-glycosylated phthalonitriles is described; which are the most used starting materials for the preparation of the corresponding glycosylated metal (mostly zinc) phthalocyanines. In the second section; the preparation of symmetric and unsymmetric mono-; tetra-; and octa- glycosylated zinc phthalocyanines are reviewed; in which the sugar is attached to the phthalocyanine macrocycle; either anomerically or via another one of its OH-groups.

Role of Flippases in Protein Glycosylation in the Endoplasmic Reticulum

PubMed Central

Rush, Jeffrey S.

2015-01-01

Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane. The glycans are first assembled as precursor glycolipids, on the cytosolic surface of the ER, which are tethered to the membrane by attachment to a long-chain polyisoprenyl phosphate (dolichol) containing a reduced α-isoprene. The lipid-anchored building blocks then migrate transversely (flip) across the ER membrane to the lumenal surface, where final assembly of the glycan is completed. This strategy allows the cell to export high-energy biosynthetic intermediates as lipid-bound glycans, while constraining the glycosyl donors to the site of assembly on the membrane surface. This review focuses on the flippases that participate in protein glycosylation in the ER. PMID:26917968

Site-specific characterization of envelope protein N-glycosylation on Sanofi Pasteur's tetravalent CYD dengue vaccine.

PubMed

Dubayle, Jean; Vialle, Sandrine; Schneider, Diane; Pontvianne, Jérémy; Mantel, Nathalie; Adam, Olivier; Guy, Bruno; Talaga, Philippe

2015-03-10

Recently, several virus studies have shown that protein glycosylation play a fundamental role in the virus-host cell interaction. Glycosylation characterization of the envelope proteins in both insect and mammalian cell-derived dengue virus (DENV) has established that two potential glycosylation residues, the asparagine 67 and 153 can potentially be glycosylated. Moreover, it appears that the glycosylation of these two residues can influence dramatically the virus production and the infection spreading in either mosquito or mammalian cells. The Sanofi Pasteur tetravalent dengue vaccine (CYD) consists of four chimeric viruses produced in mammalian vero cells. As DENV, the CYDs are able to infect human monocyte-derived dendritic cells in vitro via C-type lectins cell-surface molecules. Despite the importance of this interaction, the specific glycosylation pattern of the DENV has not been clearly documented so far. In this paper, we investigated the structure of the N-linked glycans in the four CYD serotypes. Using MALDI-TOF analysis, the N-linked glycans of CYDs were found to be a mix of high-mannose, hybrid and complex glycans. Site-specific N-glycosylation analysis of CYDs using nanoLC-ESI-MS/MS demonstrates that both asparagine residues 67 and 153 are glycosylated. Predominant glycoforms at asparagine 67 are high mannose-type structures while mainly complex- and hybrid-type structures are detected at asparagine 153. In vitro studies have shown that the immunological consequences of infection by the CYD dengue viruses 1-4 versus the wild type parents are comparable in human monocyte-derived dendritic cells. Our E-protein glycan characterizations of CYD are consistent with those observations from the wild type parents and thus support in vitro studies. In addition, these data provide new insights for the role of glycans in the dengue virus-host cell interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biosynthetic maturation of an ascites tumor cell surface sialomucin. Evidence for O-glycosylation of cell surface glycoprotein by the addition of new oligosaccharides during recycling.

PubMed

Hull, S R; Sugarman, E D; Spielman, J; Carraway, K L

1991-07-25

Previous biosynthetic studies of the ascites 13762 rat mammary adenocarcinoma cell surface sialomucin ASGP-1 (ascites sialoglycoprotein-1) showed that it is synthesized initially as a poorly glycosylated immature form, which is converted to a larger premature form (t1/2 30 min) and more slowly to the mature glycoprotein (t1/2 greater than 4 h). In the present study O-glycosylation of ASGP-1 polypeptide is shown to occur in two phases: an early phase complete in less than 30 min, which corresponds to the synthesis of the premature form, and a later phase that continues for hours and corresponds to the synthesis of the mature form. Pulse-chase labeling studies indicate that 95% of the ASGP-1 has moved to the cell surface in 2 h. Since transit to the cell surface is faster than the slow phase of addition of new oligosaccharides, some new oligosaccharides must be added after ASGP-1 has reached the cell surface. Initiation of new oligosaccharides on cell surface ASGP-1 was demonstrated directly using a biotinylation procedure to identify cell surface molecules. Glucosamine labeling of biotinylated ASGP-1 was shown to occur on galactosamine residues, which are linked to the polypeptide, establishing the addition of new oligosaccharides to the cell surface molecules. Finally, resialylation studies indicate that ASGP-1 rapidly recycles through a sialylating compartment. From these results we propose that ASGP-1 reaches the cell surface in an incompletely glycosylated state and that additional oligosaccharides are added to the glycoprotein in a second process involving recycling.

Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos.

PubMed

Ying, Jiezheng; Zhao, Juan; Hou, Yuxuan; Wang, Yifeng; Qiu, Jiehua; Li, Zhiyong; Tong, Xiaohong; Shi, Zhaomei; Zhu, Jun; Zhang, Jian

2017-01-01

Germination is a key event in the angiosperm life cycle. N-glycosylation of proteins is one of the most common post-translational modifications, and has been recognized to be an important regulator of the proteome of the germinating embryo. Here, we report the first N-linked glycosites mapping of rice embryos during germination by using a hydrophilic interaction chromatography (HILIC) glycopeptides enrichment strategy associated with high accuracy mass spectrometry identification. A total of 242 glycosites from 191 unique proteins was discovered. Inspection of the motifs and sequence structures involved suggested that all the glycosites were concentrated within [NxS/T] motif, while 82.3% of them were in a coil structure. N-glycosylation preferentially occurred on proteins with glycoside hydrolase activities, which were significantly enriched in the starch and sucrose metabolism pathway, suggesting that N-glycosylation is involved in embryo germination by regulating carbohydrate metabolism. Notably, protein-protein interaction analysis revealed a network with several Brassinosteroids signaling proteins, including XIAO and other BR-responsive proteins, implying that glycosylation-mediated Brassinosteroids signaling may be a key mechanism regulating rice embryo germination. In summary, this study expanded our knowledge of protein glycosylation in rice, and provided novel insight into the PTM regulation in rice seed germination.

Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos

PubMed Central

Hou, Yuxuan; Wang, Yifeng; Qiu, Jiehua; Li, Zhiyong; Tong, Xiaohong; Shi, Zhaomei; Zhu, Jun

2017-01-01

Germination is a key event in the angiosperm life cycle. N-glycosylation of proteins is one of the most common post-translational modifications, and has been recognized to be an important regulator of the proteome of the germinating embryo. Here, we report the first N-linked glycosites mapping of rice embryos during germination by using a hydrophilic interaction chromatography (HILIC) glycopeptides enrichment strategy associated with high accuracy mass spectrometry identification. A total of 242 glycosites from 191 unique proteins was discovered. Inspection of the motifs and sequence structures involved suggested that all the glycosites were concentrated within [NxS/T] motif, while 82.3% of them were in a coil structure. N-glycosylation preferentially occurred on proteins with glycoside hydrolase activities, which were significantly enriched in the starch and sucrose metabolism pathway, suggesting that N-glycosylation is involved in embryo germination by regulating carbohydrate metabolism. Notably, protein-protein interaction analysis revealed a network with several Brassinosteroids signaling proteins, including XIAO and other BR-responsive proteins, implying that glycosylation-mediated Brassinosteroids signaling may be a key mechanism regulating rice embryo germination. In summary, this study expanded our knowledge of protein glycosylation in rice, and provided novel insight into the PTM regulation in rice seed germination. PMID:28328971

O-GlcNAc in cancer: An Oncometabolism-fueled vicious cycle.

PubMed

Hanover, John A; Chen, Weiping; Bond, Michelle R

2018-06-01

Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueled by oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glycosylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of key targets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymes of intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription, signaling, proteostasis, and bioenergetics, key 'hallmarks of cancer'. In this review, we summarize accumulating evidence that many cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrient and oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis, Kreb's cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a 'vicious cycle'. Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. The elevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selective advantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

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

Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structuralmore » information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.« less

Alterations in protein glycosylation in PMA-differentiated U-937 cells exposed to mineral particles.

PubMed Central

Trabelsi, N; Greffard, A; Pairon, J C; Bignon, J; Zanetti, G; Fubini, B; Pilatte, Y

1997-01-01

Carbohydrate moieties of cell glycoconjugates play a pivotal role in molecular recognition phenomena involved in the regulation of most biological systems and the changes observed in cell surface carbohydrates during cell activation or differentiation frequently modulate certain cell functions. Consequently, some aspects of macrophage response to particle exposure might conceivably result from alterations in glycosylation. Therefore, the effect of mineral particles on protein glycosylation was investigated in phorbol myristate acetate (PMA)-differentiated U-937. Jacalin, a lectin specific for O-glycosylated structures, showed a global increase in O-glycosylation in particle-treated cells. In contrast, no significant modifications were observed with concanavalin A, a lectin that recognizes certain N-glycosylated structures. The sialic acid-specific lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin and the galactose-specific lectin Ricinus communis agglutinin revealed a complex pattern of alterations in glycoprotein glycosylation after crystalline silica or manganese dioxide treatments. Expression of sialyl Lewis(x), a glycosylated structure implicated in leukocyte trafficking, could not be detected in control or treated cells. This finding was consistent with the decrease in sialyl Lewis(x) expression observed during PMA-induced differentiation. In conclusion, various treatments used in this study induced quantitative as well as qualitative changes in protein glycosylation. Whether these changes are due to glycosidase release or to an alteration in glycosyltransferase expression remains to be determined. The potential functional implications of these changes are currently under investigation. Images Figure 1. A Figure 1. B Figure 2. A Figure 2. B Figure 3. A Figure 3. B Figure 3. C Figure 4. PMID:9400716

N-O linkage in carbohydrates and glycoconjugates.

PubMed

Chen, N; Xie, J

2016-11-29

The importance of oligosaccharides and their conjugates in various biological and pathological processes has stimulated growing interest in the development of (neo)glycoconjugates. Thanks to its high nucleophilicity, hydroxylamine has been employed as a powerful chemoselective ligation tool. Great effort has been focused on carbohydrates bearing aminooxy or N-hydroxy amino groups for organic synthesis, glycobiology and drug discovery. This review provides an overview of N-O linked carbohydrates and glycoconjugates, focusing particularly on the synthetic methodologies and chemical and physicochemical properties as well as biological and medical applications of N-glycosyl and O-glycosyl hydroxylamines, N-hydroxy amino and O-amino sugar as well as sugar aminooxy acid derivatives.

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

Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors.

PubMed

Zhan, Jinghui; Felder, Barbara; Ellison, Aaron R; Winters, Aaron; Salimi-Moosavi, Hossein; Scully, Sheila; Turk, James R; Wei, Ping

2013-06-01

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.

Variable domain glycosylation of ACPA-IgG: A missing link in the maturation of the ACPA response?

PubMed

Kempers, Ayla C; Hafkenscheid, Lise; Scherer, Hans Ulrich; Toes, René E M

2018-01-01

Anti-citrullinated Protein Antibodies (ACPA) are excellent markers for Rheumatoid arthritis (RA) and are postulated to have a pathogenic role in the disease process. A multistep model for the evolution of the ACPA response in RA was proposed in which an initial break of tolerance causes, as "first hit", "silent" production of ACPA without any clinical symptoms. The model further proposes that the ACPA immune response matures upon a certain (unknown) trigger, a "second hit", which leads to epitope spreading, an increase in ACPA titres and extended isotype usage before clinical RA manifestations. These occurrences are indicative of an expansion of the citrulline-specific B cell response, though ACPA remain of low avidity even in established disease. This persistence of low avidity is puzzling, as the typical signs of maturation of the immune response seem to be uncoupled from the classical process of affinity maturation. In fact, it suggests that B cells expressing ACPA could bypass selection mechanisms that otherwise control the expansion of auto-reactive B cells. In the established, chronic phase, we recently found that ACPA-IgG are extensively glycosylated in the variable (Fab) domain. More than 90% of ACPA-IgG molecules carry Fab glycans that are highly sialylated. This molecular feature is striking and may provide a missing link in our understanding of the maturation of the ACPA immune response. This review, therefore, describes the current knowledge about ACPA Fab glycosylation in the pathogenesis of RA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

O-Linked-N-Acetylglucosamine Cycling and Insulin Signaling Are Required for the Glucose Stress Response in Caenorhabditis elegans

PubMed Central

Mondoux, Michelle A.; Love, Dona C.; Ghosh, Salil K.; Fukushige, Tetsunari; Bond, Michelle; Weerasinghe, Gayani R.; Hanover, John A.; Krause, Michael W.

2011-01-01

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose–insulin homeostasis. PMID:21441213

Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

PubMed

Richard, S; Tamas, C; Sell, D R; Monnier, V M

1991-08-01

Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

Diversity in Protein Glycosylation among Insect Species

PubMed Central

Vandenborre, Gianni; Smagghe, Guy; Ghesquière, Bart; Menschaert, Gerben; Nagender Rao, Rameshwaram; Gevaert, Kris; Van Damme, Els J. M.

2011-01-01

Background A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. Methodology/Principal Findings In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin) affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum), the silkworm (Bombyx mori), the honeybee (Apis mellifera), the fruit fly (D. melanogaster) and the pea aphid (Acyrthosiphon pisum). To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. Conclusions/Significance The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed. PMID:21373189

N-linked glycan truncation causes enhanced clearance of plasma-derived von Willebrand factor.

PubMed

O'Sullivan, J M; Aguila, S; McRae, E; Ward, S E; Rawley, O; Fallon, P G; Brophy, T M; Preston, R J S; Brady, L; Sheils, O; Chion, A; O'Donnell, J S

2016-12-01

Essentials von Willebrands factor (VWF) glycosylation plays a key role in modulating in vivo clearance. VWF glycoforms were used to examine the role of specific glycan moieties in regulating clearance. Reduction in sialylation resulted in enhanced VWF clearance through asialoglycoprotein receptor. Progressive VWF N-linked glycan trimming resulted in increased macrophage-mediated clearance. Click to hear Dr Denis discuss clearance of von Willebrand factor in a free presentation from the ISTH Academy SUMMARY: Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF -/- mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis.

PubMed

Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

2015-01-01

Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N=66) and prostatitis patients (N=36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (P<0.001). Urinary bacteria count allowed for discriminating prostatitis patients from HV (P<0.001). Total amount of biantennary structures (urinary 2A/MA marker) was significantly lower in prostatitis patients compared to HV (P<0.001). Combining the urinary 2A/MA marker and urinary WBC count resulted in an AUC of 0.79, 95% confidence interval (CI)=(0.70-0.89) which was significantly better than urinary WBC count (AUC=0.70, 95% CI=[0.59-0.82], P=0.042) as isolated test. We have demonstrated the diagnostic value of urinary N-glycosylation profiling, which shows great potential as biomarker for prostatitis. Further research is required to unravel the developmental course of prostatic inflammation.

N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity.

PubMed

Dotsenko, Anna S; Gusakov, Alexander V; Volkov, Pavel V; Rozhkova, Aleksandra M; Sinitsyn, Arkady P

2016-02-01

Cellobiohydrolase I from Penicillium verruculosum (PvCel7A) has four potential N-glycosylation sites at its catalytic module: Asn45, Asn194, Asn388, and Asn430. In order to investigate how the N-glycosylation influences the activity and other properties of the enzyme, the wild type (wt) PvCel7A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens PCA10 (niaD-) strain, a fungal host for production of heterologous proteins. The rPvCel7A-wt and N45A, N194A, N388A mutants were successfully expressed and purified for characterization, whereas the expression of N430A mutant was not achieved. The MALDI-TOF mass spectrometry fingerprinting of peptides, obtained as a result of digestion of rPvCel7A forms with specific proteases, showed that the N-linked glycans represent variable high-mannose oligosaccharides and the products of their sequential enzymatic trimming, according to the formula (Man)0-13 (GlcNAc)2 , or a single GlcNAc residue. Mutations had no notable effect on pH-optimum of PvCel7A activity and enzyme thermostability. However, the mutations influenced both the enzyme adsorption ability on Avicel and its activity against natural and synthetic substrates. In particular, the N45A mutation led to a significant increase in the rate of Avicel and milled aspen wood hydrolysis, while the substrate digestion rates in the case of N194A and N388A mutants were notably lower relative to rPvCel7A-wt. These data, together with data of 3D structural modeling of the PvCel7A catalytic module, indicate that the N-linked glycans are an important part of the processive catalytic machinery of PvCel7A. © 2015 Wiley Periodicals, Inc.

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating*

PubMed Central

Lo, Wen-yi; Lagrange, Andre H.; Hernandez, Ciria C.; Harrison, Rebecca; Dell, Anne; Haslam, Stuart M.; Sheehan, Jonathan H.; Macdonald, Robert L.

2010-01-01

γ-Aminobutyric acid type A (GABAA) receptors are heteropentameric glycoproteins. Based on consensus sequences, the GABAA receptor β2 subunit contains three potential N-linked glycosylation sites, Asn-32, Asn-104, and Asn-173. Homology modeling indicates that Asn-32 and Asn-104 are located before the α1 helix and in loop L3, respectively, near the top of the subunit-subunit interface on the minus side, and that Asn-173 is located in the Cys-loop near the bottom of the subunit N-terminal domain. Using site-directed mutagenesis, we demonstrated that all predicted β2 subunit glycosylation sites were glycosylated in transfected HEK293T cells. Glycosylation of each site, however, produced specific changes in α1β2 receptor surface expression and function. Although glycosylation of Asn-173 in the Cys-loop was important for stability of β2 subunits when expressed alone, results obtained with flow cytometry, brefeldin A treatment, and endo-β-N-acetylglucosaminidase H digestion suggested that glycosylation of Asn-104 was required for efficient α1β2 receptor assembly and/or stability in the endoplasmic reticulum. Patch clamp recording revealed that mutation of each site to prevent glycosylation decreased peak α1β2 receptor current amplitudes and altered the gating properties of α1β2 receptor channels by reducing mean open time due to a reduction in the proportion of long open states. In addition to functional heterogeneity, endo-β-N-acetylglucosaminidase H digestion and glycomic profiling revealed that surface β2 subunit N-glycans at Asn-173 were high mannose forms that were different from those of Asn-32 and N104. Using a homology model of the pentameric extracellular domain of α1β2 channel, we propose mechanisms for regulation of GABAA receptors by glycosylation. PMID:20639197

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

PubMed Central

Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

2012-01-01

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

PubMed Central

Mahdavi, Jafar; Pirinccioglu, Necmettin; Oldfield, Neil J.; Carlsohn, Elisabet; Stoof, Jeroen; Aslam, Akhmed; Self, Tim; Cawthraw, Shaun A.; Petrovska, Liljana; Colborne, Natalie; Sihlbom, Carina; Borén, Thomas; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

2014-01-01

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis. PMID:24451549

Glycosylation and Processing of Pro-B-type Natriuretic Peptide in Cardiomyocytes

PubMed Central

Peng, Jianhao; Jiang, Jingjing; Wang, Wei; Qi, Xiaofei; Sun, Xue-Long; Wu, Qingyu

2011-01-01

B-type natriuretic peptide (BNP) and its related peptides are biomarkers for the diagnosis of heart failure. Recent studies identified several O-glycosylation sites, including Thr-71, on human pro-BNP but the functional significance was unclear. In this study, we analyzed glycosylation and proteolytic processing of pro-BNP in cardiomyocytes. Human pro-BNP wild-type (WT) and mutants were expressed in HEK 293 cells and murine HL-1 cardiomyocytes. Pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting. Glycosidases and glycosylation inhibitors were used to examine carbohydrates on pro-BNP. The effects of furin and corin expression on pro-BNP processing in cells also were examined. We found that in HEK 293 cells, recombinant pro-BNP contained significant amounts of O-glycans with terminal oligosialic acids. Mutation at Thr-71 reduced O-glycans on pro-BNP and increased pro-BNP processing. In HL-1 cardiomyocytes, residue Thr-71 contained little O-glycans, and pro-BNP WT and T71A mutant were processed similarly. In HEK 293 cells, pro-BNP was processed by furin. Mutations at Arg-73 and Arg-76, but not Lys-79, prevented pro-BNP processing. In HL-1 cardiomyocytes, which express furin and corin, single or double mutations at Arg-73, Arg-76 and Lys-79 did not prevent pro-BNP processing. Only when all these three residues were mutated, was pro-BNP processing completely blocked. Our data indicate that pro-BNP glycosylation in cardiomyocytes differed significantly from that in HEK 293 cells. In HEK 293 cells, furin cleaved pro-BNP at Arg-76 whereas in cardiomyocytes corin cleaved pro-BNP at multiple residues including Arg-73, Arg-76 and Lys-79. PMID:21763278

Making Home Sweet and Sturdy: Toxoplasma gondii ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity.

PubMed

Tomita, Tadakimi; Sugi, Tatsuki; Yakubu, Rama; Tu, Vincent; Ma, Yanfen; Weiss, Louis M

2017-01-10

The protozoan intracellular parasite Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world's population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the brain cyst. Our studies provide new

Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation, Growth and Development in Arabidopsis.

PubMed

Basu, Debarati; Wang, Wuda; Ma, Siyi; DeBrosse, Taylor; Poirier, Emily; Emch, Kirk; Soukup, Eric; Tian, Lu; Showalter, Allan M

2015-01-01

Hydroxyproline-O-galactosyltransferase (GALT) initiates O-glycosylation of arabinogalactan-proteins (AGPs). We previously characterized GALT2 (At4g21060), and now report on functional characterization of GALT5 (At1g74800). GALT5 was identified using heterologous expression in Pichia and an in vitro GALT assay. Product characterization showed GALT5 specifically adds galactose to hydroxyproline in AGP protein backbones. Functions of GALT2 and GALT5 were elucidated by phenotypic analysis of single and double mutant plants. Allelic galt5 and galt2 mutants, and particularly galt2 galt5 double mutants, demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type. Mutant plants showed pleiotropic growth and development phenotypes (defects in root hair growth, root elongation, pollen tube growth, flowering time, leaf development, silique length, and inflorescence growth), which were most severe in the double mutants. Conditional mutant phenotypes were also observed, including salt-hypersensitive root growth and root tip swelling as well as reduced inhibition of pollen tube growth and root growth in response to β-Yariv reagent. These mutants also phenocopy mutants for an AGP, SOS5, and two cell wall receptor-like kinases, FEI1 and FEI2, which exist in a genetic signaling pathway. In summary, GALT5 and GALT2 function as redundant GALTs that control AGP O-glycosylation, which is essential for normal growth and development.

Biosynthesis and N-glycosylation of human interferon-gamma. Asn25 and Asn97 differ markedly in how efficiently they are glycosylated and in their oligosaccharide composition.

PubMed

Sareneva, T; Mørtz, E; Tölö, H; Roepstorff, P; Julkunen, I

1996-12-01

Interferon-gamma (IFN-gamma) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N-glycosylation, and secretion of IFN-gamma in human CD8+ T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-gamma mRNA levels were found as early as 1 h after stimulation. Maximal IFN-gamma protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-gamma mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-gamma from T cells was very rapid, the secretion half-time being approximately 20-25 min. Inhibition of N-glycosylation by tunicamycin dramatically reduced the expression of IFN-gamma, but did not block its secretion. Natural IFN-gamma is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-gamma constructs lacking either one of the potential glycosylation sites suggested that Asn25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-gamma by glycosidase treatment followed by matrix-assisted-laser-desorption-ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Asn25 consisted of fucosylated, mainly complex-type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high-mannose structures. Our results emphasize the essential role of N-linked glycans in the biology of IFN-gamma and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.

Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells.

PubMed

Nojima, Kuniharu; Hochegger, Helfrid; Saberi, Alihossein; Fukushima, Toru; Kikuchi, Koji; Yoshimura, Michio; Orelli, Brian J; Bishop, Douglas K; Hirano, Seiki; Ohzeki, Mioko; Ishiai, Masamichi; Yamamoto, Kazuhiko; Takata, Minoru; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Yamazoe, Mitsuyoshi; Kawamoto, Takuo; Araki, Kasumi; Takahashi, Jun A; Hashimoto, Nobuo; Takeda, Shunichi; Sonoda, Eiichiro

2005-12-15

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

N-Glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris.

PubMed

Zou, Shuping; Huang, Shen; Kaleem, Imdad; Li, Chun

2013-03-10

Recombinant β-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of β-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes. Copyright © 2013 Elsevier B.V. All rights reserved.

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis

PubMed Central

Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie

2015-01-01

Introduction Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Materials and methods Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N = 66) and prostatitis patients (N = 36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Results Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (P < 0.001). Urinary bacteria count allowed for discriminating prostatitis patients from HV (P < 0.001). Total amount of biantennary structures (urinary 2A/MA marker) was significantly lower in prostatitis patients compared to HV (P < 0.001). Combining the urinary 2A/MA marker and urinary WBC count resulted in an AUC of 0.79, 95% confidence interval (CI) = (0.70–0.89) which was significantly better than urinary WBC count (AUC = 0.70, 95% CI = [0.59–0.82], P = 0.042) as isolated test. Conclusions We have demonstrated the diagnostic value of urinary N-glycosylation profiling, which shows great potential as biomarker for prostatitis. Further research is required to unravel the developmental course of prostatic inflammation. PMID:26526330

N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function.

PubMed

Tamir, Adi; Eichler, Jerry

2017-03-15

N-Glycosylation, the covalent linkage of glycans to select Asn residues of target proteins, is an almost universal posttranslational modification in archaea. However, whereas roles for N-glycosylation have been defined in eukarya and bacteria, the function of archaeal N-glycosylation remains unclear. Here, the impact of perturbed N-glycosylation on the structure and physiology of the haloarchaeon Haloferax volcanii was considered. Cryo-electron microscopy was used to examine right-side-out membrane vesicles prepared from cells of a parent strain and from strains lacking genes encoding glycosyltransferases involved in assembling the N-linked pentasaccharide decorating the surface layer (S-layer) glycoprotein, the sole component of the S-layer surrounding H. volcanii cells. Whereas a regularly repeating S-layer covered the entire surface of vesicles prepared from parent strain cells, vesicles from the mutant cells were only partially covered. To determine whether such N-glycosylation-related effects on S-layer assembly also affected cell function, the secretion of a reporter protein was addressed in the parent and N-glycosylation mutant strains. Compromised S-layer glycoprotein N-glycosylation resulted in impaired transfer of the reporter past the S-layer and into the growth medium. Finally, an assessment of S-layer glycoprotein susceptibility to added proteases in the mutants revealed that in cells lacking AglD, which is involved in adding the final pentasaccharide sugar, a distinct S-layer glycoprotein conformation was assumed in which the N-terminal region was readily degraded. Perturbed N-glycosylation thus affects S-layer glycoprotein folding. These findings suggest that H. volcanii could adapt to changes in its surroundings by modulating N-glycosylation so as to affect S-layer architecture and function. IMPORTANCE Long held to be a process unique to eukaryotes, it is now accepted that bacteria and archaea also perform N-glycosylation, namely, the covalent

Multiple Pathways Linking Racism to Health Outcomes

PubMed Central

Harrell, Camara Jules P.; Burford, Tanisha I.; Cage, Brandi N.; Nelson, Travette McNair; Shearon, Sheronda; Thompson, Adrian; Green, Steven

2012-01-01

This commentary discusses advances in the conceptual understanding of racism and selected research findings in the social neurosciences. The traditional stress and coping model holds that racism constitutes a source of aversive experiences that, when perceived by the individual, eventually lead to poor health outcomes. Current evidence points to additional psychophysiological pathways linking facets of racist environments with physiological reactions that contribute to disease. The alternative pathways emphasize prenatal experiences, subcortical emotional neural circuits, conscious and preconscious emotion regulation, perseverative cognitions, and negative affective states stemming from racist cognitive schemata. Recognition of these pathways challenges change agents to use an array of cognitive and self-controlling interventions in mitigating racism’s impact. Additionally, it charges policy makers to develop strategies that eliminate deep-seated structural aspects of racism in society. PMID:22518195

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase.

PubMed

Matsumoto, Misaki; Katsuyama, Masato; Iwata, Kazumi; Ibi, Masakazu; Zhang, Jia; Zhu, Kai; Nauseef, William M; Yabe-Nishimura, Chihiro

2014-03-01

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60kDa accompanied by a broad band of 60-90kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn(161) and Asn(241) on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study. Copyright © 2013 Elsevier Inc. All rights reserved.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

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

Palmieri, D.; Valli, M.; Viglio, S.

2010-03-10

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase ofmore » maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.« less

Hallmarks of glycosylation in cancer.

PubMed

Munkley, Jennifer; Elliott, David J

2016-06-07

Aberrant glycosylation plays a fundamental role in key pathological steps of tumour development and progression. Glycans have roles in cancer cell signalling, tumour cell dissociation and invasion, cell-matrix interactions, angiogenesis, metastasis and immune modulation. Aberrant glycosylation is often cited as a 'hallmark of cancer' but is notably absent from both the original hallmarks of cancer and from the next generation of emerging hallmarks. This review discusses how glycosylation is clearly an enabling characteristic that is causally associated with the acquisition of all the hallmark capabilities. Rather than aberrant glycosylation being itself a hallmark of cancer, another perspective is that glycans play a role in every recognised cancer hallmark.

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

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

Tartaglio, Virginia; Rennie, Emilie A.; Cahoon, Rebecca

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescuemore » construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. In conclusion, this study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important role s for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.« less

Effect of alternative glycosylation on insulin receptor processing.

PubMed

Hwang, J B; Frost, S C

1999-08-06

The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two alpha- and two beta-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved alpha- and beta-subunits, migrating as a 190-kDa glycoprotein. To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation. Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization. Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into alpha- and beta-subunits (t((1)/(2)) = 1.3 +/- 0.6 h), while the alternative proreceptor was degraded (t((1)/(2)) = 5.1 +/- 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This "intermediate" form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the alpha- and beta-subunits. These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.

Increased collagen-linked pentosidine levels and advanced glycosylation end products in early diabetic nephropathy.

PubMed Central

Beisswenger, P J; Moore, L L; Brinck-Johnsen, T; Curphey, T J

1993-01-01

RATIONALE: Advanced glycosylation end products (AGEs) may play an important role in the development of diabetic vascular sequelae. An AGE cross-link, pentosidine, is a sensitive and specific marker for tissue levels of AGEs. OBJECTIVES: To evaluate the role of AGEs in the development of diabetic nephropathy and retinopathy, we studied pentosidine levels and the clinical characteristics of 48 subjects with insulin-dependent diabetes mellitus. Diabetic nephropathy was classified as normal, microalbuminuria, or gross proteinuria, and retinopathy was graded as none, background, or proliferative. NEWLY OBSERVED FINDINGS: Significant elevation of pentosidine (P = 0.025) was found in subjects with microalbuminuria or gross proteinuria (73.03 +/- 9.47 vs 76.46 +/- 6.37 pmol/mg col) when compared with normal (56.96 +/- 3.26 pmol/mg col). Multivariate analysis to correct for age, duration of diabetes, and gender did not modify the results. Elevated pentosidine levels were also found in those with proliferative when compared with those with background retinopathy (75.86 +/- 5.66 vs 60.42 +/- 5.98 pmol/mg col) (P < 0.05). CONCLUSIONS: Microalbuminuria is associated with elevated levels of pentosidine similar to those found in overt diabetic nephropathy suggesting that elevated AGE levels are already present during the earliest detectable phase of diabetic nephropathy. Images PMID:8325987

Engineering N-Glycosylation Pathway in Insect Cells: Suppression of β-N-Acetylglucosaminidase and Expression of β-1,4-Galactosyltransferase.

PubMed

Kim, Yeon Kyu; Cha, Hyung Joon

2015-01-01

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. It has been proposed that β-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation profile in several insect cells including Drosophila S2. Here, we describe GlcNAcase suppression strategy using RNA interference (RNAi) to avoid the formation of paucimannosidic glycans in insect S2 cells. In addition, we describe coexpression of β(1,4)-galactosyltransferase (GalT) as a strategy to improve N-glycosylation pattern and enable recombinant therapeutic proteins to be produced in S2 cells with more complex N-glycans.

The O-GlcNAc Modification of CDK5 Involved in Neuronal Apoptosis Following In Vitro Intracerebral Hemorrhage.

PubMed

Ning, Xiaojin; Tao, Tao; Shen, Jianhong; Ji, Yuteng; Xie, Lili; Wang, Hongmei; Liu, Ning; Xu, Xide; Sun, Chi; Zhang, Dongmei; Shen, Aiguo; Ke, Kaifu

2017-04-01

Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in regulating mammalian CNS development. Studies of CDK5 have focused on its phosphorylation, although the diversity of CDK5 functions in the brain suggests additional forms of regulation. Here we expanded on the functional roles of CDK5 glycosylation in neurons. We showed that CDK5 was dynamically modified with O-GlcNAc in response to neuronal activity and that glycosylation represses CDK5-dependent apoptosis by impairing its association with p53 pathway. Blocking glycosylation of CDK5 alters cellular function and increases neuronal apoptosis in the cell model of the ICH. Our findings demonstrated a new role for O-glycosylation in neuronal apoptosis and provided a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identified a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development, and apoptosis.

Making Home Sweet and Sturdy: Toxoplasma gondii ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity

PubMed Central

Tomita, Tadakimi; Sugi, Tatsuki; Yakubu, Rama; Tu, Vincent; Ma, Yanfen

2017-01-01

ABSTRACT The protozoan intracellular parasite Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. PMID:28074022

Hallmarks of glycosylation in cancer

PubMed Central

Munkley, Jennifer; Elliott, David J.

2016-01-01

Aberrant glycosylation plays a fundamental role in key pathological steps of tumour development and progression. Glycans have roles in cancer cell signalling, tumour cell dissociation and invasion, cell-matrix interactions, angiogenesis, metastasis and immune modulation. Aberrant glycosylation is often cited as a ‘hallmark of cancer’ but is notably absent from both the original hallmarks of cancer and from the next generation of emerging hallmarks. This review discusses how glycosylation is clearly an enabling characteristic that is causally associated with the acquisition of all the hallmark capabilities. Rather than aberrant glycosylation being itself a hallmark of cancer, another perspective is that glycans play a role in every recognised cancer hallmark. PMID:27007155

N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A.

PubMed

Yao, Guorui; Zhang, Sicai; Mahrhold, Stefan; Lam, Kwok-Ho; Stern, Daniel; Bagramyan, Karine; Perry, Kay; Kalkum, Markus; Rummel, Andreas; Dong, Min; Jin, Rongsheng

2016-07-01

Botulinum neurotoxin serotype A1 (BoNT/A1), a licensed drug widely used for medical and cosmetic applications, exerts its action by invading motoneurons. Here we report a 2.0-Å-resolution crystal structure of the BoNT/A1 receptor-binding domain in complex with its neuronal receptor, glycosylated human SV2C. We found that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan-which is conserved in all SV2 isoforms across vertebrates-is essential for BoNT/A1 binding to neurons and for its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an antibotulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications, thereby achieving highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors.

N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A

PubMed Central

Yao, Guorui; Zhang, Sicai; Mahrhold, Stefan; Lam, Kwok-ho; Stern, Daniel; Bagramyan, Karine; Perry, Kay; Kalkum, Markus; Rummel, Andreas; Dong, Min; Jin, Rongsheng

2016-01-01

Botulinum neurotoxin serotype A1 (BoNT/A1) is one of the most dangerous potential bioterrorism agents, and exerts its action by invading motoneurons. It is also a licensed drug widely used for medical and cosmetic applications. Here we report a 2.0 Å resolution crystal structure of BoNT/A1 receptor-binding domain in complex with its neuronal receptor, the glycosylated human SV2C. We find that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an anti-botulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications to achieve highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors. PMID:27294781

O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

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

Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika

2012-03-02

Highlights: Black-Right-Pointing-Pointer We characterized A130022J15Rik (Eogt1)-a mouse gene homologous to Drosophila Eogt. Black-Right-Pointing-Pointer Eogt1 encodes EGF domain O-GlcNAc transferase. Black-Right-Pointing-Pointer Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. Black-Right-Pointing-Pointer O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shownmore » whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.« less

Cystic fibrosis and bacterial colonization define the sputum N-glycosylation phenotype.

PubMed

Venkatakrishnan, Vignesh; Thaysen-Andersen, Morten; Chen, Sharon C A; Nevalainen, Helena; Packer, Nicolle H

2015-01-01

Although mucin O-glycosylation of sputum from individuals suffering from cystic fibrosis (CF) is known to be altered relative to their unaffected counterparts, protein N-glycosylation of CF sputum remains structurally and functionally under-characterized. We report the first N-glycome of soluble proteins in sputum derived from five CF patients, two pathogen-free and two pathogen-infected/colonized non-CF individuals suffering from other pulmonary conditions. N-Glycans were profiled using porous graphitized carbon-liquid chromatography-negative ion-tandem mass spectrometry following enzymatic release from sputum proteins. The composition, topology and linkage isomers of 68 N-glycans were characterized and relatively quantified. Recurring structural features in all sputum N-glycomes were terminal α2,6-sialylation, α1,6-core fucosylation, β1,4-bisecting GlcNAcylation and compositions indicating paucimannosylation. Despite covering different genotypes, age, gender and microbial flora, the sputum N-glycomes showed little interpatient and longitudinal variation within CF patients. Comparative N-glycome analysis between inter-patient group revealed that lung infection/colonization, in general, extensively enriches the CF sputum N-glycosylation phenotype with paucimannose with simultaneous over-sialylation/fucosylation and under-bisecting GlcNAcylation of complex/hybrid N-glycans. In contrast, the sputum from CF patients had only slightly increased abundance of paucimannose N-glycans relative to pathogen-infected/colonized non-CF individuals. Similar to mucin O-glycosylation, protein N-glycosylation appears to be regulated primarily as a secondary effect of bacterial infection and inflammation rather than the CF pathogenesis in sputum. This study provides new structural N-glycan information to help understand the complex cellular and molecular environment of the CF affected respiratory tract. © The Author 2014. Published by Oxford University Press. All rights reserved

21 CFR 864.7470 - Glycosylated hemoglobin assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glycosylated hemoglobin assay. 864.7470 Section... Glycosylated hemoglobin assay. (a) Identification. A glycosylated hemoglobin assay is a device used to measure the glycosylated hemoglobins (A1a, A1b, and A1c) in a patient's blood by a column chromatographic...

Investigations of Scope and Mechanism of Nickel-Catalyzed Transformations of Glycosyl Trichloroacetimidates to Glycosyl Trichloroacetamides and Subsequent, Atom-Economical, One-Step Conversion to α-Urea-Glycosides

PubMed Central

McKay, Matthew J.; Park, Nathaniel H.; Nguyen, Hien M.

2014-01-01

The development and mechanistic investigation of a highly stereoselective methodology for preparing α-linked-urea neo-glycoconjugates and pseudo-oligosaccharides is described. This two-step procedure begins with the selective nickel-catalyzed conversion of glycosyl trichloroacetimidates to the corresponding α-trichloroacetamides. The α-selective nature of the conversion is controlled with a cationic nickel(II) catalyst, Ni(dppe)(OTf)2. Mechanistic studies have identified the coordination of the nickel catalyst with the equatorial C2-ether functionality of the α-glycosyl trichloroacetimidate to be paramount for achieving an α-stereoselective transformation. A cross-over experiment has indicated that the reaction does not proceed in an exclusively-intramolecular fashion. The second step in this sequence is the direct conversion of α-glycosyl trichloroacetamide products into the corresponding α-urea glycosides by reacting them with a wide variety of amine nucleophiles in presence of cesium carbonate. Only α-urea-product formation is observed, as the reaction proceeds with complete retention of stereochemical integrity at the anomeric C-N bond. PMID:24905328

Mucin glycosylating enzyme GALNT2 suppresses malignancy in gastric adenocarcinoma by reducing MET phosphorylation

PubMed Central

Liu, Shin-Yun; Shun, Chia-Tung; Hung, Kuan-Yu; Juan, Hsueh-Fen; Hsu, Chia-Lang; Huang, Min-Chuan; Lai, I-Rue

2016-01-01

Glycosylation affects malignancy in cancer. Here, we report that N- acetylgalactosaminyltransferase 2 (GALNT2), an enzyme that mediates the initial step of mucin type-O glycosylation, suppresses malignant phenotypes in gastric adenocarcinoma (GCA) by modifying MET (Hepatocyte growth factor receptor) activity. GALNT2 mRNA and protein were downregulated in GCAs, and this reduction was associated with more advanced disease stage and shorter recurrence-free survival. Suppressing GALNT2 expression in GCA cells increased cell growth, migration, and invasion in vitro, and tumor metastasis in vivo. GALNT2 knockdown enhanced phosphorylation of MET and decreased expression of the Tn antigen on MET. Inhibiting MET activity with PHA665752 decreased the malignant phenotypes caused by GALNT2 knockdown in GCA cells. Our results indicate that GALNT2 suppresses the malignant potential of GCA cells and provide novel insights into the significance of O-glycosylation in MET activity and GCA progression. PMID:26848976

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

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

Kilcoyne, Michelle; Sharma, Shashank; McDevitt, Niamh

2012-04-13

Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellularmore » matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8

N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM.

PubMed

Yurist-Doutsch, Sophie; Magidovich, Hilla; Ventura, Valeria V; Hitchen, Paul G; Dell, Anne; Eichler, Jerry

2010-02-01

Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.

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

Significant rate accelerated synthesis of glycosyl azides and glycosyl 1,2,3-triazole conjugates.

PubMed

Kumar, Rishi; Maulik, Prakas R; Misra, Anup Kumar

2008-10-01

An efficient and significantly rapid access of a series of glycosyl azides and glycosyl 1,2,3-triazole conjugates is reported using modified one-pot reaction conditions. In both cases yields were excellent and single diastereomers were obtained.

Biogenesis of C-Glycosyl Flavones and Profiling of Flavonoid Glycosides in Lotus (Nelumbo nucifera)

PubMed Central

Li, Shan-Shan; Wu, Jie; Chen, Li-Guang; Du, Hui; Xu, Yan-Jun; Wang, Li-Jing; Zhang, Hui-Jin; Zheng, Xu-Chen; Wang, Liang-Sheng

2014-01-01

Flavonoids in nine tissues of Nelumbo nucifera Gaertner were identified and quantified by high-performance liquid chromatography with diode array detector (HPLC-DAD) and HPLC-electrospray ionization-mass spectrometry (HPLC-ESI-MSn). Thirty-eight flavonoids were identified; eleven C-glycosides and five O-glycosides were discovered for the first time in N. nucifera. Most importantly, the C-glycosyl apigenin or luteolin detected in lotus plumules proved valuable for deep elucidation of flavonoid composition in lotus tissues and for further utilization as functional tea and medicine materials. Lotus leaves possessed the significantly highest amount of flavonoids (2.06E3±0.08 mg 100 g−1 FW) and separating and purifying the bioactive compound, quercetin 3-O-glucuronide, from leaves showed great potential. In contrast, flavonoids in flower stalks, seed coats and kernels were extremely low. Simultaneously, the optimal picking time was confirmed by comparing the compound contents in five developmental phases. Finally, we proposed the putative flavonoid biosynthesis pathway in N. nucifera. PMID:25279809

Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, Function in Arabinogalactan-Protein Glycosylation, Growth and Development in Arabidopsis

PubMed Central

Basu, Debarati; Showalter, Allan M.

2015-01-01

Hydroxyproline-O-galactosyltransferase (GALT) initiates O-glycosylation of arabinogalactan-proteins (AGPs). We previously characterized GALT2 (At4g21060), and now report on functional characterization of GALT5 (At1g74800). GALT5 was identified using heterologous expression in Pichia and an in vitro GALT assay. Product characterization showed GALT5 specifically adds galactose to hydroxyproline in AGP protein backbones. Functions of GALT2 and GALT5 were elucidated by phenotypic analysis of single and double mutant plants. Allelic galt5 and galt2 mutants, and particularly galt2 galt5 double mutants, demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type. Mutant plants showed pleiotropic growth and development phenotypes (defects in root hair growth, root elongation, pollen tube growth, flowering time, leaf development, silique length, and inflorescence growth), which were most severe in the double mutants. Conditional mutant phenotypes were also observed, including salt-hypersensitive root growth and root tip swelling as well as reduced inhibition of pollen tube growth and root growth in response to β-Yariv reagent. These mutants also phenocopy mutants for an AGP, SOS5, and two cell wall receptor-like kinases, FEI1 and FEI2, which exist in a genetic signaling pathway. In summary, GALT5 and GALT2 function as redundant GALTs that control AGP O-glycosylation, which is essential for normal growth and development. PMID:25974423

A Quantative Adverse Outcome Pathway Linking Aromatase Inhibition in Fathead Minnows with Population Dynamics

EPA Science Inventory

A Quantitative Adverse Outcome Pathway Linking Aromatase Inhibition in Fathead Minnows with Population DynamicsAn adverse outcome pathway (AOP) is a qualitative description linking a molecular initiating event (MIE) with measureable key events leading to an adverse outcome (AO). ...

Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue.

PubMed

Coddeville, Bernadette; Wu, Sz-Wei; Fabre, Emeline; Brassart, Colette; Rombouts, Yoann; Burguière, Adeline; Kremer, Laurent; Khoo, Kay-Hooi; Elass-Rochard, Elisabeth; Guérardel, Yann

2012-10-22

The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model. Copyright © 2012 Elsevier B.V. All rights reserved.

The effect of glycosylation on plasma N-terminal proBNP-76 levels in patients with heart or renal failure.

PubMed

Nishikimi, Toshio; Ikeda, Masashi; Takeda, Yosuke; Ishimitsu, Toshihiko; Shibasaki, Ikuko; Fukuda, Hirotsugu; Kinoshita, Hideyuki; Nakagawa, Yasuaki; Kuwahara, Koichiro; Nakao, Kazuwa

2012-01-01

Pro-brain natriuretic peptide (proBNP)-108 and N-terminal proBNP-76 (NT-BNP) contain seven sites for O-linked oligosaccharide attachment. Currently, levels of glycosylated NT-BNP are probably underestimated because it is not recognised by one antibody in the sandwich assay system. The pathophysiological significance of cardiac and plasma levels of non-glycosylated (nonglyNT-BNP) and glycosylated NT-BNP (glyNT-BNP) in heart failure (HF) and chronic renal failure (CRF) was investigated. Plasma samples from 186 patients with HF and 76 patients with CRF on haemodialysis were studied, together with 11 atrial tissue samples. To measure nonglyNT-BNP and glyNT-BNP, samples were incubated with or without deglycosylating enzymes and NT-BNP was measured using Roche Elecsys proBNP I. The percentage glyNT-BNP was calculated as glyNT-BNP/(glyNT-BNP + nonglyNT-BNP). In HF, plasma BNP, nonglyNT-BNP and glyNT-BNP levels all increased with increasing disease severity (New York Heart Association class; p<0.0001), though the molar ratio remained constant (molar ratio, BNP:nonglyNT-BNP:glyNT-BNP = 1:2.4:9.6). Before haemodialysis for CRF, plasma BNP and nonglyNT-BNP were somewhat elevated, and glyNT-BNP was markedly increased (molar ratio, BNP:nonglyNT-BNP:glyNT-BNP = 1:8.5:82). After haemodialysis, plasma BNP, nonglyNT-BNP, atrial natriuretic protein and cGMP all declined (p<0.0001), but glyNT-BNP was unchanged. Notably, the percentage of glyNT-BNP was elevated before haemodialysis, and was further increased after haemodialysis (p<0.0001). Atrial tissue levels of BNP, nonglyNT-BNP and glyNT-BNP were similar. THE findings suggest that most endogenous plasma NT-BNP is glycosylated and therefore undetectable with the current assay system, and that the relative glycosylation level is increased by haemodialysis.

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

PubMed Central

Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

Single-center experience of N-linked Congenital Disorders of Glycosylation with a Summary of Molecularly Characterized Cases in Arabs.

PubMed

Bastaki, Fatma; Bizzari, Sami; Hamici, Sana; Nair, Pratibha; Mohamed, Madiha; Saif, Fatima; Malik, Ethar Mustafa; Al-Ali, Mahmoud Taleb; Hamzeh, Abdul Rezzak

2018-01-01

Congenital disorders of glycosylation (CDG) represent an expanding group of conditions that result from defects in protein and lipid glycosylation. Different subgroups of CDG display considerable clinical and genetic heterogeneity due to the highly complex nature of cellular glycosylation. This is further complicated by ethno-geographic differences in the mutational landscape of each of these subgroups. Ten Arab CDG patients from Latifa Hospital in Dubai, United Arab Emirates, were assessed using biochemical (glycosylation status of transferrin) and molecular approaches (next-generation sequencing [NGS] and Sanger sequencing). In silico tools including CADD and PolyPhen-2 were used to predict the functional consequences of uncovered mutations. In our sample of patients, five novel mutations were uncovered in the genes: MPDU1, PMM2, MAN1B1, and RFT1. In total, 9 mutations were harbored by the 10 patients in 7 genes. These are missense and nonsense mutations with deleterious functional consequences. This article integrates a single-center experience within a list of reported CDG mutations in the Arab world, accompanied by full molecular and clinical details pertaining to the studied cases. It also sheds light on potential ethnic differences that were not noted before in regards to CDG in the Arab world. © 2017 John Wiley & Sons Ltd/University College London.

Exploring Site-Specific N-Glycosylation Microheterogeneity of Haptoglobin using Glycopeptide CID Tandem Mass Spectra and Glycan Database Search

PubMed Central

Chandler, Kevin Brown; Pompach, Petr; Goldman, Radoslav

2013-01-01

Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something which detached N-glycan and de-glycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy which takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false-discovery-rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at false-discovery-rate 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at false-discovery-rate 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

PubMed

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides

NASA Astrophysics Data System (ADS)

Chen, Yajing; Xiong, Zhichao; Zhang, Lingyi; Zhao, Jiaying; Zhang, Quanqing; Peng, Li; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

2015-02-01

Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g-1), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have

Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics.

PubMed

Price, Joshua L; Shental-Bechor, Dalit; Dhar, Apratim; Turner, Maurice J; Powers, Evan T; Gruebele, Martin; Levy, Yaakov; Kelly, Jeffery W

2010-11-03

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible

Testing pathways linking exposure to community violence and sexual behaviors among African American youth.

PubMed

Voisin, Dexter R; Hotton, Anna L; Neilands, Torsten B

2014-09-01

Exposure to community violence and HIV sexual risks are two major public health concerns among youth. This study tests various pathways linking exposure to community violence and sexual behaviors among African American adolescents. Using a sample of 563 (61% females) African American youth attending high school we examined whether problematic psychological symptoms, low school engagement, and/or negative perceptions of peer norms about safer sex functioned as pathways linking exposure to community violence and sexual behaviors. Major findings indicated that, for boys, the relationship between exposure to community violence and sexual début and sexual risk behaviors were linked by aggression. In addition, the relationship between exposure to community violence and sexual risk behaviors were linked by negative perceptions of peer attitudes about safer sex. For girls, the relationship between exposure to community violence and sexual début was linked by aggression and negative perceptions of peer attitudes about safer sex. These findings provide support for pathways linking exposure to community violence to sexual behaviors.

SignaLink 2 - a signaling pathway resource with multi-layered regulatory networks.

PubMed

Fazekas, Dávid; Koltai, Mihály; Türei, Dénes; Módos, Dezső; Pálfy, Máté; Dúl, Zoltán; Zsákai, Lilian; Szalay-Bekő, Máté; Lenti, Katalin; Farkas, Illés J; Vellai, Tibor; Csermely, Péter; Korcsmáros, Tamás

2013-01-18

Signaling networks in eukaryotes are made up of upstream and downstream subnetworks. The upstream subnetwork contains the intertwined network of signaling pathways, while the downstream regulatory part contains transcription factors and their binding sites on the DNA as well as microRNAs and their mRNA targets. Currently, most signaling and regulatory databases contain only a subsection of this network, making comprehensive analyses highly time-consuming and dependent on specific data handling expertise. The need for detailed mapping of signaling systems is also supported by the fact that several drug development failures were caused by undiscovered cross-talk or regulatory effects of drug targets. We previously created a uniformly curated signaling pathway resource, SignaLink, to facilitate the analysis of pathway cross-talks. Here, we present SignaLink 2, which significantly extends the coverage and applications of its predecessor. We developed a novel concept to integrate and utilize different subsections (i.e., layers) of the signaling network. The multi-layered (onion-like) database structure is made up of signaling pathways, their pathway regulators (e.g., scaffold and endocytotic proteins) and modifier enzymes (e.g., phosphatases, ubiquitin ligases), as well as transcriptional and post-transcriptional regulators of all of these components. The user-friendly website allows the interactive exploration of how each signaling protein is regulated. The customizable download page enables the analysis of any user-specified part of the signaling network. Compared to other signaling resources, distinctive features of SignaLink 2 are the following: 1) it involves experimental data not only from humans but from two invertebrate model organisms, C. elegans and D. melanogaster; 2) combines manual curation with large-scale datasets; 3) provides confidence scores for each interaction; 4) operates a customizable download page with multiple file formats (e.g., Bio

Extensive Crosstalk between O-GlcNAcylation and Phosphorylation Regulates Akt Signaling

PubMed Central

Sun, Danni; Xin, Xianliang; Pan, Qiuming; Peng, Shuying; Liang, Zhongjie; Luo, Cheng; Yang, Yiming; Jiang, Hualiang; Huang, Min; Chai, Wengang; Ding, Jian; Geng, Meiyu

2012-01-01

O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling. PMID:22629392

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies.

PubMed

Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20-40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development▿

PubMed Central

Greig, Kylie T.; Antonchuk, Jennifer; Metcalf, Donald; Morgan, Phillip O.; Krebs, Danielle L.; Zhang, Jian-Guo; Hacking, Douglas F.; Bode, Lars; Robb, Lorraine; Kranz, Christian; de Graaf, Carolyn; Bahlo, Melanie; Nicola, Nicos A.; Nutt, Stephen L.; Freeze, Hudson H.; Alexander, Warren S.; Hilton, Douglas J.; Kile, Benjamin T.

2007-01-01

Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity. PMID:17548465

Plant Aquaporin AtPIP1;4 Links Apoplastic H2O2 Induction to Disease Immunity Pathways.

PubMed

Tian, Shan; Wang, Xiaobing; Li, Ping; Wang, Hao; Ji, Hongtao; Xie, Junyi; Qiu, Qinglei; Shen, Dan; Dong, Hansong

2016-07-01

Hydrogen peroxide (H2O2) is a stable component of reactive oxygen species, and its production in plants represents the successful recognition of pathogen infection and pathogen-associated molecular patterns (PAMPs). This production of H2O2 is typically apoplastic but is subsequently associated with intracellular immunity pathways that regulate disease resistance, such as systemic acquired resistance and PAMP-triggered immunity. Here, we elucidate that an Arabidopsis (Arabidopsis thaliana) aquaporin (i.e. the plasma membrane intrinsic protein AtPIP1;4) acts to close the cytological distance between H2O2 production and functional performance. Expression of the AtPIP1;4 gene in plant leaves is inducible by a bacterial pathogen, and the expression accompanies H2O2 accumulation in the cytoplasm. Under de novo expression conditions, AtPIP1;4 is able to mediate the translocation of externally applied H2O2 into the cytoplasm of yeast (Saccharomyces cerevisiae) cells. In plant cells treated with H2O2, AtPIP1;4 functions as an effective facilitator of H2O2 transport across plasma membranes and mediates the translocation of externally applied H2O2 from the apoplast to the cytoplasm. The H2O2-transport role of AtPIP1;4 is essentially required for the cytoplasmic import of apoplastic H2O2 induced by the bacterial pathogen and two typical PAMPs in the absence of induced production of intracellular H2O2 As a consequence, cytoplasmic H2O2 quantities increase substantially while systemic acquired resistance and PAMP-triggered immunity are activated to repress the bacterial pathogenicity. By contrast, loss-of-function mutation at the AtPIP1;4 gene locus not only nullifies the cytoplasmic import of pathogen- and PAMP-induced apoplastic H2O2 but also cancels the subsequent immune responses, suggesting a pivotal role of AtPIP1;4 in apocytoplastic signal transduction in immunity pathways. © 2016 American Society of Plant Biologists. All Rights Reserved.

Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability.

PubMed

Zhang, Dongmei; van der Wel, Hanke; Johnson, Jennifer M; West, Christopher M

2012-01-13

Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.

Total chemical synthesis and biological activities of glycosylated and non-glycosylated forms of the chemokines CCL1 and Ser-CCL1.

PubMed

Okamoto, Ryo; Mandal, Kalyaneswar; Ling, Morris; Luster, Andrew D; Kajihara, Yasuhiro; Kent, Stephen B H

2014-05-12

CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T-cells and acts as a chemoattractant for monocytes. Originally, CCL1 was identified as a 73 amino acid protein having one N-glycosylation site, and a variant 74 residue non-glycosylated form, Ser-CCL1, has also been described. There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser-CCL1. Here we report the total chemical syntheses of both N-glycosylated and non-glycosylated forms of (Ser-)CCL1, by convergent native chemical ligation. We used an N-glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptide-(α) thioester building block. Chemotaxis assays of these glycoproteins and the corresponding non-glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser-)CCL1 using homogeneous N-glycosylated protein molecules of defined covalent structure. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

PubMed Central

Kim, Paul; Jang, Yo Han; Kwon, Soon Bin; Lee, Chung Min; Han, Gyoonhee; Seong, Baik Lin

2018-01-01

Glycosylation of the hemagglutinin (HA) and neuraminidase (NA) of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG) sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness. PMID:29642453

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

PubMed

Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

2017-01-01

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D 100 ), IV (F 220 ) and V (F 264 ) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Genetics and the environment converge to dysregulate N-glycosylation in multiple sclerosis

PubMed Central

Mkhikian, Haik; Grigorian, Ani; Li, Carey F.; Chen, Hung-Lin; Newton, Barbara; Zhou, Raymond W.; Beeton, Christine; Torossian, Sevan; Tatarian, Gevork Grikor; Lee, Sung-Uk; Lau, Ken; Walker, Erin; Siminovitch, Katherine A.; Chandy, K. George; Yu, Zhaoxia; Dennis, James W.; Demetriou, Michael

2011-01-01

How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice, N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis, spontaneous inflammatory demyelination and neurodegeneration, the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D3, including genetic variants in interleukin-7 receptor-α (IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1 (IVAVT−T) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IVAVT−T) and vitamin D3, optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation. PMID:21629267

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation

PubMed Central

Park, Julien H.; Hogrebe, Max; Grüneberg, Marianne; DuChesne, Ingrid; von der Heiden, Ava L.; Reunert, Janine; Schlingmann, Karl P.; Boycott, Kym M.; Beaulieu, Chandree L.; Mhanni, Aziz A.; Innes, A. Micheil; Hörtnagel, Konstanze; Biskup, Saskia; Gleixner, Eva M.; Kurlemann, Gerhard; Fiedler, Barbara; Omran, Heymut; Rutsch, Frank; Wada, Yoshinao; Tsiakas, Konstantinos; Santer, René; Nebert, Daniel W.; Rust, Stephan; Marquardt, Thorsten

2015-01-01

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders. PMID:26637979

Testing Pathways Linking Exposure to Community Violence and Sexual Behaviors Among African American Youth

PubMed Central

Hotton, Anna L.; Neilands, Torsten B.

2014-01-01

Exposure to community violence and HIV sexual risks are two major public health concerns among youth. This study tests various pathways linking exposure to community violence and sexual behaviors among African American adolescents. Using a sample of 563 (61 % females) African American youth attending high school we examined whether problematic psychological symptoms, low school engagement, and/or negative perceptions of peer norms about safer sex functioned as pathways linking exposure to community violence and sexual behaviors. Major findings indicated that, for boys, the relationship between exposure to community violence and sexual début and sexual risk behaviors were linked by aggression. In addition, the relationship between exposure to community violence and sexual risk behaviors were linked by negative perceptions of peer attitudes about safer sex. For girls, the relationship between exposure to community violence and sexual début was linked by aggression and negative perceptions of peer attitudes about safer sex. These findings provide support for pathways linking exposure to community violence to sexual behaviors. PMID:24327295

Congenital Disorders of Glycosylation and Intellectual Disability

ERIC Educational Resources Information Center

Wolfe, Lynne A.; Krasnewich, Donna

2013-01-01

The congenital disorders of glycosylation (CDG) are a rapidly growing group of inborn errors of metabolism that result from defects in the synthesis of glycans. Glycosylation is a major post-translational protein modification and an estimated 2% of the human genome encodes proteins for glycosylation. The molecular bases for the current 60…

Modified immunoglobulin G glycosylation pattern during turpentine-induced acute inflammation in rats.

PubMed

Canellada, Andrea; Margni, Ricardo A

2002-01-01

Alterations in the pattern of protein glycosylation have been described during inflammation. In chronic parasitic and tumoral diseases we have reported an increase in the proportion of serum Immunoglobulin G (IgG) molecules possessing an altered Fab glycosylation pattern designated asymmetric antibodies. The alteration results in augmented concanavalin A affinity and functional univalence of the antibody. In addition, Fc agalactosylation has been described as occurring in chronically autoimmune diseases. Therefore, the aim of this paper was to evaluate by analyzing sera whether during an acute inflammatory response in rats produced by subcutaneous inoculation of turpentine oil, there was an alteration in the synthesis and glycosylation of IgG (as revealed by concanavalin A binding). We found that during acute inflammation there was a decrease in the synthesis of IgG which was not affected by prior oral administration of dexamethasone; however, the turpentine-induced increase in IgG binding to concanavalin A was found to be inhibited upon prior administration of the anti-inflammatory agent. As with turpentine, the corticoid used induced an increase in the interleukin-6 levels detected in sera by ELISA. Although we have described an improvement in asymmetric antibody synthesis by low dose of interleukin-6 previously, here we found no correlation between the observed glycosylation pattern of IgG and interleukin-6 concentration assessed in sera of treated rats, probably due to a different dexamethasone mediated pathway.

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

PubMed Central

Qiu, Z; Hobman, T C; McDonald, H L; Seto, N O; Gillam, S

1992-01-01

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site. Images PMID:1583721

The Integrated Role of Wnt/β-Catenin, N-Glycosylation, and E-Cadherin-Mediated Adhesion in Network Dynamics

PubMed Central

Vargas, Diego A.; Sun, Meng; Sadykov, Khikmet; Kukuruzinska, Maria A.; Zaman, Muhammad H.

2016-01-01

The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene, DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability

Glycosylation and Activities of Natural Products.

PubMed

Huang, Gangliang; Lv, Meijiao; Hu, Jinchuan; Huang, Kunlin; Xu, Hong

2016-01-01

Natural products are widely found in nature, their number and variety are numerous, the structures are complex and diverse. These natural products have many physiological and pharmacological activities. Glycosylation can increase the diversity of structure and function of natural product, it has become the focus of drug research and development. The impacts of glycosylation of natural products to water solubility, pharmacological activities, bioavailability, or others were described in this review, which provides a reference for the development and application of glycosylated natural products.

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

PubMed

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

PubMed Central

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

Chemical O‐Glycosylations: An Overview

PubMed Central

2016-01-01

Abstract The development of glycobiology relies on the sources of particular oligosaccharides in their purest forms. As the isolation of the oligosaccharide structures from natural sources is not a reliable option for providing samples with homogeneity, chemical means become pertinent. The growing demand for diverse oligosaccharide structures has prompted the advancement of chemical strategies to stitch sugar molecules with precise stereo‐ and regioselectivity through the formation of glycosidic bonds. This Review will focus on the key developments towards chemical O‐glycosylations in the current century. Synthesis of novel glycosyl donors and acceptors and their unique activation for successful glycosylation are discussed. This Review concludes with a summary of recent developments and comments on future prospects. PMID:27777833

Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension

PubMed Central

Lima, Luís; Azevedo, Rita; Soares, Janine; Cotton, Sofia; Parreira, Beatriz; Neves, Manuel; Amaro, Teresina; Tavares, Ana; Teixeira, Filipe; Palmeira, Carlos; Rangel, Maria; Silva, André M.N.; Reis, Celso A.; Santos, Lúcio Lara; Oliveira, Maria José; Ferreira, José Alexandre

2016-01-01

Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may

Unravelling Immunoglobulin G Fc N-Glycosylation: A Dynamic Marker Potentiating Predictive, Preventive and Personalised Medicine.

PubMed

Russell, Alyce; Adua, Eric; Ugrina, Ivo; Laws, Simon; Wang, Wei

2018-01-29

Multiple factors influence immunoglobulin G glycosylation, which in turn affect the glycoproteins' function on eliciting an anti-inflammatory or pro-inflammatory response. It is prudent to underscore these processes when considering the use of immunoglobulin G N -glycan moieties as an indication of disease presence, progress, or response to therapeutics. It has been demonstrated that the altered expression of genes that encode enzymes involved in the biosynthesis of immunoglobulin G N -glycans, receptors, or complement factors may significantly modify immunoglobulin G effector response, which is important for regulating the immune system. The immunoglobulin G N -glycome is highly heterogenous; however, it is considered an interphenotype of disease (a link between genetic predisposition and environmental exposure) and so has the potential to be used as a dynamic biomarker from the perspective of predictive, preventive, and personalised medicine. Undoubtedly, a deeper understanding of how the multiple factors interact with each other to alter immunoglobulin G glycosylation is crucial. Herein we review the current literature on immunoglobulin G glycoprotein structure, immunoglobulin G Fc glycosylation, associated receptors, and complement factors, the downstream effector functions, and the factors associated with the heterogeneity of immunoglobulin G glycosylation.

Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.

PubMed

Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere

2011-12-01

The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.

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.

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing.

PubMed

Timal, Sharita; Hoischen, Alexander; Lehle, Ludwig; Adamowicz, Maciej; Huijben, Karin; Sykut-Cegielska, Jolanta; Paprocka, Justyna; Jamroz, Ewa; van Spronsen, Francjan J; Körner, Christian; Gilissen, Christian; Rodenburg, Richard J; Eidhof, Ilse; Van den Heuvel, Lambert; Thiel, Christian; Wevers, Ron A; Morava, Eva; Veltman, Joris; Lefeber, Dirk J

2012-10-01

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included the sequential application of biochemical methods in blood samples and fibroblasts. In genetically unsolved cases, homozygosity mapping has been applied in consanguineous families. Altogether, this time-consuming diagnostic strategy led to the identification of defects in 17 different CDG-I genes. Here, we applied whole-exome sequencing (WES) in combination with the knowledge of the protein N-glycosylation pathway for gene identification in our remaining group of six unsolved CDG-I patients from unrelated non-consanguineous families. Exome variants were prioritized based on a list of 76 potential CDG-I candidate genes, leading to the rapid identification of one known and two novel CDG-I gene defects. These included the first X-linked CDG-I due to a de novo mutation in ALG13, and compound heterozygous mutations in DPAGT1, together the first two steps in dolichol-PP-glycan assembly, and mutations in PGM1 in two cases, involved in nucleotide sugar biosynthesis. The pathogenicity of the mutations was confirmed by showing the deficient activity of the corresponding enzymes in patient fibroblasts. Combined with these results, the gene defect has been identified in 98% of our CDG-I patients. Our results implicate the potential of WES to unravel disease genes in the CDG-I in newly diagnosed singleton families.

Clusterin Facilitates Exchange of Glycosyl Phosphatidylinositol-Linked SPAM1 Between Reproductive Luminal Fluids and Mouse and Human Sperm Membranes1

PubMed Central

Griffiths, Genevieve S.; Galileo, Deni S.; Aravindan, Rolands G.; Martin-DeLeon, Patricia A.

2009-01-01

Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals. PMID:19357365

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation.

PubMed

Park, Julien H; Hogrebe, Max; Grüneberg, Marianne; DuChesne, Ingrid; von der Heiden, Ava L; Reunert, Janine; Schlingmann, Karl P; Boycott, Kym M; Beaulieu, Chandree L; Mhanni, Aziz A; Innes, A Micheil; Hörtnagel, Konstanze; Biskup, Saskia; Gleixner, Eva M; Kurlemann, Gerhard; Fiedler, Barbara; Omran, Heymut; Rutsch, Frank; Wada, Yoshinao; Tsiakas, Konstantinos; Santer, René; Nebert, Daniel W; Rust, Stephan; Marquardt, Thorsten

2015-12-03

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

[The alterations of proteins glycosylation in rheumatic diseases].

PubMed

Chludzińska, Anna; Chrostek, Lech; Cylwik, Bogdan

2012-08-01

The alterations in glycosylation of serum glycoproteins were reported in several pathological conditions including rheumatic diseases. The many studies demonstrated the occurrence of some differentially glycosylated plasma immunoglobulins, especially IgG in rheumatoid arthritis. The most characteristic features are the decrease in galactose content, the presence of N-acetylglucosamine and the increase in fucose content. The structure of oligosaccharides attached to the antibody Fc region affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. The changes in immunoglobulin glycosylation was suggested to be important in the etiology of rheumatoid athritis and correlated with the disease severity. In addition to impaired glycosylation of imunoglubulins, in rheumatic diseases exist the disturbances in glycosylation of both acute-phase and non acute-phase response, such as alpha-1 acid glycoprotein, haptoglobin and alpha-2 macroglobulin. The alterations in glycosylation of these glycoproteins were also correlated with the disease activity.

Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides.

PubMed

Chen, Yajing; Xiong, Zhichao; Zhang, Lingyi; Zhao, Jiaying; Zhang, Quanqing; Peng, Li; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

2015-02-21

Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g(-1)), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.

Causal pathways linking Farm to School to childhood obesity prevention.

PubMed

Joshi, Anupama; Ratcliffe, Michelle M

2012-08-01

Farm to School programs are rapidly gaining attention as a potential strategy for preventing childhood obesity; however, the causal linkages between Farm to School activities and health outcomes are not well documented. To capitalize on the increased interest in and momentum for Farm to School, researchers and practitioners need to move from developing and implementing evidence informed programs and policies to ones that are evidence-based. The purpose of this article is to outline a framework for facilitating an evidence base for Farm to School programs and policies through a systematic and coordinated approach. Employing the concepts of causal pathways, the authors introduce a proposed framework for organizing and systematically testing out multiple hypotheses (or potential causal links) for how, why, and under what conditions Farm to School Inputs and Activities may result in what Outputs, Effects, and Impacts. Using the causal pathways framework may help develop and test competing hypotheses, identify multicausality, strength, and interactions of causes, and discern the difference between catalysts and causes. In this article, we introduce causal pathways, present menus of potential independent and dependent variables from which to create and test causal pathways linking Farm to School interventions and their role in preventing childhood obesity, discuss their applicability to Farm to School research and practice, and outline proposed next steps for developing a coordinated research framework for Farm to School programs.

BID links ferroptosis to mitochondrial cell death pathways.

PubMed

Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

2017-08-01

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Cytosolic-free oligosaccharides are predominantly generated by the degradation of dolichol-linked oligosaccharides in mammalian cells.

PubMed

Harada, Yoichiro; Masahara-Negishi, Yuki; Suzuki, Tadashi

2015-11-01

During asparagine (N)-linked protein glycosylation, eukaryotic cells generate considerable amounts of free oligosaccharides (fOSs) in the cytosol. It is generally assumed that such fOSs are produced by the deglycosylation of misfolded N-glycoproteins that are destined for proteasomal degradation or as the result of the degradation of dolichol-linked oligosaccharides (DLOs), which serve as glycan donor substrates in N-glycosylation reactions. The findings reported herein show that the majority of cytosolic fOSs are generated by a peptide:N-glycanase (PNGase) and an endo-β-N-acetylglucosaminidase (ENGase)-independent pathway in mammalian cells. The ablation of the cytosolic deglycosylating enzymes, PNGase and ENGase, in mouse embryonic fibroblasts had little effect on the amount of cytosolic fOSs generated. Quantitative analyses of fOSs using digitonin-permeabilized cells revealed that they are generated by the degradation of fully assembled Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) in the lumen of the endoplasmic reticulum. Because the degradation of Glc3Man9GlcNAc2-PP-Dol is greatly inhibited in the presence of an N-glycosylation acceptor peptide that is recognized by the oligosaccharyltransferase (OST), the OST-mediated hydrolysis of DLO is the most likely mechanism responsible for the production of a large fraction of the cytosolic fOSs. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Modeling the mechanism of glycosylation reactions between ethanol, 1,2-ethanediol and methoxymethanol.

PubMed

Azofra, Luis Miguel; Alkorta, Ibon; Toro-Labbé, Alejandro; Elguero, José

2013-09-07

The mechanism of the S(N)2 model glycosylation reaction between ethanol, 1,2-ethanediol and methoxymethanol has been studied theoretically at the B3LYP/6-311+G(d,p) computational level. Three different types of reactions have been explored: (i) the exchange of hydroxyl groups between these model systems; (ii) the basic catalysis reactions by combination of the substrates as glycosyl donors (neutral species) and acceptors (enolate species); and (iii) the effect on the reaction profile of an explicit H2O molecule in the reactions considered in (ii). The reaction force, the electronic chemical potential and the reaction electronic flux have been characterized for the reaction path in each case. Energy calculations show that methoxymethanol is the worst glycosyl donor model among the ones studied here, while 1,2-ethanediol is the best, having the lowest activation barrier of 74.7 kJ mol(-1) for the reaction between this one and the ethanolate as the glycosyl acceptor model. In general, the presence of direct interactions between the atoms involved in the penta-coordinated TS increases the activation energies of the processes.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

PubMed Central

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

2016-01-01

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

Protein O-linked ß-N-acetylglucosamine: A novel effector of cardiomyocyte metabolism and function

PubMed Central

Darley-Usmar, Victor M.; Ball, Lauren E.; Chatham, John C.

2014-01-01

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is emerging as an important mechanism for the regulation of numerous biological processes critical for normal cell function. Active synthesis of O-GlcNAc is essential for cell viability and acute activation of pathways resulting in increased protein O-GlcNAc levels improves the tolerance of cells to a wide range of stress stimuli. Conversely sustained increases in O-GlcNAc levels have been implicated in numerous chronic disease states, especially as a pathogenic contributor to diabetic complications. There has been increasing interest in the role of O-GlcNAc in the heart and vascular system and acute activation of O-GlcNAc levels have been shown to reduce ischemia/reperfusion injury attenuate vascular injury responses as well mediate some of the detrimental effects of diabetes and hypertension on cardiac and vascular function. Here we provide an overview of our current understanding of pathways regulating protein O-GlcNAcylation, summarize the different methodologies for identifying and characterizing O-GlcNAcylated proteins and subsequently focus on two emerging areas: 1) the role of O-GlcNAc as a potential regulator of cardiac metabolism and 2) the cross talk between O-GlcNAc and reactive oxygen species. PMID:21878340

Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

NASA Astrophysics Data System (ADS)

Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

2015-05-01

Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

Elucidation of differences in N-glycosylation between different molecular weight forms of recombinant CLEC-2 by LC MALDI tandem MS.

PubMed

Zhou, Lei; Qian, Yifan; Zhang, Xingwang; Ruan, Yuanyuan; Ren, Shifang; Gu, Jianxin

2015-01-30

C-type lectin-like receptor 2 (CLEC-2) is a newly identified receptor expressed on the platelet surface. It has been reported that CLEC-2 exists as a higher molecular weight (HMW) and a lower molecular weight (LMW) form, which share the same protein core but differ in glycans. The two forms appear to have different ligand-binding abilities, indicating that the differential glycosylation of CLEC-2 possibly produces functionally distinct glycoforms. This study aimed to explore an easy method to directly elucidate the N-glycosylation difference by employing a glycoproteomics approach. The off-line coupling of nano-LC with a MALDI-QIT-TOF mass spectrometer was demonstrated to be capable of sensitive and direct elucidation of the glycosylation difference between HMW and LMW CLEC-2, simultaneously providing information about their oligosaccharide structures and the glycosylation sites. The results reveal that a specific glycosylation site, Asn 134, is differently glycosylated in the two forms, with complex types of bi-antennary, tri-antennary and tetra-antennary, N-linked, fucosylated glycans identified at this site in the HMW form but not in the LMW form. The observed difference in glycosylation might provide new insights into the underlying mechanisms of biological functions of CLEC-2. Because of its simplicity and sensitivity, the method explored in this work suggests that it holds promise as a method of elucidating differences in direct N-glycosylation of target glycoprotein, even in small amount of samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

A sweet new role for LCP enzymes in protein glycosylation

DOE PAGES

Amer, Brendan R.; Clubb, Robert T.

2014-11-21

The peptidoglycan that surrounds Gram-positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytRCpsA-Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton-That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromoleculesmore » to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Furthermore, since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell-based tool to discover inhibitors of this important enzyme family.« less

O-linked N-acetyl-glucosamine deposition in placental proteins varies according to maternal glycemic levels.

PubMed

Dela Justina, Vanessa; Dos Passos Junior, Rinaldo R; Bressan, Alecsander F; Tostes, Rita C; Carneiro, Fernando S; Soares, Thaigra S; Volpato, Gustavo T; Lima, Victor Vitorino; Martin, Sebastian San; Giachini, Fernanda R

2018-05-07

Hyperglycemia increases glycosylation with O-linked N‑acetyl‑glucosamine (O-GlcNAc) contributing to placental dysfunction and fetal growth impairment. Our aim was to determine how O-GlcNAc levels are affected by hyperglycemia and the O-GlcNAc distribution in different placental regions. Female Wistar rats were divided into the following groups: severe hyperglycemia (>300 mg/dL; n = 5); mild hyperglycemia (>140 mg/dL, at least than two time points during oral glucose tolerance test; n = 7) or normoglycemia (<120 mg/dL; n = 6). At 21 days of pregnancy, placental tissue was collected and processed for morphometry and immunohistochemistry analyses, or properly stored at -80 °C for protein quantification by western blot. Placental index was increased only in severe hyperglycemic rats. Morphometric analysis showed increased junctional zone and decreased labyrinth region in placentas exclusively from the severe hyperglycemic group. Proteins targeted by O-GlcNAc were detected in all regions, with increased O-GlcNAc levels in the hyperglycemic group compared to control and mild hyperglycemic rats. Proteins in endothelial and trophoblast cells were the main target for O-GlcNAc. Whereas no changes in O-GlcNAc transferase (OGT) expression were detected, O-GlcNAcase (OGA) expression was reduced in placentas from the severe hyperglycemic group and augmented in placentas from the mild hyperglycemic group, compared with their respective control groups. Placental O-GlcNAc overexpression may contribute to placental dysfunction, as indicated by the placental index. Additionally, morphometric alterations, occurring simultaneously with increased O-GlcNAc accumulation in the placental tissue may contribute to placental dysfunction during hyperglycemia. Copyright © 2017. Published by Elsevier Inc.

SignaLink 2 – a signaling pathway resource with multi-layered regulatory networks

PubMed Central

2013-01-01

Background Signaling networks in eukaryotes are made up of upstream and downstream subnetworks. The upstream subnetwork contains the intertwined network of signaling pathways, while the downstream regulatory part contains transcription factors and their binding sites on the DNA as well as microRNAs and their mRNA targets. Currently, most signaling and regulatory databases contain only a subsection of this network, making comprehensive analyses highly time-consuming and dependent on specific data handling expertise. The need for detailed mapping of signaling systems is also supported by the fact that several drug development failures were caused by undiscovered cross-talk or regulatory effects of drug targets. We previously created a uniformly curated signaling pathway resource, SignaLink, to facilitate the analysis of pathway cross-talks. Here, we present SignaLink 2, which significantly extends the coverage and applications of its predecessor. Description We developed a novel concept to integrate and utilize different subsections (i.e., layers) of the signaling network. The multi-layered (onion-like) database structure is made up of signaling pathways, their pathway regulators (e.g., scaffold and endocytotic proteins) and modifier enzymes (e.g., phosphatases, ubiquitin ligases), as well as transcriptional and post-transcriptional regulators of all of these components. The user-friendly website allows the interactive exploration of how each signaling protein is regulated. The customizable download page enables the analysis of any user-specified part of the signaling network. Compared to other signaling resources, distinctive features of SignaLink 2 are the following: 1) it involves experimental data not only from humans but from two invertebrate model organisms, C. elegans and D. melanogaster; 2) combines manual curation with large-scale datasets; 3) provides confidence scores for each interaction; 4) operates a customizable download page with multiple file formats

Surveying N2O-producing pathways in bacteria.

PubMed

Stein, Lisa Y

2011-01-01

Nitrous oxide (N(2)O) is produced by bacteria as an intermediate of both dissimilatory and detoxification pathways under a range of oxygen levels, although the majority of N(2)O is released in suboxic to anoxic environments. N(2)O production under physiologically relevant conditions appears to require the reduction of nitric oxide (NO) produced from the oxidation of hydroxylamine (nitrification), reduction of nitrite (denitrification), or by host cells of pathogenic bacteria. In a single bacterial isolate, N(2)O-producing pathways can be complex, overlapping, involve multiple enzymes with the same function, and require multiple layers of regulatory machinery. This overview discusses how to identify known N(2)O-producing inventory and regulatory sequences within bacterial genome sequences and basic physiological approaches for investigating the function of that inventory. A multitude of review articles have been published on individual enzymes, pathways, regulation, and environmental significance of N(2)O-production encompassing a large diversity of bacterial isolates. The combination of next-generation deep sequencing platforms, emerging proteomics technologies, and basic microbial physiology can be used to expand what is known about N(2)O-producing pathways in individual bacterial species to discover novel inventory and unifying features of pathways. A combination of approaches is required to understand and generalize the function and control of N(2)O production across a range of temporal and spatial scales within natural and host environments. Copyright © 2011 Elsevier Inc. All rights reserved.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1).

PubMed

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W; Prestegard, James H

2016-09-16

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC'C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC'C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, 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

Perspectives on Glycosylation and Its Congenital Disorders.

PubMed

Ng, Bobby G; Freeze, Hudson H

2018-06-01

Congenital disorders of glycosylation (CDG) are a rapidly expanding group of metabolic disorders that result from abnormal protein or lipid glycosylation. They are often difficult to clinically diagnose because they broadly affect many organs and functions and lack clinical uniformity. However, recent technological advances in next-generation sequencing have revealed a treasure trove of new genetic disorders, expanded the knowledge of known disorders, and showed a critical role in infectious diseases. More comprehensive genetic tools specifically tailored for mammalian cell-based models have revealed a critical role for glycosylation in pathogen-host interactions, while also identifying new CDG susceptibility genes. We highlight recent advancements that have resulted in a better understanding of human glycosylation disorders, perspectives for potential future therapies, and mysteries for which we continue to seek new insights and solutions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Plant Aquaporin AtPIP1;4 Links Apoplastic H2O2 Induction to Disease Immunity Pathways1[OPEN

PubMed Central

Tian, Shan; Wang, Xiaobing; Li, Ping; Wang, Hao; Ji, Hongtao; Xie, Junyi; Qiu, Qinglei

2016-01-01

Hydrogen peroxide (H2O2) is a stable component of reactive oxygen species, and its production in plants represents the successful recognition of pathogen infection and pathogen-associated molecular patterns (PAMPs). This production of H2O2 is typically apoplastic but is subsequently associated with intracellular immunity pathways that regulate disease resistance, such as systemic acquired resistance and PAMP-triggered immunity. Here, we elucidate that an Arabidopsis (Arabidopsis thaliana) aquaporin (i.e. the plasma membrane intrinsic protein AtPIP1;4) acts to close the cytological distance between H2O2 production and functional performance. Expression of the AtPIP1;4 gene in plant leaves is inducible by a bacterial pathogen, and the expression accompanies H2O2 accumulation in the cytoplasm. Under de novo expression conditions, AtPIP1;4 is able to mediate the translocation of externally applied H2O2 into the cytoplasm of yeast (Saccharomyces cerevisiae) cells. In plant cells treated with H2O2, AtPIP1;4 functions as an effective facilitator of H2O2 transport across plasma membranes and mediates the translocation of externally applied H2O2 from the apoplast to the cytoplasm. The H2O2-transport role of AtPIP1;4 is essentially required for the cytoplasmic import of apoplastic H2O2 induced by the bacterial pathogen and two typical PAMPs in the absence of induced production of intracellular H2O2. As a consequence, cytoplasmic H2O2 quantities increase substantially while systemic acquired resistance and PAMP-triggered immunity are activated to repress the bacterial pathogenicity. By contrast, loss-of-function mutation at the AtPIP1;4 gene locus not only nullifies the cytoplasmic import of pathogen- and PAMP-induced apoplastic H2O2 but also cancels the subsequent immune responses, suggesting a pivotal role of AtPIP1;4 in apocytoplastic signal transduction in immunity pathways. PMID:26945050

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

PubMed

Peters, J; Rudolf, S; Oschkinat, H; Mengele, R; Sumper, M; Kellermann, J; Lottspeich, F; Baumeister, W

1992-04-01

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.

The glycosylation and characterization of the candidate Gc macrophage activating factor.

PubMed

Ravnsborg, Tina; Olsen, Dorthe T; Thysen, Anna Hammerich; Christiansen, Maja; Houen, Gunnar; Højrup, Peter

2010-04-01

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood. Copyright 2010 Elsevier B.V. All rights reserved.

GlycoPP: A Webserver for Prediction of N- and O-Glycosites in Prokaryotic Protein Sequences

PubMed Central

Chauhan, Jagat S.; Bhat, Adil H.; Raghava, Gajendra P. S.; Rao, Alka

2012-01-01

Glycosylation is one of the most abundant post-translational modifications (PTMs) required for various structure/function modulations of proteins in a living cell. Although elucidated recently in prokaryotes, this type of PTM is present across all three domains of life. In prokaryotes, two types of protein glycan linkages are more widespread namely, N- linked, where a glycan moiety is attached to the amide group of Asn, and O- linked, where a glycan moiety is attached to the hydroxyl group of Ser/Thr/Tyr. For their biologically ubiquitous nature, significance, and technology applications, the study of prokaryotic glycoproteins is a fast emerging area of research. Here we describe new Support Vector Machine (SVM) based algorithms (models) developed for predicting glycosylated-residues (glycosites) with high accuracy in prokaryotic protein sequences. The models are based on binary profile of patterns, composition profile of patterns, and position-specific scoring matrix profile of patterns as training features. The study employ an extensive dataset of 107 N-linked and 116 O-linked glycosites extracted from 59 experimentally characterized glycoproteins of prokaryotes. This dataset includes validated N-glycosites from phyla Crenarchaeota, Euryarchaeota (domain Archaea), Proteobacteria (domain Bacteria) and validated O-glycosites from phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria (domain Bacteria). In view of the current understanding that glycosylation occurs on folded proteins in bacteria, hybrid models have been developed using information on predicted secondary structures and accessible surface area in various combinations with training features. Using these models, N-glycosites and O-glycosites could be predicted with an accuracy of 82.71% (MCC 0.65) and 73.71% (MCC 0.48), respectively. An evaluation of the best performing models with 28 independent prokaryotic glycoproteins confirms the suitability of these models in predicting N- and O

Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia.

PubMed

Sou, Si Nga; Jedrzejewski, Philip M; Lee, Ken; Sellick, Christopher; Polizzi, Karen M; Kontoravdi, Cleo

2017-07-01

Despite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb ) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc-glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N-linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N-linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc-glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570-1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

Linking Family Economic Hardship to Early Childhood Health: An Investigation of Mediating Pathways.

PubMed

Hsu, Hui-Chin; Wickrama, Kandauda A S

2015-12-01

The underlying mechanisms through which family economic adversity influences child health are less understood. Taking a process-oriented approach, this study examined maternal mental health and investment in children, child health insurance, and child healthcare as mediators linking family economic hardship (FEH) to child health. A structural equation modeling was applied to test the hypothesized mediating model. After adjustment for sociodemographic risk factors, results revealed: (1) a significant direct path linking FEH to poor child health (effect size = .372), and (2) six significant mediating pathways (total effect size = .089). In two mediating pathways, exposures to FEH undermined mothers' mental health: in the first pathway poor maternal mental health led to decreased parental investment, which, in turn, contributed to poor child health, whereas in the second pathway the adverse effect of poor maternal mental health was cascaded through child unmet healthcare need, which resulted in poor child health. One pathway involved child insurance status, where the effect of FEH increased the likelihood to be uninsured, which led to unmet healthcare need, and, in turn, to poor health. Three pathways involved preventive care: in one pathway FEH contributed to poor preventive care, which led to unmet healthcare need and then to poor health; in the other two pathways where poor preventive care respectively gave rise to decreased investment in children or poor maternal mental health, which further contributed to poor child health. Results suggest that the association between FEH and children's health is mediated by multiple pathways.

FoxO Transcription Factors and Regenerative Pathways in Diabetes Mellitus

PubMed Central

Maiese, Kenneth

2015-01-01

Mammalian forkhead transcription factors of the O class (FoxO) are exciting targets under consideration for the development of new clinical entities to treat metabolic disorders and diabetes mellitus (DM). DM, a disorder that currently affects greater than 350 million individuals globally, can become a devastating disease that leads to cellular injury through oxidative stress pathways and affects multiple systems of the body. FoxO proteins can regulate insulin signaling, gluconeogenesis, insulin resistance, immune cell migration, and cell senescence. FoxO proteins also control cell fate through oxidative stress and pathways of autophagy and apoptosis that either lead to tissue regeneration or cell demise. Furthermore, FoxO signaling can be dependent upon signal transduction pathways that include silent mating type information regulation 2 homolog 1 (S. cerevisiae) (SIRT1), Wnt, and Wnt1 inducible signaling pathway protein 1 (WISP1). Cellular metabolic pathways driven by FoxO proteins are complex, can lead to variable clinical outcomes, and require in-depth analysis of the epigenetic and post-translation protein modifications that drive FoxO protein activation and degradation. PMID:26256004

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

A new glycosylated dihydrophaseic acid from cacao germs (Theobroma cacao L.).

PubMed

Sannohe, Yumiko; Gomi, Shuichi; Murata, Takashi; Ohyama, Makoto; Yonekura, Kumiko; Kanegae, Minoru; Koga, Jinichiro

2011-01-01

Cacao beans are composed of cacao nibs and germs. Although numerous chemical and physiological studies on cacao nib compounds have been reported, there is little information on cacao germ compounds. We therefore analyzed an extract from the cacao germ, and found two compounds that were specific to the germ. One of these two compounds was identified as the new glycosylated abscisic acid metabolite, dihydrophaseic acid-4'-O-6″-(β-ribofuranosyl)-β-glucopyranoside, and the other as the known compound, dihydrophaseic acid-4'-O-β-D-glucopyranoside.

N-glycosylation of plant recombinant pharmaceuticals.

PubMed

Bardor, Muriel; Cabrera, Gleysin; Stadlmann, Johannes; Lerouge, Patrice; Cremata, José A; Gomord, Véronique; Fitchette, Anne-Catherine

2009-01-01

N-glycosylation is a maturation event necessary for the correct function, efficiency, and stability of a high number of biopharmaceuticals. This chapter presented here proposes various methods to determine whether, how, and where a plant pharmaceutical is N-glycosylated. These methods rely on blot detection with glycan-specific probes, specific deglycosylation of glycoproteins followed by mass spectrometry, N-glycan profile analysis, and glycopeptide identification by LC-MS.

Glycosylated polyacrylate nanoparticles by emulsion polymerization

PubMed Central

Abeylath, Sampath C.; Turos, Edward

2007-01-01

A selection of glycosylated polyacrylate nanoparticles has been prepared by radical-initiated emulsion polymerization in aqueous media. Using ethyl acrylate as a co-monomer, carbohydrate acrylates were incorporated into the poly(ethyl acrylate) framework to give stable emulsions of glyconanoparticles with an average particle size of around 40 nm. Using this technique a variety of glyconanoparticles were prepared from 3-O-acryloyl-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, 1-O-acryloyl-2,3:5,6-di-O-isopropylidene-α-D-mannofuranose, 6-O-acryloyl-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose, 2-N-acryloyl-1,3,4,6-tetra-O-acetyl-β-D-glucosamine, 5-O-acryloyl-2,3-isopropylidene-1-methoxy-β-D-ribofuranose and 4-N-acetyl-5’-O-acryloyl-2’,3’-O-isopropylidene cytidine. Scanning electron microscopy, dynamic light scattering and proton NMR analysis of the emulsions indicated essentially 100% incorporation of the carbohydrate acrylate monomer into the polymer with the exception of O-benzyl- and O-benzoyl-protected carbohydrate acrylates, which gave incomplete incorporation. Formation of larger glyconanoparticles of ~80nm with (unprotected) 3-O-acryloyl-D-glucose and 5-O-acryloyl-1-methoxy-β-D-ribofuranose revealed the influence of free hydroxyl groups in the monomer on the particle size during polymerization, a feature which is also apparently dependent on the amount of carbohydrate in the matrix. This methodology allows for a new, simple route to the synthesis of polymeric glyconanoparticles with potential applications in targeted drug delivery and materials development. PMID:18677404

Room-temperature ionic liquids enhanced green synthesis of β-glycosyl 1-ester.

PubMed

Cui, Yanli; Xu, Minghan; Yao, Weirong; Mao, Jianwei

2015-04-30

We herein report an efficient synthesis of β-glycosyl 1-ester in room-temperature ionic liquids (RTILs) promoted via silver salt and quaternary ammonium salt (PTC) with good or excellent yields. All products were isolated exclusively as the β-anomers. Four different RTILs, eight metal salts and four quaternary ammonium salts were screened in the glycosylation reaction. The synergistic effect of C6mim·OTf, Ag2O and tetrabutylammonium iodine gave the best results. Their promotion to the system was integral. Thorough study provided insight into the catalytic activity of ionic liquid structure, metal salts and quaternary ammonium salt to these reactions. It is worth mentioning that the yield of aliphatic compound 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl butyrate (3l) was highly improved when using C6mim·OTf as solvent compared with the normal volatile solvents under the same catalysts. This green approach has been proved to be practical and compatible with a wide range from aliphatic to aromatic substrates. Copyright © 2015 Elsevier Ltd. All rights reserved.

A general strategy for stereoselective glycosylations.

PubMed

Kim, Jin-Hwan; Yang, Hai; Park, Jin; Boons, Geert-Jan

2005-08-31

The principal challenge that the synthesis of oligosaccharides of biological importance presents is the development of a general approach for the stereoselective introduction of a glycosidic linkage. It is shown here that a (1S)-phenyl-2-(phenylsulfanyl)ethyl moiety at C-2 of a glycosyl donor can perform neighboring group participation to give a quasi-stable anomeric sulfonium ion. Due to steric and electronic factors, the sulfonium ion is formed as a trans-decalin ring system. Displacement of the sulfonium ion by a hydroxyl leads to the stereoselective formation of alpha-glycosides. NMR experiments were employed to show convincingly the presence of the beta-linked sulfonium ion intermediate. The (1S)-phenyl-2-(phenylsulfanyl)ethyl moiety could be introduced by reaction of a sugar alcohol with acetic acid (1S)-phenyl-2-(phenylsulfanyl)ethyl ester in the presence of BF(3)-OEt(2). Furthermore, it could be removed by conversion into acetate by treatment with BF(3)-OEt(2) in acetic anhydride. The introduction as well as the cleavage reaction proceeds through the formation of an intermediate episulfonium ion. The use of the new methodology in combination with traditional neighboring group participation by esters to introduce beta-glycosides makes it possible, for the first time, to synthesize a wide variety of oligosaccharides by routine procedures. The latter was demonstrated by the synthesis of the Galili trisaccharide, which has been identified as an epitope that can trigger acute rejections in xeno-transplantations, by the one-pot two-step glycosylation sequence.

Genetics Home Reference: DOLK-congenital disorder of glycosylation

MedlinePlus

... called glycosylation, which attaches groups of sugar molecules (oligosaccharides) to proteins. Glycosylation changes proteins in ways that ... to dolichol phosphate in order to build the oligosaccharide chain. Once the chain is formed, dolichol phosphate ...

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

PubMed Central

Krambeck, Frederick J.; Bennun, Sandra V.; Betenbaugh, Michael J.

2017-01-01

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of

Targeted methods for quantitative analysis of protein glycosylation

PubMed Central

Goldman, Radoslav; Sanda, Miloslav

2018-01-01

Quantification of proteins by LC-MS/MS-MRM has become a standard method with broad projected clinical applicability. MRM quantification of protein modifications is, however, far less utilized, especially in the case of glycoproteins. This review summarizes current methods for quantitative analysis of protein glycosylation with a focus on MRM methods. We describe advantages of this quantitative approach, analytical parameters that need to be optimized to achieve reliable measurements, and point out the limitations. Differences between major classes of N- and O-glycopeptides are described and class-specific glycopeptide assays are demonstrated. PMID:25522218

Surface glycosylation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) membrane for selective adsorption of low-density lipoprotein.

PubMed

Wang, Wei; Lan, Ping

2014-01-01

A novel method of constructing a glycosylated surface on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] membrane surface for the selective adsorption of low-density lipoprotein (LDL) was developed, which involved the photoinduced graft polymerization of acrylic acid followed by the chemical binding of carboxyl groups with glucosamine in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride and N-hydroxy-succinimide. The chemical structures of the fabricated membranes were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Zeta potential and water contact angle measurements were performed to investigate the surface charge and wettability of the membranes, respectively. An enzyme linked immunosorbent assay was used to measure the LDL adsorption on the plain and modified membrane surfaces. It was found that the surface glycosylation of P(3HB-co-4HB) membrane greatly enhanced the affinity interactions with LDL and the absorbed LDL could be easily desorbed with eluents, indicating a specific and reversible binding of LDL to the surface. Furthermore, the hemocompatibility of glycosylated membrane was improved as examined by platelet adhesion. The results suggest that the glycosylated P(3HB-co-4HB) membrane is promising for application in LDL apheresis therapy.

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.

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

PubMed Central

Sumper, M; Berg, E; Mengele, R; Strobel, I

1990-01-01

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988). Images PMID:2123862

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

PubMed

Sumper, M; Berg, E; Mengele, R; Strobel, I

1990-12-01

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).

Cellobiohydrolase I enzymes

DOEpatents

Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

2014-01-28

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN

PubMed Central

Menck, Kerstin; Scharf, Christian; Bleckmann, Annalen; Dyck, Lydia; Rost, Ulrike; Wenzel, Dirk; Dhople, Vishnu M.; Siam, Laila; Pukrop, Tobias; Binder, Claudia; Klemm, Florian

2015-01-01

Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN. PMID:25503107

[Non-enzymatic glycosylation of dietary protein in vitro].

PubMed

Bednykh, B S; Evdokimov, I A; Sokolov, A I

2015-01-01

Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

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

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.

An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

PubMed

Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

2016-11-18

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

Glycosylated haemoglobin: measurement and clinical use.

PubMed

Peacock, I

1984-08-01

The discovery, biochemistry, laboratory determination, and clinical application of glycosylated haemoglobins are reviewed. Sources of error are discussed in detail. No single assay method is suitable for all purposes, and in the foreseeable future generally acceptable standards and reference ranges are unlikely to be agreed. Each laboratory must establish its own. Nevertheless, the development of glycosylated haemoglobin assays is an important advance. They offer the best available means of assessing diabetic control.

Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

PubMed

Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

2017-10-31

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Tissue-nonspecific alkaline phosphatase is activated in enterocytes by oxidative stress via changes in glycosylation.

PubMed

López-Posadas, Rocío; González, Raquel; Ballester, Isabel; Martínez-Moya, Patricia; Romero-Calvo, Isabel; Suárez, María Dolores; Zarzuelo, Antonio; Martínez-Augustin, Olga; Sánchez de Medina, Fermín

2011-02-01

Intestinal inflammation produces an induction of alkaline phosphatase (AP) activity that is attributable in part to augmented expression, accompanied by a change in isoform, in epithelial cells. This study focuses on induction of AP in intestinal epithelial cells in vitro. Treatment with the oxidants H2O2, monochloramine, or tButOOH increases AP activity in vitro in Caco-2, HT29, and IEC18 cells. We selected IEC18 cells for further testing. Basal AP activity in IEC18 cells is of the tissue-nonspecific (bone-liver-kidney) type, as indicated by Northern and Western blot analysis. Oxidative stress augments AP activity and the sensitivity of the enzyme to levamisole, homoarginine, and heat in IEC18 cells. Increased immunoreactivity to tissue-nonspecific AP antibodies suggests an isoform shift from liver to either kidney or bone type. This effect occurs without changes at the mRNA level and is sensitive to tunicamycin, an inhibitor of N-glycosylation, and neuraminidase digestion. Saponin and deoxycholate produce similar effects to oxidants. Butyrate but not proinflammatory cytokines or LPS can induce a similar effect but without toxicity. The AP increase is not prevented by modulators of the MAPK, NF-κB, calcium, and cyclic adenosine monophosphate (cAMP) pathways, and is actually enhanced by actinomycin D via higher cell stress. Oxidative stress causes a distinct increase in enterocyte AP activity together with cell toxicity via changes in the glycosylation of the enzyme that correspond to a shift in isotype within the tissue-nonspecific paradigm. We speculate that this may have physiological implication for gut defense.

N-Glycosylation of Campylobacter jejuni Surface Proteins Promotes Bacterial Fitness

PubMed Central

Nothaft, Harald; Zheng, Jing

2013-01-01

Campylobacter jejuni is the etiologic agent of human bacterial gastroenteritis worldwide. In contrast, despite heavy colonization, C. jejuni maintains a commensal mode of existence in chickens. The consumption of contaminated chicken products is thought to be the principal mode of C. jejuni transmission to the human population. C. jejuni harbors a system for N-linked protein glycosylation that has been well characterized and modifies more than 60 periplasmic and membrane-bound proteins. However, the precise role of this modification in the biology of C. jejuni remains unexplored. We hypothesized that the N-glycans protect C. jejuni surface proteins from the action of gut proteases. The C. jejuni pglB mutant, deficient in the expression of the oligosaccharyltransferase, exhibited reduced growth in medium supplemented with chicken cecal contents (CCC) compared with that of wild-type (WT) cells. Inactivation of the cecal proteases by heat treatment or with protease inhibitors completely restored bacterial viability and partially rescued bacterial growth. Physiological concentrations of trypsin, but not chymotrypsin, also reduced C. jejuni pglB mutant CFU. Live or dead staining indicated that CCC preferentially influenced C. jejuni growth as opposed to bacterial viability. We identified multiple chicken cecal proteases by mass fingerprinting. The use of protease inhibitors that target specific classes indicated that both metalloproteases and serine proteases were involved in the attenuated growth of the oligosaccharyltransferase mutant. In conclusion, protein N-linked glycosylation of surface proteins may enhance C. jejuni fitness by protecting bacterial proteins from cleavage due to gut proteases. PMID:23460522

Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles.

PubMed

Hossler, Patrick; Racicot, Christopher; Chumsae, Christopher; McDermott, Sean; Cochran, Keith

2017-03-01

Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N-glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N-glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under-appreciated for these under-utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511-522, 2017. © 2017 American Institute of Chemical Engineers.

Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes.

PubMed

Yu, Seon-Mi; Kim, Song-Ja

2010-11-30

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG- modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.

The effect of simulated space radiation on the N-glycosylation of human immunoglobulin G1.

PubMed

Szarka, Mate; Szilasi, Szabolcs; Donczo, Boglarka; Sarkozy, Daniel; Rajta, Istvan; Guttman, Andras

2018-05-18

On a roundtrip to Mars, astronauts are expectedly exposed to an approximate amount of radiation that exceeds the lifetime limits on Earth. This elevated radiation dose is mainly due to Galactic Cosmic Rays and Solar Particle Events. Specific patterns of the N-glycosylation of human Igs have already been associated with various ailments such as autoimmune diseases, malignant transformation, chronic inflammation, and ageing. The focus of our work was to investigate the effect of low-energy proton irradiation on the IgG N-glycosylation profile with the goal if disease associated changes could be detected during space travel and not altered by space radiation. Two ionization sources were used during the experiments, a Van de Graaff generator for the irradiation of solidified hIgG samples in vacuum, and a Tandetron accelerator to irradiate hIgG samples in aqueous solution form. Structural carbohydrate analysis was accomplished by CE with laser induced fluorescent detection to determine the effects of simulated space radiation on N-glycosylation of hIgG1 samples. Our results revealed that even several thousand times higher radiation doses that of astronauts can suffer during long duration missions beyond the shielding environment of Low Earth Orbit, no changes were observed in hIgG1 N-glycosylation. Consequently, changes in N-linked carbohydrate profile of IgG1 can be used as molecular diagnostic tools in space. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Importance of N-Glycosylation on CD147 for Its Biological Functions

PubMed Central

Bai, Yang; Huang, Wan; Ma, Li-Tian; Jiang, Jian-Li; Chen, Zhi-Nan

2014-01-01

Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation. PMID:24739808

Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), A New Post-translational Modification in Mammals.

PubMed

Maynard, Jason C; Burlingame, Alma L; Medzihradszky, Katalin F

2016-11-01

Intracellular GlcNAcylation of Ser and Thr residues is a well-known and widely investigated post-translational modification. This post-translational modification has been shown to play a significant role in cell signaling and in many regulatory processes within cells. O-GlcNAc transferase is the enzyme responsible for glycosylating cytosolic and nuclear proteins with a single GlcNAc residue on Ser and Thr side-chains. Here we report that the same enzyme may also be responsible for S-GlcNAcylation, i.e. for linking the GlcNAc unit to the peptide by modifying a cysteine side-chain. We also report that O-GlcNAcase, the enzyme responsible for removal of O-GlcNAcylation does not appear to remove the S-linked sugar. Such Cys modifications have been detected and identified in mouse and rat samples. This work has established the occurrence of 14 modification sites assigned to 11 proteins unambiguously. We have also identified S-GlcNAcylation from human Host Cell Factor 1 isolated from HEK-cells. Although these site assignments are primarily based on electron-transfer dissociation mass spectra, we also report that S-linked GlcNAc is more stable under collisional activation than O-linked GlcNAc derivatives. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fluorescence Spectroscopic Studies on the Complexation of Antidiabetic Drugs with Glycosylated Serum Albumin

NASA Astrophysics Data System (ADS)

Seedher, N.; Kanojia, M.

2013-11-01

Glycosylation decreases the association constant values and hence the binding affinity of human serum albumin (HSA) for the antidiabetic drugs under study. The percentage of HAS-bound drug at physiological temperature was only about 21-38 % as compared to 46-74 % for non-glycosylated HSA. Thus the percentage of free drug available for an antihyperglycemic effect was about double (62-79 %) compared to the values for non-glycosylated HSA. Much higher free drug concentrations available for pharmacological effect can lead to the risk of hypoglycemia. Hydrophobic interactions were predominantly involved in the binding. In the binding of gliclazide, hydrogen bonding and electrostatic interactions were involved. Site specificity for glycosylated HSA was the same as that for non-glycosylated HSA; gliclazide and repaglinide bind only at site II whereas glimepiride and glipizide bind at both sites I and II. Glycosylation, however, caused conformational changes in albumin, and the binding region within site II was different for glycosylated and non-glycosylated albumin. Stern-Volmer analysis also indicated the conformational changes in albumin as a result of glycosylation and showed that the dynamic quenching mechanism was valid for fluorescence of both glycosylated and non-glycosylated HSA.

General synthesis of C-glycosyl amino acids via proline-catalyzed direct electrophilic alpha-amination of C-glycosylalkyl aldehydes.

PubMed

Nuzzi, Andrea; Massi, Alessandro; Dondoni, Alessandro

2008-10-16

Non-natural axially and equatorially linked C-glycosyl alpha-amino acids (glycines, alanines, and CH2-serine isosteres) with either S or R alpha-configuration were prepared by D- and L-proline-catalyzed (de >95%) alpha-amination of C-glycosylalkyl aldehydes using dibenzyl azodicarboxylate as the electrophilic reagent.

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism.

PubMed

Laitinen, Teresa; Morreel, Kris; Delhomme, Nicolas; Gauthier, Adrien; Schiffthaler, Bastian; Nickolov, Kaloian; Brader, Günter; Lim, Kean-Jin; Teeri, Teemu H; Street, Nathaniel R; Boerjan, Wout; Kärkönen, Anna

2017-07-01

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H 2 O 2 ) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce ( Picea abies ) was used as a research model. Scavenging of apoplastic H 2 O 2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H 2 O 2 -scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H 2 O 2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H 2 O 2 production in addition to potential H 2 O 2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism. © 2017 American Society of Plant Biologists. All Rights Reserved.

Selective control of oligosaccharide transfer efficiency for the N-glycosylation sequon by a point mutation in oligosaccharyltransferase.

PubMed

Igura, Mayumi; Kohda, Daisuke

2011-04-15

Asn-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria. Oligosaccharyltransferase (OST) catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. Inefficient oligosaccharide transfer results in glycoprotein heterogeneity, which is particularly bothersome in pharmaceutical glycoprotein production. Amino acid variation at the X position of the Asn-X-Ser/Thr sequon is known to modulate the glycosylation efficiency. The best amino acid at X is valine, for an archaeal Pyrococcus furiosus OST. We performed a systematic alanine mutagenesis study of the archaeal OST to identify the essential and dispensable amino acid residues in the three catalytic motifs. We then investigated the effects of the dispensable mutations on the amino acid preference in the N-glycosylation sequon. One residue position was found to selectively affect the amino acid preference at the X position. This residue is located within the recently identified DXXKXXX(M/I) motif, suggesting the involvement of this motif in N-glycosylation sequon recognition. In applications, mutations at this position may facilitate the design of OST variants adapted to particular N-glycosylation sites to reduce the heterogeneity of glycan occupancy. In fact, a mutation at this position led to 9-fold higher activity relative to the wild-type enzyme, toward a peptide containing arginine at X in place of valine. This mutational approach is potentially applicable to eukaryotic and eubacterial OSTs for the production of homogenous glycoproteins in engineered mammalian and Escherichia coli cells.

Selective Control of Oligosaccharide Transfer Efficiency for the N-Glycosylation Sequon by a Point Mutation in Oligosaccharyltransferase*

PubMed Central

Igura, Mayumi; Kohda, Daisuke

2011-01-01

Asn-linked glycosylation is the most ubiquitous posttranslational protein modification in eukaryotes and archaea, and in some eubacteria. Oligosaccharyltransferase (OST) catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. Inefficient oligosaccharide transfer results in glycoprotein heterogeneity, which is particularly bothersome in pharmaceutical glycoprotein production. Amino acid variation at the X position of the Asn-X-Ser/Thr sequon is known to modulate the glycosylation efficiency. The best amino acid at X is valine, for an archaeal Pyrococcus furiosus OST. We performed a systematic alanine mutagenesis study of the archaeal OST to identify the essential and dispensable amino acid residues in the three catalytic motifs. We then investigated the effects of the dispensable mutations on the amino acid preference in the N-glycosylation sequon. One residue position was found to selectively affect the amino acid preference at the X position. This residue is located within the recently identified DXXKXXX(M/I) motif, suggesting the involvement of this motif in N-glycosylation sequon recognition. In applications, mutations at this position may facilitate the design of OST variants adapted to particular N-glycosylation sites to reduce the heterogeneity of glycan occupancy. In fact, a mutation at this position led to 9-fold higher activity relative to the wild-type enzyme, toward a peptide containing arginine at X in place of valine. This mutational approach is potentially applicable to eukaryotic and eubacterial OSTs for the production of homogenous glycoproteins in engineered mammalian and Escherichia coli cells. PMID:21357684

The O-β-linked N-acetylglucosaminylation of the Lamin B receptor and its impact on DNA binding and phosphorylation.

PubMed

Smet-Nocca, Caroline; Page, Adeline; Cantrelle, François-Xavier; Nikolakaki, Eleni; Landrieu, Isabelle; Giannakouros, Thomas

2018-04-01

Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-β-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-β-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation. Copyright © 2018 Elsevier B.V. All rights reserved.

Unraveling the Mechanism Underlying the Glycosylation and Methylation of Anthocyanins in Peach1[C][W

PubMed Central

Cheng, Jun; Wei, Guochao; Zhou, Hui; Gu, Chao; Vimolmangkang, Sornkanok; Liao, Liao; Han, Yuepeng

2014-01-01

Modification of anthocyanin plays an important role in increasing its stability in plants. Here, six anthocyanins were identified in peach (Prunus persica), and their structural diversity is attributed to glycosylation and methylation. Interestingly, peach is quite similar to the wild species Prunus ferganensis but differs from both Prunus davidiana and Prunus kansueasis in terms of anthocyanin composition in flowers. This indicates that peach is probably domesticated from P. ferganensis. Subsequently, genes responsible for both methylation and glycosylation of anthocyanins were identified, and their spatiotemporal expression results in different patterns of anthocyanin accumulation in flowers, leaves, and fruits. Two tandem-duplicated genes encoding flavonoid 3-O-glycosyltransferase (F3GT) in peach, PpUGT78A1 and PpUGT78A2, showed different activity toward anthocyanin, providing an example of divergent evolution of F3GT genes in plants. Two genes encoding anthocyanin O-methyltransferase (AOMT), PpAOMT1 and PpAOMT2, are expressed in leaves and flowers, but only PpAOMT2 is responsible for the O-methylation of anthocyanins at the 3′ position in peach. In addition, our study reveals a novel branch of UGT78 genes in plants that lack the highly conserved intron 2 of the UGT gene family, with a great variation of the amino acid residue at position 22 of the plant secondary product glycosyltransferase box. Our results not only provide insights into the mechanisms underlying anthocyanin glycosylation and methylation in peach but will also aid in future attempts to manipulate flavonoid biosynthesis in peach as well as in other plants. PMID:25106821

Comparison of HPLC/ESI-FTICR MS Versus MALDI-TOF/TOF MS for Glycopeptide Analysis of a Highly Glycosylated HIV Envelope Glycoprotein

PubMed Central

Irungu, Janet; Go, Eden P.; Zhang, Ying; Dalpathado, Dilusha S.; Liao, Hua-Xin; Haynes, Barton F.; Desaire, Heather

2013-01-01

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140ΔCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis. PMID:18565761

25-Hydroxycholesterol Inhibition of Lassa Virus Infection through Aberrant GP1 Glycosylation.

PubMed

Shrivastava-Ranjan, Punya; Bergeron, Éric; Chakrabarti, Ayan K; Albariño, César G; Flint, Mike; Nichol, Stuart T; Spiropoulou, Christina F

2016-12-20

Lassa virus (LASV) infection is a major public health concern due to high fatality rates and limited effective treatment. The interferon-stimulated gene cholesterol 25-hydroxylase (CH25H) encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC). 25HC is involved in regulating cholesterol biosynthesis and has recently been identified as a potent antiviral targeting enveloped virus entry. Here, we show a previously unrecognized role of CH25H in inhibiting LASV glycoprotein glycosylation and the production of infectious virus. Overexpression of CH25H or treatment with 25HC decreased LASV G1 glycoprotein N-glycan maturation and reduced the production of infectious LASV. Depletion of endogenous CH25H using small interfering RNA (siRNA) enhanced the levels of fully glycosylated G1 and increased infectious LASV production. Finally, LASV particles produced from 25HC-treated cells were found to be less infectious, to incorporate aberrantly glycosylated GP1 species, and to be defective in binding alpha-dystroglycan, an attachment and entry receptor. Our findings identify a novel role for CH25H in controlling LASV propagation and indicate that manipulation of the expression of CH25H or the administration of 25HC may be a useful anti-LASV therapy. Lassa fever is an acute viral hemorrhagic fever in humans caused by Lassa virus (LASV). No vaccine for LASV is currently available. Treatment is limited to the administration of ribavirin, which is only effective when given early in the course of illness. Cholesterol 25-hydroxylase (CH25H) is a recently identified interferon-stimulated gene (ISG); it encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC), which inhibits several viruses. Here, we identify a novel antiviral mechanism of 25HC that is dependent on inhibiting the glycosylation of Lassa virus (LASV) glycoprotein and reducing the infectivity of LASV as a means of suppressing viral replication. Since N-linked glycosylation is a

Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress

PubMed Central

Mustafa, Ghazala; Komatsu, Setsuko

2014-01-01

Flooding stress has a negative impact on soybean cultivation because it severely impairs growth and development. To understand the flooding responsive mechanism in early stage soybeans, a glycoproteomic technique was used. Two-day-old soybeans were treated with flooding for 2 days and roots were collected. Globally, the accumulation level of glycoproteins, as revealed by cross-reaction with concanavalin A decreased by 2 days of flooding stress. Glycoproteins were enriched from total protein extracts using concanavalin A lectin resin and analyzed using a gel-free proteomic technique. One-hundred eleven and 69 glycoproteins were identified without and with 2 days of flooding stress, respectively. Functional categorization of these identified glycoproteins indicated that the accumulation level of proteins related to protein degradation, cell wall, and glycolysis increased, while stress-related proteins decreased under flooding stress. Also the accumulation level of glycoproteins localized in the secretory pathway decreased under flooding stress. Out of 23 common glycoproteins between control and flooding conditions, peroxidases and glycosyl hydrolases were decreased by 2 days of flooding stress. mRNA expression levels of proteins in the endoplasmic reticulum and N-glycosylation related proteins were downregulated by flooding stress. These results suggest that flooding might negatively affect the process of N-glycosylation of proteins related to stress and protein degradation; however glycoproteins involved in glycolysis are activated. PMID:25477889

Stress transgenerationally programs metabolic pathways linked to altered mental health.

PubMed

Kiss, Douglas; Ambeskovic, Mirela; Montina, Tony; Metz, Gerlinde A S

2016-12-01

Stress is among the primary causes of mental health disorders, which are the most common reason for disability worldwide. The ubiquity of these disorders, and the costs associated with them, lends a sense of urgency to the efforts to improve prediction and prevention. Down-stream metabolic changes are highly feasible and accessible indicators of pathophysiological processes underlying mental health disorders. Here, we show that remote and cumulative ancestral stress programs central metabolic pathways linked to mental health disorders. The studies used a rat model consisting of a multigenerational stress lineage (the great-great-grandmother and each subsequent generation experienced stress during pregnancy) and a transgenerational stress lineage (only the great-great-grandmother was stressed during pregnancy). Urine samples were collected from adult male F4 offspring and analyzed using 1 H NMR spectroscopy. The results of variable importance analysis based on random variable combination were used for unsupervised multivariate principal component analysis and hierarchical clustering analysis, as well as metabolite set enrichment analysis (MSEA) and pathway analysis. We identified distinct metabolic profiles associated with the multigenerational and transgenerational stress phenotype, with consistent upregulation of hippurate and downregulation of tyrosine, threonine, and histamine. MSEA and pathway analysis showed that these metabolites are involved in catecholamine biosynthesis, immune responses, and microbial host interactions. The identification of metabolic signatures linked to ancestral programming assists in the discovery of gene targets for future studies of epigenetic regulation in pathogenic processes. Ultimately, this research can lead to biomarker discovery for better prediction and prevention of mental health disorders.

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.

The Effect of Linked Learning Certified Pathways on Selected Student Outcomes

ERIC Educational Resources Information Center

Fitzgerald, Robert; Ottem, Randolph; Hufford, Justine

2016-01-01

This report examines outcomes for grade-12 students in academic years (AY) 2010-11, 2011-12, and 2012-13 who were enrolled in a Linked Learning certified pathway (LLCP) in California. Outcomes include student engagement in learning, measured by high school attendance and discipline events, as well as college readiness and postsecondary enrollment.…

Food-grade TiO2 is trapped by intestinal mucus in vitro but does not impair mucin O-glycosylation and short-chain fatty acid synthesis in vivo: implications for gut barrier protection.

PubMed

Talbot, Pauline; Radziwill-Bienkowska, Joanna M; Kamphuis, Jasper B J; Steenkeste, Karine; Bettini, Sarah; Robert, Véronique; Noordine, Marie-Louise; Mayeur, Camille; Gaultier, Eric; Langella, Philippe; Robbe-Masselot, Catherine; Houdeau, Eric; Thomas, Muriel; Mercier-Bonin, Muriel

2018-06-19

Titanium dioxide (TiO 2 ) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO 2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO 2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO 2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO 2 particles accumulated inside "patchy" regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular "islands" located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO 2 particles was attributed to this mucus patchy structure. We compared TiO 2 -mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. Food-grade TiO 2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under "healthy gut

Prion propagation in cells expressing PrP glycosylation mutants.

PubMed

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Dom34 Links Translation to Protein O-mannosylation.

PubMed

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S; Lagadec, Quentin; Cantero, Pilar D; Pfeifer, Eugen; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F

2016-10-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

Distinct roles of N- and O-glycans in cellulase activity and stability

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

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Distinct roles of N- and O-glycans in cellulase activity and stability

DOE PAGES

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.; ...

2017-12-11

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

O-GlcNAcylation in Cancer Biology: Linking Metabolism and Signaling.

PubMed

Ferrer, Christina M; Sodi, Valerie L; Reginato, Mauricio J

2016-08-14

The hexosamine biosynthetic pathway (HBP) is highly dependent on multiple metabolic nutrients including glucose, glutamine, and acetyl-CoA. Increased flux through HBP leads to elevated post-translational addition of β-D-N-acetylglucosamine sugars to nuclear and cytoplasmic proteins. Increased total O-GlcNAcylation is emerging as a general characteristic of cancer cells, and recent studies suggest that O-GlcNAcylation is a central communicator of nutritional status to control key signaling and metabolic pathways that regulate multiple cancer cell phenotypes. This review summarizes our current understanding of changes of O-GlcNAc cycling enzymes in cancer, the role of O-GlcNAcylation in tumorigenesis, and the current challenges in targeting this pathway therapeutically. Copyright © 2016 Elsevier Ltd. All rights reserved.

Integrative pathway analysis of a genome-wide association study of V̇o2max response to exercise training

PubMed Central

Vivar, Juan C.; Sarzynski, Mark A.; Sung, Yun Ju; Timmons, James A.; Bouchard, Claude; Rankinen, Tuomo

2013-01-01

We previously reported the findings from a genome-wide association study of the response of maximal oxygen uptake (V̇o2max) to an exercise program. Here we follow up on these results to generate hypotheses on genes, pathways, and systems involved in the ability to respond to exercise training. A systems biology approach can help us better establish a comprehensive physiological description of what underlies V̇o2maxtrainability. The primary material for this exploration was the individual single-nucleotide polymorphism (SNP), SNP-gene mapping, and statistical significance levels. We aimed to generate novel hypotheses through analyses that go beyond statistical association of single-locus markers. This was accomplished through three complementary approaches: 1) building de novo evidence of gene candidacy through informatics-driven literature mining; 2) aggregating evidence from statistical associations to link variant enrichment in biological pathways to V̇o2max trainability; and 3) predicting possible consequences of variants residing in the pathways of interest. We started with candidate gene prioritization followed by pathway analysis focused on overrepresentation analysis and gene set enrichment analysis. Subsequently, leads were followed using in silico analysis of predicted SNP functions. Pathways related to cellular energetics (pantothenate and CoA biosynthesis; PPAR signaling) and immune functions (complement and coagulation cascades) had the highest levels of SNP burden. In particular, long-chain fatty acid transport and fatty acid oxidation genes and sequence variants were found to influence differences in V̇o2max trainability. Together, these methods allow for the hypothesis-driven ranking and prioritization of genes and pathways for future experimental testing and validation. PMID:23990238

N-Glycosylation of the Na+-Taurocholate Cotransporting Polypeptide (NTCP) Determines Its Trafficking and Stability and Is Required for Hepatitis B Virus Infection.

PubMed

Appelman, Monique D; Chakraborty, Anindita; Protzer, Ulrike; McKeating, Jane A; van de Graaf, Stan F J

2017-01-01

The sodium/bile acid cotransporter NTCP was recently identified as a receptor for hepatitis B virus (HBV). NTCP is glycosylated and the role of glycans in protein trafficking or viral receptor activity is not known. NTCP contains two N-linked glycosylation sites and asparagine amino acid residues N5 and N11 were mutated to a glutamine to generate NTCP with a single glycan (NTCP-N5Q or NTCP- N11Q) or no glycans (NTCP- N5,11Q). HepG2 cells expressing NTCP with a single glycan supported HBV infection at a comparable level to NTCP-WT. The physiological function of NTCP, the uptake of bile acids, was also not affected in cells expressing these single glycosylation variants, consistent with their trafficking to the plasma membrane. However, glycosylation-deficient NTCP (NTCP-N5,11Q) failed to support HBV infection, showed minimal cellular expression and was degraded in the lysosome. This affected the physiological bile acid transporter function of NTCP-N5,11Q in a similar fashion. In conclusion, N-glycosylation is required for efficient NTCP localization at the plasma membrane and subsequent HBV infection and these characteristics are preserved in NTCP carrying a single carbohydrate moiety.

N-Glycosylation of the Na+-Taurocholate Cotransporting Polypeptide (NTCP) Determines Its Trafficking and Stability and Is Required for Hepatitis B Virus Infection

PubMed Central

Appelman, Monique D.; Chakraborty, Anindita; Protzer, Ulrike; McKeating, Jane A.

2017-01-01

The sodium/bile acid cotransporter NTCP was recently identified as a receptor for hepatitis B virus (HBV). NTCP is glycosylated and the role of glycans in protein trafficking or viral receptor activity is not known. NTCP contains two N-linked glycosylation sites and asparagine amino acid residues N5 and N11 were mutated to a glutamine to generate NTCP with a single glycan (NTCP-N5Q or NTCP- N11Q) or no glycans (NTCP- N5,11Q). HepG2 cells expressing NTCP with a single glycan supported HBV infection at a comparable level to NTCP-WT. The physiological function of NTCP, the uptake of bile acids, was also not affected in cells expressing these single glycosylation variants, consistent with their trafficking to the plasma membrane. However, glycosylation-deficient NTCP (NTCP-N5,11Q) failed to support HBV infection, showed minimal cellular expression and was degraded in the lysosome. This affected the physiological bile acid transporter function of NTCP-N5,11Q in a similar fashion. In conclusion, N-glycosylation is required for efficient NTCP localization at the plasma membrane and subsequent HBV infection and these characteristics are preserved in NTCP carrying a single carbohydrate moiety. PMID:28125599

A small multigene hydroxyproline-O-galactosyltransferase family functions in arabinogalactan-protein glycosylation, growth and development in Arabidopsis.

PubMed

Basu, Debarati; Tian, Lu; Wang, Wuda; Bobbs, Shauni; Herock, Hayley; Travers, Andrew; Showalter, Allan M

2015-12-21

Arabinogalactan-proteins (AGPs) are ubiquitous components of cell walls throughout the plant kingdom and are extensively post translationally modified by conversion of proline to hydroxyproline (Hyp) and by addition of arabinogalactan polysaccharides (AG) to Hyp residues. AGPs are implicated to function in various aspects of plant growth and development, but the functional contributions of AGP glycans remain to be elucidated. Hyp glycosylation is initiated by the action of a set of Hyp-O-galactosyltransferase (Hyp-O-GALT) enzymes that remain to be fully characterized. Three members of the GT31 family (GALT3-At3g06440, GALT4-At1g27120, and GALT6-At5g62620) were identified as Hyp-O-GALT genes by heterologous expression in tobacco leaf epidermal cells and examined along with two previously characterized Hyp-O-GALT genes, GALT2 and GALT5. Transcript profiling by real-time PCR of these five Hyp-O-GALTs revealed overlapping but distinct expression patterns. Transiently expressed GALT3, GALT4 and GALT6 fluorescent protein fusions were localized within Golgi vesicles. Biochemical analysis of knock-out mutants for the five Hyp-O-GALT genes revealed significant reductions in both AGP-specific Hyp-O-GALT activity and β-Gal-Yariv precipitable AGPs. Further phenotypic analysis of these mutants demonstrated reduced root hair growth, reduced seed coat mucilage, reduced seed set, and accelerated leaf senescence. The mutants also displayed several conditional phenotypes, including impaired root growth, and defective anisotropic growth of root tips under salt stress, as well as less sensitivity to the growth inhibitory effects of β-Gal-Yariv reagent in roots and pollen tubes. This study provides evidence that all five Hyp-O-GALT genes encode enzymes that catalyze the initial steps of AGP galactosylation and that AGP glycans play essential roles in both vegetative and reproductive plant growth.

Investigating the benefits of scene linking for a pathway HMD: from laboratory flight experiments to flight tests

NASA Astrophysics Data System (ADS)

Schmerwitz, Sven; Többen, Helmut; Lorenz, Bernd; Iijima, Tomoko; Kuritz-Kaiser, Anthea

2006-05-01

Pathway-in-the-sky displays enable pilots to accurately fly difficult trajectories. However, these displays may drive pilots' attention to the aircraft guidance task at the expense of other tasks particularly when the pathway display is located head-down. A pathway HUD may be a viable solution to overcome this disadvantage. Moreover, the pathway may mitigate the perceptual segregation between the static near domain and the dynamic far domain and hence, may improve attention switching between both sources. In order to more comprehensively overcome the perceptual near-to-far domain disconnect alphanumeric symbols could be attached to the pathway leading to a HUD design concept called 'scene-linking'. Two studies are presented that investigated this concept. The first study used a simplified laboratory flight experiment. Pilots (N=14) flew a curved trajectory through mountainous terrain and had to detect display events (discrete changes in a command speed indicator to be matched with current speed) and outside scene events (hostile SAM station on ground). The speed indicators were presented in superposition to the scenery either in fixed position or scene-linked to the pathway. Outside scene event detection was found improved with scene linking, however, flight-path tracking was markedly deteriorated. In the second study a scene-linked pathway concept was implemented on a monocular retinal scanning HMD and tested in real flights on a Do228 involving 5 test pilots. The flight test mainly focused at usability issues of the display in combination with an optical head tracker. Visual and instrument departure and approach tasks were evaluated comparing HMD navigation with standard instrument or terrestrial navigation. The study revealed limitations of the HMD regarding its see-through capability, field of view, weight and wearing comfort that showed to have a strong influence on pilot acceptance rather than rebutting the approach of the display concept as such.

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism1[OPEN

PubMed Central

Laitinen, Teresa

2017-01-01

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H2O2-scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H2O2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism. PMID:28522458

Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2

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

Vita, N.; Magazin, M.; Marchese, E.

We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with (35S)-methionine, or with (3H)-glucosamine and (3H)-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the (35S)-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and themore » structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2.« less

Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC.

PubMed

Totten, Sarah M; Feasley, Christa L; Bermudez, Abel; Pitteri, Sharon J

2017-03-03

Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

PubMed Central

Go, Eden P.; Ding, Haitao; Zhang, Shijian; Ringe, Rajesh P.; Nicely, Nathan; Hua, David; Steinbock, Robert T.; Golabek, Michael; Alin, James; Alam, S. Munir; Cupo, Albert; Haynes, Barton F.; Kappes, John C.; Moore, John P.; Sodroski, Joseph G.

2017-01-01

ABSTRACT HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells. IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following

Equitable Access by Design. A Conceptual Framework for Integrated Student Supports within Linked Learning Pathways

ERIC Educational Resources Information Center

de Velasco, Jorge Ruiz; Newman, Elizabeth; Borsato, Graciela

2016-01-01

This report proposes a conceptual framework for defining and implementing a system of integrated student supports that provides equitable access to college and career readiness via Linked Learning pathways in high schools. The framework emphasizes the central commitment of the Linked Learning approach to challenge prevailing norms of…

A mouse model of a human congenital disorder of glycosylation caused by loss of PMM2

PubMed Central

Chan, Barden; Clasquin, Michelle; Smolen, Gromoslaw A.; Histen, Gavin; Powe, Josh; Chen, Yue; Lin, Zhizhong; Lu, Chenming; Liu, Yan; Cang, Yong; Yan, Zhonghua; Xia, Yuanfeng; Thompson, Ryan; Singleton, Chris; Dorsch, Marion; Silverman, Lee; Su, Shin-San Michael; Freeze, Hudson H.; Jin, Shengfang

2016-01-01

The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease. PMID:27053713

Flagellin glycosylation with pseudaminic acid in Campylobacter and Helicobacter: prospects for development of novel therapeutics.

PubMed

Salah Ud-Din, Abu Iftiaf Md; Roujeinikova, Anna

2018-04-01

Many pathogenic bacteria require flagella-mediated motility to colonise and persist in their hosts. Helicobacter pylori and Campylobacter jejuni are flagellated epsilonproteobacteria associated with several human pathologies, including gastritis, acute diarrhea, gastric carcinoma and neurological disorders. In both species, glycosylation of flagellin with an unusual sugar pseudaminic acid (Pse) plays a crucial role in the biosynthesis of functional flagella, and thereby in bacterial motility and pathogenesis. Pse is found only in pathogenic bacteria. Its biosynthesis via six consecutive enzymatic steps has been extensively studied in H. pylori and C. jejuni. This review highlights the importance of flagella glycosylation and details structural insights into the enzymes in the Pse pathway obtained via a combination of biochemical, crystallographic, and mutagenesis studies of the enzyme-substrate and -inhibitor complexes. It is anticipated that understanding the underlying structural and molecular basis of the catalytic mechanisms of the Pse-synthesising enzymes will pave the way for the development of novel antimicrobials.

The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

PubMed

Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

1998-04-17

The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

Surface Glycosylation Profiles of Urine Extracellular Vesicles

PubMed Central

Gerlach, Jared Q.; Krüger, Anja; Gallogly, Susan; Hanley, Shirley A.; Hogan, Marie C.; Ward, Christopher J.

2013-01-01

Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications. PMID:24069349

Hepatic Inflammation and Fibrosis: Functional Links and Key Pathways

PubMed Central

Seki, Ekihiro; Schwabe, Robert F.

2014-01-01

Inflammation is one of the most characteristic features of chronic liver disease of viral, alcoholic, fatty and autoimmune origin. Inflammation is typically present in all disease stages, and associated with the development of fibrosis, cirrhosis and hepatocellular carcinoma. In the past decade, numerous studies have contributed to improved understanding of the links between hepatic inflammation and fibrosis. Here, we review mechanisms that link inflammation with the development of liver fibrosis, focusing on the role of inflammatory mediators in hepatic stellate cell (HSC) activation and HSC survival during fibrogenesis and fibrosis regression. We will summarize the contributions of different inflammatory cells, including hepatic macrophages, T- and B-lymphocytes, NK cells and platelets, as well as key effectors such as cytokines, chemokines, and damage-associated molecular patterns. Furthermore, we will discuss the relevance of inflammatory signaling pathways for clinical liver disease and for the development of anti-fibrogenic strategies. PMID:25066777

Collagen structural microheterogeneity and a possible role for glycosylated hydroxylysine in type I collagen

PubMed Central

Yamauchi, Mitsuo; Noyes, Claudia; Kuboki, Yoshinori; Mechanic, Gerald L.

1982-01-01

A three-chained peptide from type I collagen, crosslinked by hydroxyaldolhistidine, has been isolated from a tryptic digest of 5 M guanidine·HCl-insoluble bovine skin collagen (a small but as yet unknown percentage of the total collagen in whole skin). OsO4/NaIO4 specifically cleaved the crosslink at its double bond into a two-chained crosslink peptide and a single peptide. The sequence of the two-chained peptide containing the bifunctional crosslink was determined after amino acid analysis of the separated peptides. The crosslink consists of an aldehyde derived from hydroxylysine-87 in the aldehyde-containing cyanogen bromide fragment α1CB5ald and an aldehyde derived from the lysine in the COOH-terminal nonhelical region of the α1CB6ald fragment. The α1CB6ald portion of the peptide exhibited structural microheterogeneity, containing the inverted sequence Ala-Lys-His instead of the normal sequence Lys-Ala-His. This indicates that another structural gene exists for α1(I) chain. The original three-chained peptide did not contain any glycosylated hydroxylysine or glycosylated hydroxyaldolhistidine. The lack of glycosylation of hydroxylysine-87 in α1CB5, which is usually glycosylated, allowed formation of the aldehyde, and this, coupled with the sequence inversion, may have allowed formation of the nonreducible crosslink hydroxyaldolhistidine. We suggest that the role of glycosylation, a posttranslational modification, of specific hydroxylysine residues is to prevent their oxidative deamination to aldehydes, thereby precluding formation of complex stable crosslinks. Complex crosslinks would decrease the rate of collagen turnover. The decrease, with time, would increase the population of stable crosslinked collagen molecules, which would eventually accumulate with age. PMID:6961443

Leading High School Transformation for College and Career Success: A Guide for Developing a System of Linked Learning Pathways

ERIC Educational Resources Information Center

Stearns, Roman

2014-01-01

This ConnectEd Guide for Developing a System of Linked Learning Pathways will introduce school district leaders and their community partners to Linked Learning and a system of quality pathways that can transform high schools, instructional practice, and the student experience. Not intended to be prescriptive, this document can and should be…

Quantifying risk of penile prosthesis infection with elevated glycosylated hemoglobin.

PubMed

Wilson, S K; Carson, C C; Cleves, M A; Delk, J R

1998-05-01

Elevation of glycosylated hemoglobin above levels of 11.5 mg.% has been considered a contraindication to penile prosthesis implantation in diabetic patients. We determine the predictive value of glycosylated hemoglobin A1C in penile prosthesis infections in diabetic and nondiabetic patients to confirm or deny this prevalent opinion. We conducted a 2-year prospective study of 389 patients, including 114 diabetics, who underwent 3-piece penile prosthesis implantation. All patients had similar preoperative preparation without regard to diabetic status, control or glycosylated hemoglobin A1C level. Risk of infection was statistically analyzed for diabetics versus nondiabetics, glycosylated hemoglobin A1C values above and below 11.5 mg.%, insulin dependent versus oral medication diabetics, and fasting blood sugars above and below 180 mg.%. Prosthesis infections developed in 10 diabetics (8.7%) and 11 nondiabetics (4.0%). No increased infection rate was observed in diabetics with high fasting sugars or diabetics on insulin. There was no statistically significant increased infection risk with increased levels of glycosylated hemoglobin A1C among all patients or among only the diabetics. In fact, there was no meaningful difference in the median or mean level of glycosylated hemoglobin A1C in the infected and noninfected patients regardless of diabetes. Use of glycosylated hemoglobin A1C values to identify and exclude surgical candidates with increased risk of infections is not proved by this study. Elevation of fasting sugar or insulin dependence also does not increase risk of infection in diabetics undergoing prosthesis implantation.

N-Glycosylation Determines Ionic Permeability and Desensitization of the TRPV1 Capsaicin Receptor*

PubMed Central

Veldhuis, Nicholas A.; Lew, Michael J.; Abogadie, Fe C.; Poole, Daniel P.; Jennings, Ernest A.; Ivanusic, Jason J.; Eilers, Helge; Bunnett, Nigel W.; McIntyre, Peter

2012-01-01

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca2+]i) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1−/− mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission. PMID:22570472

A Novel Functional Role of Collagen Glycosylation

PubMed Central

Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

2011-01-01

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

A novel Phex mutation with defective glycosylation causes hypophosphatemia and rickets in mice.

PubMed

Xiong, Xiwen; Qi, Xin; Ge, Xiaomei; Gu, Pengyu; Zhao, Jing; Zhao, Qingshun; Gao, Xiang

2008-01-01

N-ethyl-N-nitrosourea (ENU) mutagenesis is a phenotype-driven approach with potential to assign function to every locus in the mouse genome. In this article, we describe a new mutation, Pug, as a mouse model for X-linked hypophosphatemic rickets (XLH) in human. Mice carrying the Pug mutation exhibit abnormal phenotypes including growth retardation, hypophosphatemia and decreased bone mineral density (BMD). The new mutation was mapped to X-chromosome between 65.4 cM and 66.6 cM, where Phex gene resides. Sequence analysis revealed a unique T-to-C transition mutation resulting in Phe-to-Ser substitution at amino acid 80 of PHEX protein. In vitro studies of Pug mutation demonstrated that PHEX(pug) was incompletely glycosylated and sequestrated in the endoplasmic reticulum region of cell, whereas wild-type PHEX could be fully glycosylated and transported to the plasma membrane to exert its function as an endopeptidase. Taken together, the Pug mutant directly confirms the role of Phex in phosphate homeostasis and normal skeletal development and may serves as a new disease model of human hypophosphatemic rickets.

Integration and Validation of the Genome-Scale Metabolic Models of Pichia pastoris: A Comprehensive Update of Protein Glycosylation Pathways, Lipid and Energy Metabolism.

PubMed

Tomàs-Gamisans, Màrius; Ferrer, Pau; Albiol, Joan

2016-01-01

Genome-scale metabolic models (GEMs) are tools that allow predicting a phenotype from a genotype under certain environmental conditions. GEMs have been developed in the last ten years for a broad range of organisms, and are used for multiple purposes such as discovering new properties of metabolic networks, predicting new targets for metabolic engineering, as well as optimizing the cultivation conditions for biochemicals or recombinant protein production. Pichia pastoris is one of the most widely used organisms for heterologous protein expression. There are different GEMs for this methylotrophic yeast of which the most relevant and complete in the published literature are iPP668, PpaMBEL1254 and iLC915. However, these three models differ regarding certain pathways, terminology for metabolites and reactions and annotations. Moreover, GEMs for some species are typically built based on the reconstructed models of related model organisms. In these cases, some organism-specific pathways could be missing or misrepresented. In order to provide an updated and more comprehensive GEM for P. pastoris, we have reconstructed and validated a consensus model integrating and merging all three existing models. In this step a comprehensive review and integration of the metabolic pathways included in each one of these three versions was performed. In addition, the resulting iMT1026 model includes a new description of some metabolic processes. Particularly new information described in recently published literature is included, mainly related to fatty acid and sphingolipid metabolism, glycosylation and cell energetics. Finally the reconstructed model was tested and validated, by comparing the results of the simulations with available empirical physiological datasets results obtained from a wide range of experimental conditions, such as different carbon sources, distinct oxygen availability conditions, as well as producing of two different recombinant proteins. In these simulations, the

You are what you eat: O-linked N-acetylglucosamine in disease, development and epigenetics.

PubMed

Olivier-Van Stichelen, Stéphanie; Hanover, John A

2015-07-01

The O-linked N-acetylglucosamine (O-GlcNAc) modification is both responsive to nutrient availability and capable of altering intracellular cellular signalling. We summarize data defining a role for O-GlcNAcylation in metabolic homeostasis and epigenetic regulation of development in the intrauterine environment. O-GlcNAc transferase (OGT) catalyzes nutrient-driven O-GlcNAc addition and is subject to random X-inactivation. OGT plays key roles in growth factor signalling, stem cell biology, epigenetics and possibly imprinting. The O-GlcNAcase, which removes O-GlcNAc, is subject to tight regulation by higher order chromatin structure. O-GlcNAc cycling plays an important role in the intrauterine environment wherein OGT expression is an important biomarker of placental stress. Regulation of O-GlcNAc cycling by X-inactivation, epigenetic regulation and nutrient-driven processes makes it an ideal candidate for a nutrient-dependent epigenetic regulator of human disease. In addition, O-GlcNAc cycling influences chromatin modifiers critical to the regulation and timing of normal development including the polycomb repression complex and the ten-eleven translocation proteins mediating DNA methyl cytosine demethylation. The pathway also impacts the hypothalamic-pituitary-adrenal axis critical to intrauterine programming influencing disease susceptibility in later life.

Effect of glycosylation on an immunodominant region in the V1V2 variable domain of the HIV-1 envelope gp120 protein

DOE PAGES

Tian, Jianhui; Lopez, Cesar Augusto; Derdeyn, Cynthia A.; ...

2016-10-07

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchangemore » molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. Furthermore, these studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to

Effect of glycosylation on an immunodominant region in the V1V2 variable domain of the HIV-1 envelope gp120 protein

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

Tian, Jianhui; Lopez, Cesar Augusto; Derdeyn, Cynthia A.

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchangemore » molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. Furthermore, these studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

PubMed Central

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Influence of glycosylation of deamidated wheat gliadin on its interaction mechanism with resveratrol.

PubMed

Qiu, Chaoying; Wang, Yong; Teng, Yinglai; Zhao, Mouming

2017-04-15

Gliadin is a main composition of wheat storage protein with unique characteristics. Polyphenol with health benefits tends to form complex with protein. In this study, glycosylation of deamidated wheat gliadin (gliadin) was carried out. Fluorescence quenching was applied to evaluate their binding mechanisms with resveratrol. Results showed that glycosylation could increase the solubility and decrease the surface hydrophobicity of gliadin. Both gliadin and glycosylated gliadin have strong affinity with resveratrol. The thermodynamic parameters of binding process indicated that complexation of resveratrol with gliadin was mainly driven by hydrophobic interaction, while by hydrogen bonds with glycosylated gliadin. The hydrosolubility of resveratrol was dramatically increased especially in the presence of glycosylated gliadin. This was consistent with the higher binding constant of glycosylated gliadin with resveratrol. Therefore, gliadin and glycosylated gliadin are both effective to carry resveratrol or other bioactive compounds, and their binding mechanisms are different due to structural difference. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intergenerational Pathways Linking Childhood Sexual Abuse to HIV Risk Among Women

PubMed Central

Cavanaugh, Courtenay E.; Classen, Catherine C.

2009-01-01

Childhood sexual abuse is prevalent among women and it has been linked to a number of problems affecting women's health and functioning including women's parenting practices. Another body of literature has linked specific maternal parenting practices to daughters’ HIV risk, including mother-daughter sex communication, monitoring/knowledge about daughters’ activities, mother-daughter relationship quality, attitudes towards sex, and modeling of sexual values. This paper reviews and links these two bodies of literature to indicate how maternal histories of childhood sexual abuse may compromise mothers’ parenting practices, which may in turn impact daughters’ HIV risk. We also build upon Malow and colleagues’ model (2006) of the associations between childhood sexual abuse and HIV risk to present a model indicating potential intergenerational pathways between childhood sexual abuse and HIV risk among women. The literature supporting this model and gaps in the literature are described. PMID:19333846

Creating Knock-outs of Conserved Oligomeric Golgi complex subunits using CRISPR-mediated gene editing paired with a selection strategy based on glycosylation defects associated with impaired COG complex function

PubMed Central

Blackburn, Jessica Bailey; Lupashin, Vladimir V.

2017-01-01

Summary The Conserved Oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex’s role in glycosylation homeostasis to obtain COG complex subunit knock-outs (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis. PMID:27632008

Dom34 Links Translation to Protein O-mannosylation

PubMed Central

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S.; Lagadec, Quentin; Cantero, Pilar D.; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F.

2016-01-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5′-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3′-UTR of transcripts. PMID:27768707

Understanding the impact of Fc glycosylation on its conformational changes by molecular dynamics simulations and bioinformatics.

PubMed

Zhang, Yubo

2015-12-01

N-linked glycosylation of Fc at N297 plays an important role in its effector function, aberrance of which would cause disease pathogenesis. Here, we performed all-atom molecular dynamics simulations to explore the effects of Fc glycosylation on its dynamics behaviors. Firstly, equilibrium simulations suggested that Fc deglycosylation was able to induce residual flexibility in its CH2 domain. Besides, the free energy landscape revealed three minimum energy wells in deglycosylated Fc, representing its "open", "semi-closed" and "closed" states. However, we could only observe the "open" state of glycosylated Fc. Supportively, principal component analysis emphasized the prominent motion of delyclosylated Fc and dynamically depicted how it changed from the "open" state to its "closed" state. Secondly, we studied the recognition mechanism of the Fc binding to its partners. Energy decomposition analysis identified key residues of Fc to recognize its two partners P13 and P34. Evidently, electrostatic potential surfaces showed that electrostatic attraction helped to stabilize the interaction between Fc and its partners. Also, relative binding free energies explained different binding affinities in Fc-P13 and Fc-P34. Collectively, these results together provided the structural basis for understanding conformational changes of deglycosylated Fc and the recognition mechanism of the Fc binding to its partners.

Muscular dystrophies due to defective glycosylation of dystroglycan

PubMed Central

Muntoni, F; Brockington, M; Godfrey, C; Ackroyd, M; Robb, S.; Manzur, A; Kinali, M; Mercuri, E; Kaluarachchi, M; Feng, L; Jimenez-Mallebrera, C.; Clement, E; Torelli, S; Sewry, CA; Brown, SC

2007-01-01

Summary Muscular dystrophies are a clinically and genetically heterogeneous group of disorders. Until recently most of the proteins associated with muscular dystrophies were believed to be proteins of the sarcolemma associated with reinforcing the plasma membrane or in facilitating its re-sealing following injury. In the last few years a novel and frequent pathogenic mechanism has been identified that involves the abnormal glycosylation of alpha-dystroglycan (ADG). This peripheral membrane protein undergoes complex and crucial glycosylation steps that enable it to interact with LG domain containing extracellular matrix proteins such as laminins, agrin and perlecan. Mutations in six genes (POMT1, POMT2, POMGnT1, fukutin, FKRP and LARGE) have been identified in patients with reduced glycosylation of ADG. While initially a clear correlation between gene defect and phenotype was observed for each of these 6 genes (for example, Walker Warburg syndrome was associated with mutations in POMT1 and POMT2, Fukuyama congenital muscular dystrophy associated with fukutin mutations, and Muscle Eye Brain disease associated with POMGnT1 mutations), we have recently demonstrated that allelic mutations in each of these 6 genes can result in a much wider spectrum of clinical conditions. Thus, the crucial aspect in determining the phenotypic severity is not which gene is primarily mutated, but how severely the mutation affects the glycosylation of ADG. Systematic mutation analysis of these 6 glycosyltransferases in patients with a dystroglycan glycosylation disorder identifies mutations in approximately 65% suggesting that more genes have yet to be identified. PMID:18646561