Glycoprotein changes in non-insulin dependent diabetic rats: effect of N-benzoyl-D-phenylalanine and metformin.

PubMed

Pari, Leelavinothan; Ashokkumar, Natarajan

2006-01-01

The effect of N-benzoyl-D-phenylalanine (NBDP) and metformin on neonatal streptozotocin (nSTZ) induced diabetes has been studied on plasma and tissue glycoproteins. In some pathological conditions, such as cancer, rheumatoid arthritis and diabetes, there is an abnormal glycosylation of acute phase serum proteins. As most serum proteins are produced in the liver, we have examined glycoprotein metabolism in diabetic condition. To induce non-insulin-dependent diabetes mellitus (NIDDM) a single dose of streptozotocin (100 mg/kg body weight) was injected into two day old rats. After 10-12 weeks, rats weighing above 150 g were selected for NIDDM model. In these rat, blood glucose and plasma glycoproteins were significantly increased whereas plasma insulin was significantly decreased. There was a significant decrease in the level of sialic acid and elevated levels of hexose, hexosamine and fucose in tissues. Oral administration of NBDP and metformin to diabetic rats decreased blood glucose and plasma glycoproteins. Plasma insulin and tissue sialic acid were increased whereas tissue concentrations of hexose, hexosamine and fucose were near normal. Our study suggests that NBDP and metformin possess a significant beneficial effect on glycoproteins in addition to their antidiabetic effect.

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

PubMed Central

Karav, Sercan; Le Parc, Annabelle; Leite Nobrega de Moura Bell, Juliana Maria; Frese, Steven A.; Kirmiz, Nina; Block, David E.; Barile, Daniela

2016-01-01

ABSTRACT Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as

Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway

PubMed Central

Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Horimoto, Satoshi; Sugimoto, Takehiro; Ishikawa, Tokiro; Takeda, Shunichi; Yamamoto, Takashi; Suzuki, Tadashi; Kamiya, Yukiko

2015-01-01

Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ–ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ–ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER. PMID:26572623

The contact site A glycoprotein of Dictyostelium discoideum carries a phospholipid anchor of a novel type.

PubMed Central

Stadler, J; Keenan, T W; Bauer, G; Gerisch, G

1989-01-01

The contact site A glycoprotein, a cell adhesion protein of aggregating Dictyostelium cells, was labeled with fatty acid, myo-inositol, phosphate and ethanolamine in vivo, indicating that the protein is anchored in the membrane by a lipid. This lipid was not susceptible to phosphatidyl inositol specific phospholipase C. When cleaved with nitrous acid or when subjected to acetolysis, the anchor released lipids which were different from those released from Trypanosoma variant cell surface glycoprotein, a protein with a known phosphatidyl inositol-glycan anchor. Resistance to weak and sensitivity to strong alkali indicated that the fatty acid in the contact site A glycolipid anchor was in an amide bond. On incubation with sphingomyelinase, a lipid with the chromatographic behavior of ceramide was released. These results suggest that the contact site A glycoprotein is anchored by a ceramide based lipid glycan. Images PMID:2721485

N-Glycosylation Profiling of Porcine Reproductive and Respiratory Syndrome Virus Envelope Glycoprotein 5

PubMed Central

Li, Juan; Tao, Shujuan; Orlando, Ron; Murtaugh, Michael P.

2015-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV. PMID:25726973

Structural analysis of glycoproteins: building N-linked glycans with Coot.

PubMed

Emsley, Paul; Crispin, Max

2018-04-01

Coot is a graphics application that is used to build or manipulate macromolecular models; its particular forte is manipulation of the model at the residue level. The model-building tools of Coot have been combined and extended to assist or automate the building of N-linked glycans. The model is built by the addition of monosaccharides, placed by variation of internal coordinates. The subsequent model is refined by real-space refinement, which is stabilized with modified and additional restraints. It is hoped that these enhanced building tools will help to reduce building errors of N-linked glycans and improve our knowledge of the structures of glycoproteins.

Standardization, evaluation and early-phase method validation of an analytical scheme for batch-consistency N-glycosylation analysis of recombinant produced glycoproteins.

PubMed

Zietze, Stefan; Müller, Rainer H; Brecht, René

2008-03-01

In order to set up a batch-to-batch-consistency analytical scheme for N-glycosylation analysis, several sample preparation steps including enzyme digestions and fluorophore labelling and two HPLC-methods were established. The whole method scheme was standardized, evaluated and validated according to the requirements on analytical testing in early clinical drug development by usage of a recombinant produced reference glycoprotein (RGP). The standardization of the methods was performed by clearly defined standard operation procedures. During evaluation of the methods, the major interest was in the loss determination of oligosaccharides within the analytical scheme. Validation of the methods was performed with respect to specificity, linearity, repeatability, LOD and LOQ. Due to the fact that reference N-glycan standards were not available, a statistical approach was chosen to derive accuracy from the linearity data. After finishing the validation procedure, defined limits for method variability could be calculated and differences observed in consistency analysis could be separated into significant and incidental ones.

Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers.

PubMed

Melchior, Donald L; Sharom, Frances J; Evers, Raymond; Wright, George E; Chu, Joseph W K; Wright, Stephen E; Chu, Xiaoyan; Yabut, Jocelyn

2012-03-01

P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2=0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 min, and requires minimal quantities of test drug. The method is amenable to robotics and

Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

PubMed Central

Melchior, Donald L.; Sharom, Frances J.; Evers, Raymond; Wright, George E.; Chu, Joseph W.K.; Wright, Stephen E.; Chu, Xiaoyan; Yabut, Jocelyn

2012-01-01

Introduction P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Methods Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Results Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2 = 0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. Discussion This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 minutes, and requires minimal quantities of test

A Rhomboid Protease Gene Deletion Affects a Novel Oligosaccharide N-Linked to the S-layer Glycoprotein of Haloferax volcanii*

PubMed Central

Parente, Juliana; Casabuono, Adriana; Ferrari, María Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; Giménez, María Inés

2014-01-01

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family. PMID:24596091

A rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii.

PubMed

Parente, Juliana; Casabuono, Adriana; Ferrari, María Celeste; Paggi, Roberto Alejandro; De Castro, Rosana Esther; Couto, Alicia Susana; Giménez, María Inés

2014-04-18

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.

Targeted Mass Spectrometric Approach for Biomarker Discovery and Validation with Nonglycosylated Tryptic Peptides from N-linked Glycoproteins in Human Plasma*

PubMed Central

Lee, Ju Yeon; Kim, Jin Young; Park, Gun Wook; Cheon, Mi Hee; Kwon, Kyung-Hoon; Ahn, Yeong Hee; Moon, Myeong Hee; Lee, Hyoung–Joo; Paik, Young Ki; Yoo, Jong Shin

2011-01-01

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma

Increased Bisecting N-Acetylglucosamine and Decreased Branched Chain Glycans of N-linked Glycoproteins in Expressed Prostatic Secretions Associated with Prostate Cancer Progression

PubMed Central

Nyalwidhe, Julius O.; Betesh, Lucy R.; Powers, Thomas W.; Jones, E. Ellen; White, Krista Y.; Burch, Tanya C.; Brooks, Jasmin; Watson, Megan T.; Lance, Raymond S.; Troyer, Dean A.; Semmes, O. John; Mehta, Anand; Drake, Richard R.

2013-01-01

Purpose Using prostatic fluids rich in glycoproteins like prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) , the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging mass spectrometry-based glycopeptide analysis platforms. Experimental Design A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides. Results Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data. Conclusion and clinical relevance Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis. PMID:23775902

nES GEMMA Analysis of Lectins and Their Interactions with Glycoproteins - Separation, Detection, and Sampling of Noncovalent Biospecific Complexes

NASA Astrophysics Data System (ADS)

Engel, Nicole Y.; Weiss, Victor U.; Marchetti-Deschmann, Martina; Allmaier, Günter

2017-01-01

In order to better understand biological events, lectin-glycoprotein interactions are of interest. The possibility to gather more information than the mere positive or negative response for interactions brought mass spectrometry into the center of many research fields. The presented work shows the potential of a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) to detect weak, noncovalent, biospecific interactions besides still unbound glycoproteins and unreacted lectins without prior liquid phase separation. First results for Sambucus nigra agglutinin, concanavalin A, and wheat germ agglutinin and their retained noncovalent interactions with glycoproteins in the gas phase are presented. Electrophoretic mobility diameters (EMDs) were obtained by nES GEMMA for all interaction partners correlating very well with molecular masses determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the individual molecules. Moreover, EMDs measured for the lectin-glycoprotein complexes were in good accordance with theoretically calculated mass values. Special focus was laid on complex formation for different lectin concentrations and binding specificities to evaluate the method with respect to results obtained in the liquid phase. The latter was addressed by capillary electrophoresis on-a-chip (CE-on-a-chip). Of exceptional interest was the fact that the formed complexes could be sampled according to their size onto nitrocellulose membranes after gas-phase separation. Subsequent immunological investigation further proved that the collected complex actually retained its native structure throughout nES GEMMA analysis and sampling.

Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors.

PubMed

Spiro, M J; Spiro, R G

2000-05-01

Although glucose residues in a triglucosyl sequence are essential for the N-glycosylation of proteins and in their monoglucosyl form have been implicated in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial steps in the glycoprotein processing sequence. In order to provide a probe for the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant endo-alpha-mannosidase employing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the change in molecular mass brought about by the release of glucosylated mannose (Glc(1-3)Man). With this approach the presence of two triglucosylated-N-linked oligosaccharides in vesicular stomatis virus (VSV) G protein formed by castanospermine-treated CHO cells or the glucosidase I deficient Lec23 mutant could be clearly demonstrated and an even more pronounced change in migration was observed upon endomannosidase treatment of their more heavily N-glycosylated lysosomal membrane glycoproteins. Furthermore, the G protein of the temperature sensitive VSV ts045 mutant was found to be sensitive to endomannosidase, resulting in a change in electrophoretic mobility consistent with the presence of mono-glucosylated-N-linked oligosaccharides. The finding that endomannosidase also acts effectively on oligosaccharide lipids, as assessed by SDS-PAGE or thin layer chromatography, indicated that it would be a valuable tool in assessing the glucosylation state of these biosynthetic intermediates in normal cells as well as in mutants or altered metabolic states, even if the polymannose portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during glycoprotein quality control and when used together with endo-beta-N- acetylglucosaminidase H can distinguish between those

MRM validation of targeted nonglycosylated peptides from N-glycoprotein biomarkers using direct trypsin digestion of undepleted human plasma.

PubMed

Lee, Ju Yeon; Kim, Jin Young; Cheon, Mi Hee; Park, Gun Wook; Ahn, Yeong Hee; Moon, Myeong Hee; Yoo, Jong Shin

2014-02-26

A rapid, simple, and reproducible MRM-based validation method for serological glycoprotein biomarkers in clinical use was developed by targeting the nonglycosylated tryptic peptides adjacent to N-glycosylation sites. Since changes in protein glycosylation are known to be associated with a variety of diseases, glycoproteins have been major targets in biomarker discovery. We previously found that nonglycosylated tryptic peptides adjacent to N-glycosylation sites differed in concentration between normal and hepatocellular carcinoma (HCC) plasma due to differences in steric hindrance of the glycan moiety in N-glycoproteins to tryptic digestion (Lee et al., 2011). To increase the feasibility and applicability of clinical validation of biomarker candidates (nonglycosylated tryptic peptides), we developed a method to effectively monitor nonglycosylated tryptic peptides from a large number of plasma samples and to reduce the total analysis time with maximizing the effect of steric hindrance by the glycans during digestion of glycoproteins. The AUC values of targeted nonglycosylated tryptic peptides were excellent (0.955 for GQYCYELDEK, 0.880 for FEDGVLDPDYPR and 0.907 for TEDTIFLR), indicating that these could be effective biomarkers for hepatocellular carcinoma. This method provides the necessary throughput required to validate glycoprotein biomarkers, as well as quantitative accuracy for human plasma analysis, and should be amenable to clinical use. Difficulties in verifying and validating putative protein biomarkers are often caused by complex sample preparation procedures required to determine their concentrations in a large number of plasma samples. To solve the difficulties, we developed MRM-based protein biomarker assays that greatly reduce complex, time-consuming, and less reproducible sample pretreatment steps in plasma for clinical implementation. First, we used undepleted human plasma samples without any enrichment procedures. Using nanoLC/MS/MS, we targeted

Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

PubMed Central

Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

2015-01-01

Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

Interaction of pyridostigmine bromide and N,N-diethyl-m-toluamide alone and in combination with P-glycoprotein expressed in Escherichia coli leaky mutant.

PubMed

El-Masry, Eman M; Abou-Donia, Mohamed B

2006-05-01

P-glycoprotein (P-gp), the most extensively studied ATP-binding transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of the cell. This study was carried out to determine the effect of N,N-diethyl-m-toluamide (DEET) and pyridostigmine bromide (PB), alone and in combination, on P-gp expression using Escherichia coli leaky mutant transformed with Mdr1 gene (pT5-7/mdr1), which codes for P-gp or lactose permease (pT5-7/lacY) as negative control. Also, daunomycin (a known P-gp sustrate) was used as a positive control and reserpine (a known P-gp inhibitor) served as a negative control. An in vitro cell-resistant assay was used to monitor the potential of test compounds to interact with P-gp. Following exposure of the cells to pyridostigmine bromide or daunomycin, P-gp conferred significant resistance against both compounds, while reserpine and DEET significantly inhibited the glycoprotein. Cells were grown in the presence of noncytotoxic concentrations of daunomycin, pyridostigmine bromide, reserpine, or DEET, and membrane fractions were examined by Western immunoblotting for expression of P-gp. Daunomycin induced P-gp expression quantitatively more than pyridostigmine bromide, while reserpine and DEET significantly inhibited P-gp expression in cells harboring mdr1. Photoaffinity labeling experiment performed with the P-gp ligand [125I]iodoarylazidoprazosin demonstrated that compounds that induced or inhibited P-gp transport activity also bound to P-gp. DEET was also found to be a potent inhibitor of P-gp-mediated ATPase activity, whereas pyridostigmine bromide increased P-gp ATPase activity. Cells expressing P-gp or lac permease were exposed to pyridostigmine bromide and DEET, alone and in combination. Noncytotoxic concentrations of DEET significantly inhibited P-gp-mediated resistance against pyridostigmine bromide, resulting in a reduction of the number of effective drug interactions with biological targets. An explanation of

The hydroxyapatite-binding regions of a rat salivary glycoprotein.

PubMed

Embery, G; Green, D R

1989-09-01

The regions of a salivary sulphated glycoprotein which are involved in its attachment to hydroxyapatite (Biogel HTP) have been characterised. The sulphated glycoprotein, a 35S-labelled preparation from mixed palatal and buccal minor gland secretions of the rat was bound onto hydroxyapatite and the resultant glycoprotein-hydroxyapatite complex was sequentially digested with pronase E and alpha-L-fucosidase, a treatment which released 86.8% +/- 1.7% of the radioactivity of the initially bound glycoprotein. The fragments which remained attached to the hydroxyapatite after enzymic digestion were fractionated on Sephadex G-25 and analysed for carbohydrate and amino acid components. A range of amino acids were detected which could reflect both glycosylated and non-glycosylated-binding regions. Sialic acid, although considered to be involved in the attachment process was not detected in any of the fragments remaining after enzymic digestion, a finding which provides indirect evidence that the enzymically liberated products do not subsequently re-attach to the hydroxyapatite surface. The notable feature of the fractions with average Mr estimated at 1000 or less is the high proportion of N-acetylhexosamine and N-acetylgalactosamine. It is apparent that the hexosamine residues, which normally bear the ester sulphate moieties of sulphated glycoproteins, play an important role in the attachment of sulphated glycoproteins to hydroxyapatite.

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

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

PubMed Central

Ahi, Yadvinder S.; Zhang, Shu; Thappeta, Yashna; Denman, Audrey; Feizpour, Amin; Reinhard, Bjoern; Muriaux, Delphine; Fivash, Matthew J.

2016-01-01

ABSTRACT Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes. PMID:27879338

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

PubMed

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

2018-03-21

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

Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

PubMed Central

Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.

2014-01-01

ABSTRACT The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex. PMID:25352624

Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

USDA-ARS?s Scientific Manuscript database

Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain.

PubMed

González, Silvia A; Affranchino, José L

2016-07-01

The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding. Of note, there are two contiguous and highly conserved potential N-glycosylation sites ((418)NST(420) and (422)NLT(424)) located at the C-terminal side of the V3 domain. We therefore decided to study the relevance for Env functions of these N-glycosylation motifs and found that disruption of both of them by introducing the N418Q/N422Q double amino acid substitution drastically impairs Env processing into the SU and TM subunits. Moreover, the simultaneous mutation of these N-glycosylation sites prevents Env incorporation into virions and Env-mediated cell-to-cell fusion. Notably, a recombinant soluble version of the SU glycoprotein carrying the double amino acid replacement N418Q/N422Q at the V3 C-terminal side binds to CXCR4 with an efficiency similar to that of wild-type SU.

The adhesion of Pseudomonas aeruginosa to high molecular weight human tear film species corresponds to glycoproteins reactive with Sambucus nigra lectin.

PubMed

Aristoteli, Lina Panayiota; Willcox, Mark D P

2006-11-01

Pseudomonas aeruginosa is a pathogen gaining prevalence in contact lens-related corneal ulcers. Tear outflow protects the ocular surface, where high molecular weight tear glycoproteins bind bacteria for removal from the eye. The purpose of the present study was to identify glycoproteins in human tears involved in the adhesion of ocular P. aeruginosa isolates. Basal human tears were applied to a bacterial adhesion assay involving electrophoretic separation of tear components, transfer to nitrocellulose and incubation with biotin-labelled bacteria. Glycoproteins were further characterised using lectin profiling. The results showed large-dimension agarose gels were imperative for the detection of at least four glycoproteins with a migration >200 kDa, including species not previously identified. P. aeruginosa 6294 preferentially bound to a well-defined glycoprotein near the origin of the gel that, unlike other glycoproteins >200 kDa, reacted with Sambucus nigra lectin (sialic acid alpha2-6) but not WGA lectin (N-acetylglucosamine, sialic acid alpha2-3). Adhesion did not involve free biotin label or hydrophobic interactions. Also, the pre-incubation of separated tear glycoproteins with S. nigra lectin increased subsequent adhesion of 6294 to this tear glycoprotein. The less virulent Paer1 strain showed diffuse adhesion in the S. nigra-reactive region at the gel origin. In conclusion, an overlay adhesion assay was developed that identified slow-migrating sialylated glycoprotein species in human tears preferentially bound by P. aeruginosa ocular strains, and S. nigra lectin seemed to enhance the interaction. The study provides a basis for direct investigation of bacterial adhesion to glycoproteins with an apparent migration >200 kDa in tear fluid.

Mannostatin A, a new glycoprotein-processing inhibitor.

PubMed

Tropea, J E; Kaushal, G P; Pastuszak, I; Mitchell, M; Aoyagi, T; Molyneux, R J; Elbein, A D

1990-10-30

Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal alpha-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward alpha- and beta-glucosidase and galactosidase as well as beta-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal alpha-mannosidases, with estimated IC50's of 70 nM, 450 nM, and 160 nM, respectively. The type of inhibition was competitive in nature. This compound also proved to be an effective competitive inhibitor of the glycoprotein-processing enzyme mannosidase II (IC50 of about 10-15 nM with p-nitrophenyl alpha-D-mannopyranoside as substrate, and about 90 nM with [3H]mannose-labeled GlcNAc-Man5GlcNAc as substrate). However, it was virtually inactive toward mannosidase I. The N-acetylated derivative of mannostatin A had no inhibitory activity. In cell culture studies, mannostatin A also proved to be a potent inhibitor of glycoprotein processing. Thus, in influenza virus infected Madin Darby canine kidney (MDCK) cells, mannostatin A blocked the normal formation of complex types of oligosaccharides on the viral glycoproteins and caused the accumulation of hybrid types of oligosaccharides. This observation is in keeping with other data which indicate that the site of action of mannostatin A is mannosidase II. Thus, mannostatin A represents the first nonalkaloidal processing inhibitor and adds to the growing list of chemical structures that can have important biological activity.

Introducing N-glycans into natural products through a chemoenzymatic approach.

PubMed

Huang, Wei; Ochiai, Hirofumi; Zhang, Xinyu; Wang, Lai-Xi

2008-11-24

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-beta-N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific, and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural products may bestow novel properties onto these natural products for drug discovery and development.

Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation.

PubMed

Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W Andy

2016-11-30

Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

Glycoprotein of the wall of sycamore tissue-culture cells.

PubMed

Heath, M F; Northcote, D H

1971-12-01

1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the alpha-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the alpha-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.

Square-wave voltammetry assays for glycoproteins on nanoporous gold

PubMed Central

Pandey, Binod; Bhattarai, Jay K.; Pornsuriyasak, Papapida; Fujikawa, Kohki; Catania, Rosa; Demchenko, Alexei V.; Stine, Keith J.

2014-01-01

Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A – ALP (or soybean agglutinin – ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A–ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL−1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay. PMID:24611035

N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN.

PubMed

Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E; Huante, Matthew B; Slack, Olga A L; Carpio, Victor H; Freiberg, Alexander N; Ikegami, Tetsuro

2016-05-23

Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)-any amino acid (X)-serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: "Gc-large" and "Gc-small", and N1077 was responsible for "Gc-large" band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

PubMed Central

Ferris, Sean P.; Jaber, Nikita S.; Molinari, Maurizio; Arvan, Peter; Kaufman, Randal J.

2013-01-01

Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER. PMID:23864712

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum

PubMed Central

1995-01-01

It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin- like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP- Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides. PMID:7642696

Identification of Poly-N-Acetyllactosamine-Carrying Glycoproteins from HL-60 Human Promyelocytic Leukemia Cells Using a Site-Specific Glycome Analysis Method, Glyco-RIDGE

NASA Astrophysics Data System (ADS)

Togayachi, Akira; Tomioka, Azusa; Fujita, Mika; Sukegawa, Masako; Noro, Erika; Takakura, Daisuke; Miyazaki, Michiyo; Shikanai, Toshihide; Narimatsu, Hisashi; Kaji, Hiroyuki

2018-04-01

To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE). This method used accurate masses and retention times of glycopeptides, without requiring MS2, and could be applied to complex mixtures. To increase the number of identified peptide, fractionation of sample glycopeptides for reduction of sample complexity is required. Therefore, in this study, glycopeptides were fractionated into four fractions by hydrophilic interaction chromatography, and each fraction was analyzed using the Glyco-RIDGE method. As a result, many glycopeptides having long glycans were enriched in the highest hydrophilic fraction. Based on the monosaccharide composition, these glycans were thought to be poly-N-acetyllactosamine (polylactosamine [pLN]), and 31 pLN-carrier proteins were identified in HL-60 cells. Gene ontology enrichment analysis revealed that pLN carriers included many molecules related to signal transduction, receptors, and cell adhesion. Thus, these findings provided important insights into the analysis of the glycoproteome using our novel Glyco-RIDGE method. [Figure not available: see fulltext.

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins.

PubMed

Ahi, Yadvinder S; Zhang, Shu; Thappeta, Yashna; Denman, Audrey; Feizpour, Amin; Gummuluru, Suryaram; Reinhard, Bjoern; Muriaux, Delphine; Fivash, Matthew J; Rein, Alan

2016-11-22

Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called "glycogag" (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes. Many murine leukemia viruses (MLVs) encode a protein called "glycogag." The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all

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.

N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

PubMed Central

Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E.; Huante, Matthew B.; Slack, Olga A.L.; Carpio, Victor H.; Freiberg, Alexander N.; Ikegami, Tetsuro

2016-01-01

Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)–any amino acid (X)–serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN. PMID:27223297

Dynamics of glycoprotein charge in the evolutionary history of human influenza.

PubMed

Arinaminpathy, Nimalan; Grenfell, Bryan

2010-12-30

Influenza viruses show a significant capacity to evade host immunity; this is manifest both as large occasional jumps in the antigenic phenotype of viral surface molecules and in gradual antigenic changes leading to annual influenza epidemics in humans. Recent mouse studies show that avidity for host cells can play an important role in polyclonal antibody escape, and further that electrostatic charge of the hemagglutinin glycoprotein can contribute to such avidity. We test the role of glycoprotein charge on sequence data from the three major subtypes of influenza A in humans, using a simple method of calculating net glycoprotein charge. Of all subtypes, H3N2 in humans shows a striking pattern of increasing positive charge since its introduction in 1968. Notably, this trend applies to both hemagglutinin and neuraminidase glycoproteins. In the late 1980s hemagglutinin charge reached a plateau, while neuraminidase charge started to decline. We identify key groups of amino acid sites involved in this charge trend. To our knowledge these are the first indications that, for human H3N2, net glycoprotein charge covaries strongly with antigenic drift on a global scale. Further work is needed to elucidate how such charge interacts with other immune escape mechanisms, such as glycosylation, and we discuss important questions arising for future study.

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.

Variant surface glycoprotein density defines an immune evasion threshold for African trypanosomes undergoing antigenic variation.

PubMed

Pinger, Jason; Chowdhury, Shanin; Papavasiliou, F Nina

2017-10-10

Trypanosoma brucei is a protozoan parasite that evades its host's adaptive immune response by repeatedly replacing its dense variant surface glycoprotein (VSG) coat from its large genomic VSG repertoire. While the mechanisms regulating VSG gene expression and diversification have been examined extensively, the dynamics of VSG coat replacement at the protein level, and the impact of this process on successful immune evasion, remain unclear. Here we evaluate the rate of VSG replacement at the trypanosome surface following a genetic VSG switch, and show that full coat replacement requires several days to complete. Using in vivo infection assays, we demonstrate that parasites undergoing coat replacement are only vulnerable to clearance via early IgM antibodies for a limited time. Finally, we show that IgM loses its ability to mediate trypanosome clearance at unexpectedly early stages of coat replacement based on a critical density threshold of its cognate VSGs on the parasite surface. Trypanosoma brucei evades the host immune system through replacement of a variant surface glycoprotein (VSG) coat. Here, the authors show that VSG replacement takes several days to complete, and the parasite is vulnerable to the host immune system for a short period of time during the process.

Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.

PubMed

Hirayama, Hiroto; Seino, Junichi; Kitajima, Toshihiko; Jigami, Yoshifumi; Suzuki, Tadashi

2010-04-16

In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

PubMed

Giménez, Estela; Balmaña, Meritxell; Figueras, Joan; Fort, Esther; de Bolós, Carme; Sanz-Nebot, Victòria; Peracaula, Rosa; Rizzi, Andreas

2015-03-25

In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for

Synthesis of N-(β-D-glycuronopyranosyl)alkanamides and 1-(β-D-glycuronopyranosyl)-4-phenyl-[1,2,3]-triazoles as N-glycoprotein linkage region analogs: examination of the effect of C5 substituent on the N-glycosidic torsion (ΦN) based on X-ray crystallography.

PubMed

Mathiselvam, Manoharan; Loganathan, Duraikkannu; Varghese, Babu

2013-10-18

The torsion angle around the N-glycoprotein linkage region (GlcNAc-Asn) is an important factor for presenting sugar on the cell surface which is crucial for many biological processes. Earlier studies using model and analogs showed that this important torsion angle is greatly influenced by substitutions in the sugar part. In the present work, uronic acid alkanamides and triazole derivatives have been designed and synthesized as newer analogs of N-glycoprotein linkage region to understand the influence of the carboxylic group on linkage region torsion as well as on molecular packing. Crystal structure of N-(β-D-galacturonopyranosyl)acetamide is solved with the space group of P22121. Comparison of the torsion angle and molecular packing of this compound with N-(β-D-galactopyranosyl)acetamide showed that changing the C6-hydoxymethyl group to the carboxylic acid group has minimum influence on the N-glycosidic torsion angle, ΦN and significant influence on the molecular packing. Copyright © 2013 Elsevier Ltd. All rights reserved.

Tromantadine inhibits HSV-1 induced syncytia formation and viral glycoprotein processing

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

Ickes, D.E.

1989-01-01

Tromantadine inhibits a late event in Herpes Simplex Virus Type 1 (HSV-1) replication, visualized by the inhibition of both the size and number of syncytia. Tromantadine can be added at any time between 1 and 9 h post infection with complete inhibition of syncytia formation. Glycan synthesis of the viral glycoproteins, important for syncytia formation, is incomplete due to tromantadine treatment. Tromantadine does not inhibit the initiation of glycosylation, since viral glycoproteins, gX{sub t}, synthesized in the presence of tromantadine still incorporate {sup 3}H-glucosamine. Tromantadine does not inhibit the transport of t e viral glycoproteins to the cell surface, sincemore » glycoproteins B, C, and D are expressed, as demonstrated by immunofluorescence. Tromantadine inhibition of HSV-1 glycoprotein processing is demonstrated by an increase in mobility of the radioimmunoprecipitated gX{sub t}, on SDS-PAGE. The gX{sub t} of KOS, a non-syncytial strain of HSV-1, had a similar increase in mobility, suggesting that the block in glycoprotein processing is a general effect of tromantadine treatment. Fucose, which is incorporated into oligosaccharides in the medial Golgi, is incorporated into gX{sub t}, indicating that the tromantadine block in glycoprotein processing occurs after this step. Lectin binding studies and SDS-PAGE analysis of gC processed in the presence of tromantadine, gC{sub t}, indicates that it has terminal galactose residues in both N- and O-linked glycans (binds Peanut and Ricin Agglutinins, respectively). The inhibition of sialylation of N-linked glycans by tromantadine was indicated by the extent of the increase in SDS-PAGE mobility of the G protein from Vesicular Stomatitis Virus. O-glycanase digestion and SDS-PAGE analysis of gC{sub t} indicate that the O-linked disaccharide NAcGal-Galactose is present.« less

Free Oligosaccharides to Monitor Glycoprotein Endoplasmic Reticulum-associated Degradation in Saccharomyces cerevisiae*

PubMed Central

Hirayama, Hiroto; Seino, Junichi; Kitajima, Toshihiko; Jigami, Yoshifumi; Suzuki, Tadashi

2010-01-01

In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. “Free” forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-β-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole α-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae. PMID:20150426

The Role of Conserved N-Linked Glycans on Ebola Virus Glycoprotein 2.

PubMed

Lennemann, Nicholas J; Walkner, Madeline; Berkebile, Abigail R; Patel, Neil; Maury, Wendy

2015-10-01

N-linked glycosylation is a common posttranslational modification found on viral glycoproteins (GPs) and involved in promoting expression, cellular attachment, protection from proteases, and antibody evasion. The GP subunit GP2 of filoviruses contains 2 completely conserved N-linked glycosylation sites (NGSs) at N563 and N618, suggesting that they have been maintained through selective pressures. We assessed mutants lacking these glycans for expression and function to understand the role of these sites during Ebola virus entry. Elimination of either GP2 glycan individually had a modest effect on GP expression and no impact on antibody neutralization of vesicular stomatitis virus pseudotyped with Ebola virus GP. However, loss of the N563 glycan enhanced entry by 2-fold and eliminated GP detection by a well-characterized monoclonal antibody KZ52. Loss of both sites dramatically decreased GP expression and abolished entry. Surprisingly, a GP that retained a single NGS at N563, eliminating the remaining 16 NGSs from GP1 and GP2, had detectable expression, a modest increase in entry, and pronounced sensitivity to antibody neutralization. Our findings support the importance of the GP2 glycans in GP expression/structure, transduction efficiency, and antibody neutralization, particularly when N-linked glycans are also removed from GP1. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus

USGS Publications Warehouse

Huang, C.; Chien, M.S.; Landolt, M.L.; Batts, W.; Winton, J.

1996-01-01

Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.

Rapid analysis of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed-phase ultra-performance liquid chromatography with fluorescence detection and electrospray ionization time-of-flight mass spectrometry.

PubMed

Kurihara, Takamasa; Min, Jun Zhe; Hirata, Asuka; Toyo'oka, Toshimasa; Inagaki, Shinsuke

2009-05-01

Rapid, selective and sensitive determination of N-linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The asparaginyl-oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel-permeation chromatography were labeled with 1-pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF-MS. The PSC-labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI-TOF-MS. As the results, 15, eight and four kinds of N-linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N-linked oligosaccharides in various glycoproteins seems to be possible.

Carbohydrate moieties of myelin-associated glycoprotein, major glycoprotein of the peripheral nervous system myelin and other myelin glycoproteins potentially involved in cell adhesion.

PubMed

Badache, A; Burger, D; Villarroya, H; Robert, Y; Kuchler, S; Steck, A J; Zanetta, J P

1992-01-01

The myelin-associated glycoprotein (MAG) and the major glycoprotein of the peripheral nervous system myelin (P0) are two members of the family of cell adhesion molecules (CAMs). A role in cell adhesion of the carbohydrate moiety of these molecules has been attributed to the presence of N-glycans bearing the HNK-1 carbohydrate epitope. On the other hand, it has been suggested that these glycoproteins could be ligands of an endogenous mannose-binding lectin present in myelin, the cerebellar soluble lectin (CSL). In order to further document the heterogeneity of the glycans of these two CAMs, we have used several probes: an anti-carbohydrate antibody of the HNK-1 type, called Elec-39, the plant lectin concanavalin A (ConA), and the endogenous lectin CSL involved in myelin compaction. This study shows that CSL binds to a small proportion of the polypeptide chains of MAG found in adult CNS of rats and man and the polypeptide chains of P0 molecules from adult human and rat sciatic nerve. For MAG from adult rat brain, the binding of CSL is restricted to glycans of polypeptide chains which could be separated from the others according to their solubility properties. These MAG molecular entities react also with the Elec-39 antibody and with ConA. These results confirm that P0 and MAG are heterogeneous in their carbohydrate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycoproteomic analysis of bronchoalveolar lavage (BAL) fluid identifies tumor-associated glycoproteins from lung adenocarcinoma.

PubMed

Li, Qing Kay; Shah, Punit; Li, Yan; Aiyetan, Paul O; Chen, Jing; Yung, Rex; Molena, Daniela; Gabrielson, Edward; Askin, Frederic; Chan, Daniel W; Zhang, Hui

2013-08-02

Cytological examination of cells from bronchoalveolar lavage (BAL) is commonly used for the diagnosis of lung cancer. Proteins released from lung cancer cells into BAL may serve as biomarkers for cancer detection. In this study, N-glycoproteins in eight cases of BAL fluid, as well as eight lung adenocarcinoma tissues and eight tumor-matched normal lung tissues, were analyzed using the solid-phase extraction of N-glycoprotein (SPEG), iTRAQ labeling, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of 80 glycoproteins found in BAL specimens, 32 were identified in both cancer BAL and cancer tissues, with levels of 25 glycoproteins showing at least a 2-fold difference between cancer and benign BAL. Among them, eight glycoproteins showed greater than 2-fold elevations in cancer BAL, including Neutrophil elastase (NE), Integrin alpha-M, Cullin-4B, Napsin A, lysosome-associated membrane protein 2 (LAMP2), Cathepsin D, BPI fold-containing family B member 2, and Neutrophil gelatinase-associated lipocalin. The levels of Napsin A in cancer BAL were further verified in independently collected 39 BAL specimens using an ELISA assay. Our study demonstrates that potential protein biomarkers in BAL fluid can be detected and quantified.

Isolement et caractérisation de deux subunites constitutives des glycoproteines de structure du tissu sous cutané de lapin.

PubMed

Randoux, A; CornilletStoupy, J; Desanti, M; Borel, J P

1976-09-28

Structural glycoproteins have been extracted by 8 M ureau from the insoluble residue remaining after collagenase digestion of rabbit dermis and purified by Sepharose 4 B chromatography. After reduction and alkylation, Dowex 1 x 2 chromatography allowed separation of two structural glycoproteins (D1 and D2) in an homogenous state as shown by chromatographic and electrophoretic behaviour as well as N terminal amino acid determination. These two glycoproteins have a molecular weight of about 16 000. Their amino acid compositions (very similar), are characterized by a high level of dicarboxylic amino acids and the absence of hydroxyproline and hydroxylysine. The less acidic glycoprotein (D1) has glycine for N terminal amino acid and contains 10.4 percent of bound carbohydrates. The glycoprotein D2 contains 5.1 percent of bound carbohydrates and its N terminal amino acid is glutamic acid.

Development of Glycoprotein Capture-Based Label-Free Method for the High-throughput Screening of Differential Glycoproteins in Hepatocellular Carcinoma*

PubMed Central

Chen, Rui; Tan, Yexiong; Wang, Min; Wang, Fangjun; Yao, Zhenzhen; Dong, Liwei; Ye, Mingliang; Wang, Hongyang; Zou, Hanfa

2011-01-01

A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers. PMID:21474793

Requirements for cell rounding and surface protein down-regulation by Ebola virus glycoprotein.

PubMed

Francica, Joseph R; Matukonis, Meghan K; Bates, Paul

2009-01-20

Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of beta1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

Modeling of Oligosaccharides within Glycoproteins from Free-Energy Landscapes

PubMed Central

2017-01-01

In spite of the abundance of glycoproteins in biological processes, relatively little three-dimensional structural data is available for glycan structures. Here, we study the structure and flexibility of the vast majority of mammalian oligosaccharides appearing in N- and O-glycosylated proteins using a bottom up approach. We report the conformational free-energy landscapes of all relevant glycosidic linkages as obtained from local elevation simulations and subsequent umbrella sampling. To the best of our knowledge, this represents the first complete conformational library for the construction of N- and O-glycan structures. Next, we systematically study the effect of neighboring residues, by extensively simulating all relevant trisaccharides and one tetrasaccharide. This allows for an unprecedented comparison of disaccharide linkages in large oligosaccharides. With a small number of exceptions, the conformational preferences in the larger structures are very similar as in the disaccharides. This, finally, allows us to suggest several efficient approaches to construct complete N- and O-glycans on glycoproteins, as exemplified on two relevant examples. PMID:28816453

Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter.

PubMed

Romsicki, Y; Sharom, F J

2001-06-12

The P-glycoprotein multidrug transporter acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. The protein is proposed to interact with its substrates within the hydrophobic interior of the membrane. There is indirect evidence to suggest that P-glycoprotein can also transport, or "flip", short chain fluorescent lipids between leaflets of the membrane. In this study, we use a fluorescence quenching technique to directly show that P-glycoprotein reconstituted into proteoliposomes translocates a wide variety of NBD lipids from the outer to the inner leaflet of the bilayer. Flippase activity depended on ATP hydrolysis at the outer surface of the proteoliposome, and was inhibited by vanadate. P-Glycoprotein exhibited a broad specificity for phospholipids, and translocated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Lipid derivatives that were flipped included molecules with long, short, unsaturated, and saturated acyl chains and species with the NBD group covalently linked to either acyl chains or the headgroup. The extent of lipid translocation from the outer to the inner leaflet in a 20 min period at 37 degrees C was directly estimated, and fell in the range of 0.36-1.83 nmol/mg of protein. Phospholipid flipping was inhibited in a concentration-dependent, saturable fashion by various substrates and modulators, including vinblastine, verapamil, and cyclosporin A, and the efficiency of inhibition correlated well with the affinity of binding to Pgp. Taken together, these results suggest that P-glycoprotein carries out both lipid translocation and drug transport by the same path. The transporter may be a generic flippase for hydrophobic molecules with the correct steric attributes that are present within the membrane interior.

Determination of N-linked glycosylation in viral glycoproteins by negative ion mass spectrometry and ion mobility

PubMed Central

Bitto, David; Harvey, David J.; Halldorsson, Steinar; Doores, Katie J.; Pritchard, Laura K.; Huiskonen, Juha T.; Bowden, Thomas A.; Crispin, Max

2016-01-01

Summary Glycan analysis of virion-derived glycoproteins is challenging due to the difficulties in glycoprotein isolation and low sample abundance. Here, we describe how ion mobility mass spectrometry can be used to obtain spectra from virion samples. We also describe how negative ion fragmentation of glycans can be used to probe structural features of virion glycans. PMID:26169737

Crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo‐electron microscopy

PubMed Central

Walls, Alexandra; Tortorici, M. Alejandra; Bosch, Berend‐Jan; Frenz, Brandon; Rottier, Peter J. M.; DiMaio, Frank; Rey, Felix A.

2016-01-01

Abstract The tremendous pandemic potential of coronaviruses was demonstrated twice in the last 15 years by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor‐binding and membrane fusion functions. Despite their biomedical importance, coronavirus S glycoproteins have proven difficult targets for structural characterization, precluding high‐resolution studies of the biologically relevant trimer. Recent technological developments in single particle cryo‐electron microscopy allowed us to determine the first structure of a coronavirus S glycoprotein trimer which provided a framework to understand the mechanisms of viral entry and suggested potential inhibition strategies for this family of viruses. Here, we describe the key factors that enabled this breakthrough. PMID:27667334

Release of oligomannoside-type glycans as a marker of the degradation of newly synthesized glycoproteins.

PubMed

Villers, C; Cacan, R; Mir, A M; Labiau, O; Verbert, A

1994-02-15

The N-glycosylation of proteins is accompanied by the release of soluble oligosaccharide material. Besides oligosaccharide phosphates originating from the cleavage of lipid intermediates, neutral free oligosaccharides represent the major part of this material and are heterogeneous depending on whether the reducing end has one or two N-acetylglucosamine residues. The present study focuses on the intracellular origin of neutral free oligosaccharides in a CHO cell line. Kinetic and pulse-chase experiments clearly indicate that oligosaccharides possessing a chitobiosyl unit are derived from oligosaccharide pyrophosphodolichol, whereas oligosaccharides possessing one N-acetyl-glucosamine residue are derived from newly synthesized glycoprotein. This relationship is confirmed by comparing the glycosylation pattern of lipid donors and glycoproteins with those of neutral free oligosaccharides under various incubation conditions (inhibition of protein synthesis, presence of processing inhibitors, presence or absence of glucose). Degradation of newly synthesized glycoprotein and formation of neutral oligosaccharides with one N-acetylglucosamine residue are inhibited at 16 degrees C but not affected by lysosomotropic agents such as leupeptin or NH4Cl. Together with the fact that the degradation of newly synthesized glycoproteins and the subsequent release of the glycan are recovered in permeabilized cells, these results suggest that this phenomenon occurs in the rough endoplasmic reticulum or in a closely related compartment.

Feline tetherin is characterized by a short N-terminal region and is counteracted by the feline immunodeficiency virus envelope glycoprotein.

PubMed

Celestino, Michele; Calistri, Arianna; Del Vecchio, Claudia; Salata, Cristiano; Chiuppesi, Flavia; Pistello, Mauro; Borsetti, Alessandra; Palù, Giorgio; Parolin, Cristina

2012-06-01

Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2(504) and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2(504) is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2(504) failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2(504) was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2(504) also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.

Unique N-Glycan Moieties of the 66-kDa Cell Wall Glycoprotein from the Red Microalga Porphyridium sp.

PubMed Central

Levy-Ontman, Oshrat; Arad, Shoshana (Malis); Harvey, David J.; Parsons, Thomas B.; Fairbanks, Antony; Tekoah, Yoram

2011-01-01

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man8–9Xyl1–2Me3GlcNAc2. The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins. PMID:21515680

Analysis of N-acetylaminosugars by CE: a comparative derivatization study.

PubMed

Rustighi, Isabella; Campa, Cristiana; Rossi, Marco; Semeraro, Sabrina; Vetere, Amedeo; Gamini, Amelia

2009-08-01

N-linked or O-linked glycans derived from glycoprotein processing carry, an N-acetylglucosamine or an N-acetylgalactosamine respectively, at their reducing termini. The presence of the N-acetylamino group on C-2 of reducing sugar residues has been reported to hamper the derivatization reaction with a chromophore at the anomeric centre. In this paper N-acetyllactosamine, N-acetylglucosamine, N-acetylgalactosamine and several other neutral monosaccharides are coupled to three different dyes (4-aminobenzonitrile, 2-aminopyridine, 2-aminobenzoic acid (2-AA)) by reductive amination and analysed by CE with UV detection. The 2-AA derivatives showed the lowest concentration detection limits, varying approximately in the 2-3 muM range for the saccharides tested including the N-acetamido ones. The possibility to separate and detect with the same sensitivity ten 2-AA-labelled monosaccharides mainly found in mammalian or plant glycoproteins in a single CE run is highlighted. The analysis has been carried out in less than 25 min using the borate-complexation method in CZE mode. The influence of the strength of the acid used as catalyst in the chemical modification of the sugars with 2-AA is also shortly addressed.

Identification of the glycoproteins of lymphocystis disease virus (LDV) of fish.

PubMed

Robin, J; Laperrière, A; Berthiaume, L

1986-01-01

Analysis of highly purified fish Lymphocystis Disease Virus (LDV), strain Leetown NFH, by three different methods, namely periodic Acid Schiff reaction, radiolabelling with tritiated fucose and N-acetyl-D-glucosamine and staining with three lectins, indicated that ten glycoproteins were associated with the virus structure. Six of them were detected by all of the three methods, three by both radiolabelling and lectin staining but only one by the lectin technique. Localization of these glycoproteins at the surface or inside the virion is discussed.

Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides.

PubMed

Ito, Yukishige; Takeda, Yoichi

2012-01-01

Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules.

Role of the Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

DTIC Science & Technology

2005-05-01

AD Award Number: DAMD17-03-1-0243 TITLE: Role of the Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast...Glycoprotein Processing in Breast Cancer 5b.GRANTNUMBER Cells DAAD17-03-1-0243 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Sergey N... processing of glycoproteins, exocytosis, protein delivery systems, gene expression, western and northern blot analysis, immunotiuorescence, gradient

The eicosanoid, 15-(S)-HETE, stimulates secretion of mucin-like glycoprotein by the corneal epithelium.

PubMed

Jackson , R S; Van Dyken, S J; McCartney, M D; Ubels, J L

2001-07-01

The eicosanoid, 15-(S)-hydroxyeicosa-5Z, 8Z-11Z, 13E-tetraenoic acid (15-(S)-HETE), is known to stimulate production of mucin glycoprotein by airway epithelium. This study investigated the effect of 15-(S)-HETE on the mucin glycoprotein secretion by the corneal epithelium. To determine the effect of dose, corneas of anesthetized New Zealand White rabbits were treated with 50, 500, or 5,000 nM 15-(S)-HETE in artificial tears for 120 minutes. To determine the time to onset of the response, corneas were treated with 500 or 1,000 nM 15-(S)-HETE in balanced salt solution for periods ranging from 5 to 120 minutes. Corneas were fixed for electron microscopy in fixative containing 0.5% cetylpyridinium chloride (CPC) to stabilize the layer of mucin-like glycoprotein on the corneal surface. The mucin layer thickness was measured by image analysis of electron micrographs. The layer of CPC-fixed mucin-like glycoprotein on the surface of control corneas was 0.46 +/- 0.04 microm thick. After treatment with 15-(S)-HETE, the thickness of the mucin layer increased to 0.64 +/- 0.1 microm at 50 or 5,000 nM HETE and as much as 1.02 +/- 0.2 microm in response to 500 nM HETE. Mucin thickness reached a statistical maximum of 0.59 +/- 0.1 microm after only 5 minutes of exposure to 500 or 1,000 nM HETE. Exposure of the cornea to 15-(S)-HETE causes a rapid-onset increase in the thickness of a layer of mucin-like glycoprotein on the surface of the corneal epithelium. This supports previous reports that corneal epithelial cells produce mucin and suggests that treatment with topical 15-(S)-HETE may be effective in treating ocular surface mucin deficiency in dry eye syndrome.

Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

PubMed

Mabashi-Asazuma, Hideaki; Shi, Xianzong; Geisler, Christoph; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

2013-02-01

Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

Escherichia coli as a glycoprotein production host: recent developments and challenges.

PubMed

Jaffé, Stephen R P; Strutton, Benjamin; Levarski, Zdenko; Pandhal, Jagroop; Wright, Phillip C

2014-12-01

Chinese Hamster Ovary cells are the most popular host expression system for the large-scale production of human therapeutic glycoproteins, but, the race to engineer Escherichia coli to perform glycosylation is gathering pace. The successful functional transfer of an N-glycosylation pathway from Campylobacter jejuni to Escherichia coli in 2002 can be considered as the crucial first engineering step. Here, we discuss the recent advancements in the field of N-glycosylation of recombinant therapeutic proteins in E. coli cells, from the manipulation of glycan composition, to the improvement in glycosylation efficiency, along with the challenges that remain before E. coli can be available as an industry host cell for economically viable glycoprotein production. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

PubMed

Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

2011-05-01

To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

Integrated proteomic and N-glycoproteomic analyses of doxorubicin sensitive and resistant ovarian cancer cells reveal glycoprotein alteration in protein abundance and glycosylation

PubMed Central

Hou, Junjie; Zhang, Chengqian; Xue, Peng; Wang, Jifeng; Chen, Xiulan; Guo, Xiaojing; Yang, Fuquan

2017-01-01

Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and

Purification and characterization of a soluble glycoprotein from garlic (Allium sativum) and its in vitro bioactivity.

PubMed

Wang, Yan; Zou, Tingting; Xiang, Minghui; Jin, Chenzhong; Zhang, Xuejiao; Chen, Yong; Jiang, Qiuqing; Hu, Yihong

2016-10-02

A soluble glycoprotein was purified to homogeneity from ripe garlic (Allium sativum) bulbs using ammonium sulfate precipitation, Sephadex G-100 gel filtration, and diethylaminoethyl-52 cellulose anion-exchange chromatography. A native mass of 55.7 kDa estimated on gel permeation chromatography and a molecular weight of 13.2 kDa observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis supported that the glycoprotein is a homotetramer. β-Elimination reaction result suggested that the glycoprotein is an N-linked type. Fourier-transform infrared spectroscopy proved that it contains sugar. Gas chromatography-mass spectrometer analysis showed that its sugar component was galactose. The glycoprotein has 1,1-diphenyl-2-picrylhydrazil free radical scavenging activity and the peroxidation inhibition ability to polyunsaturated fatty acid. These results indicated that the glycoprotein has potential for food additives, functional foods, and even biotechnological and medical applications.

Enrichment of Glycoproteins using Nano-scale Chelating Con A Monolithic Capillary Chromatography

PubMed Central

Feng, Shun; Yang, Na; Pennathur, Subramaniam; Goodison, Steve; Lubman, David M.

2009-01-01

Immobilized lectin chromatography can be employed for glycoprotein enrichment, but commonly used columns have limitations of yield and resolution. In order to improve efficiency and to make the technique applicable to minimal sample material, we have developed a nano-scale chelating Concanavalin A (Con A) monolithic capillary prepared using GMA-EDMA (glycidyl methacrylate–co-ethylene dimethacrylate) as polymeric support. Con A was immobilized on Cu(II)-charged iminodiacetic acid (IDA) regenerable sorbents by forming a IDA:Cu(II):Con A sandwich affinity structure that has high column capacity as well as stability. When compared with conventional Con A lectin chromatography, the monolithic capillary enabled the better reproducible detection of over double the number of unique N-glycoproteins in human urine samples. Utility for analysis of minimal biological samples was confirmed by the successful elucidation of glycoprotein profiles in mouse urine samples at the microliter scale. The improved efficiency of the nano-scale monolithic capillary will impact the analysis of glycoproteins in complex biological samples, especially where only limited material may be available. PMID:19366252

Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

PubMed Central

Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

2017-01-01

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

PubMed Central

Gitz, Eelo; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan W.; Gerritsen, Hans C.; Urbanus, Rolf T.

2012-01-01

Background Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. Design and Methods: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. Results Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. Conclusions We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future. PMID:22733027

Glycoprotein Disease Markers and Single Protein-omics*

PubMed Central

Chandler, Kevin; Goldman, Radoslav

2013-01-01

Glycoproteins are well represented among biomarkers for inflammatory and cancer diseases. Secreted and membrane-associated glycoproteins make excellent targets for noninvasive detection. In this review, we discuss clinically applicable markers of cancer diseases and methods for their analysis. High throughput discovery continues to supply marker candidates with unusual glycan structures, altered glycoprotein abundance, or distribution of site-specific glycoforms. Improved analytical methods are needed to unlock the potential of these discoveries in validated clinical assays. A new generation of targeted quantitative assays is expected to advance the use of glycoproteins in early detection of diseases, molecular disease classification, and monitoring of therapeutic interventions. PMID:23399550

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

PubMed

da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping; Schnell, Matthias J; von Messling, Veronika

2017-04-15

The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens

PubMed Central

da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping

2017-01-01

ABSTRACT The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that

Defining Simple nD Operations Based on Prosmatic nD Objects

NASA Astrophysics Data System (ADS)

Arroyo Ohori, K.; Ledoux, H.; Stoter, J.

2016-10-01

An alternative to the traditional approaches to model separately 2D/3D space, time, scale and other parametrisable characteristics in GIS lies in the higher-dimensional modelling of geographic information, in which a chosen set of non-spatial characteristics, e.g. time and scale, are modelled as extra geometric dimensions perpendicular to the spatial ones, thus creating a higher-dimensional model. While higher-dimensional models are undoubtedly powerful, they are also hard to create and manipulate due to our lack of an intuitive understanding in dimensions higher than three. As a solution to this problem, this paper proposes a methodology that makes nD object generation easier by splitting the creation and manipulation process into three steps: (i) constructing simple nD objects based on nD prismatic polytopes - analogous to prisms in 3D -, (ii) defining simple modification operations at the vertex level, and (iii) simple postprocessing to fix errors introduced in the model. As a use case, we show how two sets of operations can be defined and implemented in a dimension-independent manner using this methodology: the most common transformations (i.e. translation, scaling and rotation) and the collapse of objects. The nD objects generated in this manner can then be used as a basis for an nD GIS.

Ammonia transport in the kidney by Rhesus glycoproteins

PubMed Central

Verlander, Jill W.

2014-01-01

Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport. PMID:24647713

Evidence for glycoprotein transport into complex plastids.

PubMed

Peschke, Madeleine; Moog, Daniel; Klingl, Andreas; Maier, Uwe G; Hempel, Franziska

2013-06-25

Diatoms are microalgae that possess so-called "complex plastids," which evolved by secondary endosymbiosis and are surrounded by four membranes. Thus, in contrast to primary plastids, which are surrounded by only two membranes, nucleus-encoded proteins of complex plastids face additional barriers, i.e., during evolution, mechanisms had to evolve to transport preproteins across all four membranes. This study reveals that there exist glycoproteins not only in primary but also in complex plastids, making transport issues even more complicated, as most translocation machineries are not believed to be able to transport bulky proteins. We show that plastidal reporter proteins with artificial N-glycosylation sites are indeed glycosylated during transport into the complex plastid of the diatom Phaeodactylum tricornutum. Additionally, we identified five endogenous glycoproteins, which are transported into different compartments of the complex plastid. These proteins get N-glycosylated during transport across the outermost plastid membrane and thereafter are transported across the second, third, and fourth plastid membranes in the case of stromal proteins. The results of this study provide insights into the evolutionary pressure on translocation mechanisms and pose unique questions on the operating mode of well-known transport machineries like the translocons of the outer/inner chloroplast membranes (Toc/Tic).

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by solvent fractionation

PubMed Central

Creeth, J. Michael; Bhaskar, K. Ramakrishnan; Donald, Alastair S. R.; Morgan, Walter T. J.

1974-01-01

1. The glycoprotein components of a human ovarian-cyst fluid were isolated by a solvent [95% (w/w) phenol]-extraction procedure; the phenol-insoluble water-soluble glycoprotein was further fractionated by (NH4)2SO4 and by ethanol to yield eight fractions. 2. The fractions were analysed in terms of amino acids, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. Variations occurred, particularly in the proportion of peptide; these were partly correlated with varying extent of serological activity. 3. The fractions were characterized physicochemically in terms of buoyant density and degree of spreading in a density gradient, sedimentation velocity and molecular weight; their partial specific volumes and specific refraction increments were also determined. 4. The fractions showed wide variations in their sedimentation-velocity and density-gradient patterns, and gave evidence of pauci-dispersity in density. The fraction regarded as the most typical blood-group-specific glycoprotein sedimented as a single rapidly spreading peak and was of high molecular weight. 5. Significant correlations were observed between the physical properties of the glycoprotein fractions and the amount of their peptide component. The buoyant densities and sedimentation coefficients varied in a manner that suggested the existence of two families of glycoproteins. 6. It is suggested that variability in the extent of glycosylation, or in the degree of cross-linking, might account for the two families of glycoproteins, and that the extent of cross-linkage might also be a factor determining the solubility of these glycoproteins in hot saturated (NH4)2SO4. ImagesFig. 1.PLATE 1 PMID:4219280

Identification of a glycoprotein from rat liver mitochondrial inner membrane and demonstration of its origin in the endoplasmic reticulum.

PubMed

Chandra, N C; Spiro, M J; Spiro, R G

1998-07-31

Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-beta-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact

Resistance of herpes simplex virus type 2 to neomycin maps to the N-terminal portion of glycoprotein C.

PubMed Central

Oyan, A M; Dolter, K E; Langeland, N; Goins, W F; Glorioso, J C; Haarr, L; Crumpacker, C S

1993-01-01

Entry of herpes simplex virus (HSV) into cells is believed to be mediated by specific binding of envelope proteins to a cellular receptor. Neomycin specifically blocks this initial step in infection by HSV-1 but not HSV-2. Resistance of HSV-2 to this compound maps to a region of the genome encoding glycoprotein C (gC-2). We have studied the function of gC-2 in the initial interaction of the virus with the host cell, using HSV-2 mutants deleted for gC-2 and gC-2-rescued recombinants. Resistance to neomycin was directly linked to the presence of gC-2 within the viral genome. In addition, deletion of the gC-2 gene caused a marked delay in adsorption to cells relative to the wild-type virus. HSV-1 recombinants containing chimeric gC genes composed of HSV-1 and HSV-2 sequences were used to localize neomycin resistance within the N-terminal 223 amino acids of gC-2. This region of the glycoprotein comprises an important domain responsible for binding of HSV-2 to cell receptors in the presence of neomycin. A gC-2-negative mutant is still infectious, indicating that HSV-2 also has an alternative pathway of adsorption. Images PMID:8386261

Platelet glycoproteins associated with aspirin-treatment upon platelet activation

PubMed Central

Shah, Punit; Yang, Weiming; Sun, Shisheng; Pasay, Jered; Faraday, Nauder; Zhang, Hui

2017-01-01

Platelet glycoproteins are known to play central roles in hemostasis and vascular integrity and have pathologic roles in vascular occlusive diseases such as myocardial infarction and stroke. Characterizing glycoproteins within and secreted by platelets can provide insight into the mechanisms that underlie vascular pathologies and the therapeutic benefits or failure of anti-platelet agents. To study the impact of aspirin, which is commonly prescribed for primary and secondary cardiovascular prevention, on the platelet glycoproteome, we evaluated washed platelets from ten donors. The platelet glycoproteome, was studied using an iTRAQ in resting and stimulated states and with and without aspirin treatment. Using solid phase extraction of glycosite-containing peptides (SPEG), we were able to identify 799 unique N-linked glycosylation sites (glycosites) in platelets, representing the largest and the most comprehensive analysis to date. We were able to identity a number of glycoproteins impacted by aspirin treatment, which we validated using global proteomics analysis of platelets and their secreted proteins. In our analyses, metallopeptidase inhibitor 1 (TIMP1) was the single most significantly affected glycoprotein by aspirin treatment. ELISA assays confirmed proteomic results and validated our strategy. Functional analysis demonstrated that TIMP1 levels were highly correlated with platelet reactivity in vitro, with a correlation coefficient of −0.5. The release of TIMP1 from platelets, which was previously unknown to be affected by aspirin treatment, may play important roles in hemostasis and/or vascular integrity. If validated, our findings may be useful for developing assays that assess platelet response to aspirin or other anti-platelet therapies. PMID:27452734

Cell-wall polysaccharides and glycoproteins of parenchymatous tissues of runner bean (Phaseolus coccineus).

PubMed Central

Ryden, P; Selvendran, R R

1990-01-01

1. Polymers were solubilized from the cell walls of parenchyma from mature runner-bean pods with minimum degradation by successive extractions with cyclohexane-trans-1,2-diamine-NNN'N'-tetra-acetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue, which contained cross-linked pectic polysaccharides and Hyp-rich glycoproteins. These were solubilized with chlorite/acetic acid and cellulase. The polymers were fractionated by anion-exchange chromatography, and fractions were subjected to methylation analysis. 2. The pectic polysaccharides differed in their ease of extraction, and a small proportion were highly cross-linked. The bulk of the pectic polysaccharides solubilized by CDTA and Na2CO3 were less branched than those solubilized by KOH. There was good evidence that most of the pectic polysaccharides were not degraded during extraction. 3. The protein-containing fractions included Hyp-rich and Hyp-poor glycoproteins associated with easily extractable pectic polysaccharides, Hyp-rich glycoproteins solubilized with 4M-KOH+borate, the bulk of which were not associated with pectic polysaccharides, and highly cross-linked Hyp-rich glycoproteins. 4. Isodityrosine was not detected, suggesting that it does not have a (major) cross-linking role in these walls. Instead, it is suggested that phenolics, presumably linked to C-5 of 3,5-linked Araf residues of Hyp-rich glycoproteins, serve to cross-link some of the polymers. 5. There were two main types of xyloglucan, with different degrees of branching. The bulk of the less branched xyloglucans were solubilized by more-concentrated alkali. The anomeric configurations of the sugars in one of the highly branched xyloglucans were determined by 13C-n.m.r. spectroscopy. 6. The structural features of the cell-wall polymers and complexes are discussed in relation to the structure of the cell walls of parenchyma tissues. PMID:2167068

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.

PubMed

Katoh, Toshihiko; Katayama, Takane; Tomabechi, Yusuke; Nishikawa, Yoshihide; Kumada, Jyunichi; Matsuzaki, Yuji; Yamamoto, Kenji

2016-10-28

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-glycoproteins immunological test system....5420 Alpha-1-glycoproteins immunological test system. (a) Identification. An alpha-1-glycoproteins... alpha-1-glycoproteins (a group of plasma proteins found in the alpha-1 group when subjected to...

Solubilization of glycoproteins of envelope viruses by detergents

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

Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.

1986-11-20

The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-..beta..-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis.more » Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines.« less

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

PubMed Central

Ao, Jie; Chinnici, Jennifer L.; Maddi, Abhiram

2015-01-01

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix. PMID:26048011

A double responsive smart upconversion fluorescence sensing material for glycoprotein.

PubMed

Guo, Ting; Deng, Qiliang; Fang, Guozhen; Yun, Yaguang; Hu, Yongjin; Wang, Shuo

2016-11-15

A novel strategy was developed to prepare double responsive smart upconversion fluorescence material for highly specific enrichment and sensing of glycoprotein. The novel double responsive smart sensing material was synthesized by choosing Horse radish peroxidase (HRP) as modal protein, the grapheme oxide (GO) as support material, upconversion nanoparticles (UCNPs) as fluorescence signal reporter, N-isopropyl acrylamide (NIPAAM) and 4-vinylphenylboronic acid (VPBA) as functional monomers. The structure and component of smart sensing material was investigated by transmission electron microscopy (TEM), Scanning electron microscopy (SEM), X-ray photoelectron spectroscopic (XPS) and Fourier transform infrared (FTIR), respectively. These results illustrated the smart sensing material was prepared successfully. The recognition characterizations of smart sensing material were evaluated, and results showed that the fluorescence intensity of smart sensing material was reduced gradually, as the concentration of protein increased, and the smart sensing material showed selective recognition for HRP among other proteins. Furthermore, the recognition ability of the smart sensing material for glycoprotein was regulated by controlling the pH value and temperature. Therefore, this strategy opens up new way to construct smart material for detection of glycoprotein. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of an informed search space maximizes confidence of site-specific assignment of glycoprotein glycosylation.

PubMed

Khatri, Kshitij; Klein, Joshua A; Zaia, Joseph

2017-01-01

In order to interpret glycopeptide tandem mass spectra, it is necessary to estimate the theoretical glycan compositions and peptide sequences, known as the search space. The simplest way to do this is to build a naïve search space from sets of glycan compositions from public databases and to assume that the target glycoprotein is pure. Often, however, purified glycoproteins contain co-purified glycoprotein contaminants that have the potential to confound assignment of tandem mass spectra based on naïve assumptions. In addition, there is increasing need to characterize glycopeptides from complex biological mixtures. Fortunately, liquid chromatography-mass spectrometry (LC-MS) methods for glycomics and proteomics are now mature and accessible. We demonstrate the value of using an informed search space built from measured glycomes and proteomes to define the search space for interpretation of glycoproteomics data. We show this using α-1-acid glycoprotein (AGP) mixed into a set of increasingly complex matrices. As the mixture complexity increases, the naïve search space balloons and the ability to assign glycopeptides with acceptable confidence diminishes. In addition, it is not possible to identify glycopeptides not foreseen as part of the naïve search space. A search space built from released glycan glycomics and proteomics data is smaller than its naïve counterpart while including the full range of proteins detected in the mixture. This maximizes the ability to assign glycopeptide tandem mass spectra with confidence. As the mixture complexity increases, the number of tandem mass spectra per glycopeptide precursor ion decreases, resulting in lower overall scores and reduced depth of coverage for the target glycoprotein. We suggest use of α-1-acid glycoprotein as a standard to gauge effectiveness of analytical methods and bioinformatics search parameters for glycoproteomics studies. Graphical Abstract Assignment of site specific glycosylation from LC

Expression of N-acetyl-D-galactosamine associated epitope in synovium: a potential marker of glycoprotein production.

PubMed

El-Gabalawy, H; King, R; Bernstein, C; Ma, G; Mou, Y; Alguacil-Garcia, A; Fritzler, M; Wilkins, J

1997-07-01

To investigate synovial glycoprotein production in situ, a novel monoclonal antibody (Mab), A13D8, was used to evaluate the expression of an epitope containing N-acetyl-D-galactosamine (GalNAc) in normal and pathological synovium. Immunohistological and cytochemical analysis of synovial tissue samples was undertaken with single and double staining techniques using the A13D8 Mab, anti-CD68, vascular cell adhesion molecule-1 (VCAM-1), the hyaluronan associated enzyme uridine diphosphoglucose dehydrogenase (UDPGD), and the anti-Golgi Mab SSN/HR-1992. The specificity of the A13D8 Mab was established through blocking studies using carbohydrate residues, including GalNAc and N-acetylglucosamine (GlcNAc). A13D8 is expressed intensely in the cytoplasm of normal type B lining cells, which coexpress VCAM-1 and UDPGD, and is not expressed by CD68+ type A lining cells. In the lining layer of RA synovium, there is a negative correlation between A13D8 expression and the level of lymphocytic infiltration in the sublining areas (r = -0.43, p < 0.001). The endothelium of a subset of venules, typically in lymphocyte-rich aggregates, also stains intensely for A13D8. Pretreatment of the Mab with GalNAc completely eliminates the tissue staining, as well as the 110 kDa band seen on immunoblot, whereas pretreatment of A13D8 with GlcNAc and lactose has no effect. Double staining of HEp-2 cells with A13D8 and the anti-Golgi Mab SSN/HR-1992 reveals co-localization of the A13D8 epitope to the Golgi apparatus. Type B synovial lining cells and selected synovial endothelium express GalNAc containing epitope identified by Mab A13D8. Marked reduction in the expression of this epitope in the lining layer of inflamed RA synovium suggests that the synovial production of GalNAc containing glycoproteins, such as mucins, may be altered in this disorder.

Qualitative and Quantitative Analysis of Carbohydrate Modification on Glycoproteins from Seeds of Ginkgo biloba.

PubMed

Wang, Ting; Hu, Xiao-Chun; Cai, Zhi-Peng; Voglmeir, Josef; Liu, Li

2017-09-06

Recent progress in the relationship between carbohydrate cross-reactive determinants (CCDs) and allergic response highlights the importance of carbohydrate moieties in the innate immune system. Previous research pointed out that the protein allergen in Ginkgo biloba seeds is glycosylated, and the oligosaccharides conjugated to these proteins might also contribute to the allergy. The aim of this study was to analyze carbohydrate moieties, especially N-linked glycans, of glycoproteins from Ginkgo seeds originating from different places for detailed structures, to enable further research on the role played by N-glycans in Ginkgo-caused allergy. Results of monosaccharide composition and immunoblotting assays indicated the existence of N-glycans. Detailed structural elucidation of the N-glycans was further carried out by means of hydrophilic interaction ultraperformance liquid chromatography (HILIC-UPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 14 out of 16 structures detected by UPLC were confirmed by MALDI-TOF-MS and tandem mass spectrometry, among which complex-type N-glycans bearing Lewis A determinants and high-mannose-type N-glycans were identified from Ginkgo seeds for the first time. Precise quantification of N-glycans was performed by use of an external standard, and both the absolute amount of each N-glycan and the percentage of different types of N-glycan showed significant diversity among the samples without any pattern of geographic variation.

HIV-1 Envelope Glycoproteins from Diverse Clades Differentiate Antibody Responses and Durability among Vaccinees

PubMed Central

2018-01-01

ABSTRACT Induction of broadly cross-reactive antiviral humoral responses with the capacity to target globally diverse circulating strains is a key goal for HIV-1 immunogen design. A major gap in the field is the identification of diverse HIV-1 envelope antigens to evaluate vaccine regimens for binding antibody breadth. In this study, we define unique antigen panels to map HIV-1 vaccine-elicited antibody breadth and durability. Diverse HIV-1 envelope glycoproteins were selected based on genetic and geographic diversity to cover the global epidemic, with a focus on sexually acquired transmitted/founder viruses with a tier 2 neutralization phenotype. Unique antigenicity was determined by nonredundancy (Spearman correlation), and antigens were clustered using partitioning around medoids (PAM) to identify antigen diversity. Cross-validation demonstrated that the PAM method was better than selection by reactivity and random selection. Analysis of vaccine-elicited V1V2 binding antibody in longitudinal samples from the RV144 clinical trial revealed the striking heterogeneity among individual vaccinees in maintaining durable responses. These data support the idea that a major goal for vaccine development is to improve antibody levels, breadth, and durability at the population level. Elucidating the level and durability of vaccine-elicited binding antibody breadth needed for protection is critical for the development of a globally efficacious HIV vaccine. IMPORTANCE The path toward an efficacious HIV-1 vaccine will require characterization of vaccine-induced immunity that can recognize and target the highly genetically diverse virus envelope glycoproteins. Antibodies that target the envelope glycoproteins, including diverse sequences within the first and second hypervariable regions (V1V2) of gp120, were identified as correlates of risk for the one partially efficacious HIV-1 vaccine. To build upon this discovery, we experimentally and computationally evaluated humoral

Functional Regulation of Sugar Assimilation by N-Glycan-specific Interaction of Pancreatic α-Amylase with Glycoproteins of Duodenal Brush Border Membrane*

PubMed Central

Asanuma-Date, Kimie; Hirano, Yuki; Le, Na; Sano, Kotone; Kawasaki, Nana; Hashii, Noritaka; Hiruta, Yoko; Nakayama, Ken-ichi; Umemura, Mariko; Ishikawa, Kazuhiko; Sakagami, Hiromi; Ogawa, Haruko

2012-01-01

Porcine pancreatic α-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680–4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na+/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycan-specific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic α-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic α-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other. PMID:22584580

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

PubMed

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

2000-03-01

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

Studies on the synthesis and processing of the asparagine-linked carbohydrate units of glycoproteins.

PubMed

Spiro, R G; Spiro, M J

1982-12-24

It has become apparent in recent years from the work of a number of laboratories that the N-glycosylation of both membrane and secretory glycoproteins is effected by the transfer en bloc to nascent polypeptides of a glucose-containing oligosaccharide (Glc3Man9GlcNAc2) from a dolichyl pyrophosphoryl carrier; this is followed by a series of modifying reactions to yield the mature polymannose and complex asparagine-linked carbohydrate units. The enzymic steps involved in the assembly of the precursor oligosaccharide, its transfer to protein and its subsequent processing represent potential sites for the regulation of glycoprotein synthesis. Studies performed in our laboratory have dealt primarily with thyroid slices and particulate enzymes with special regard to the role of glucose in these events. Thyroglobulin, the major secretory glycoprotein of this tissue, has well defined complex and polymannose saccharide units, and indeed the most complete form of the latter (Man9GlcNAc2) has the same structure as the lipid-linked oligosaccharide without the glucose. Our studies indicate that effective N-glycosylation requires a complete glucose chain (Glc3) and that the glucose sequence is assembled from dolichol-P-glucose in a stepwise manner through the concerted action of at least two transferases in a fashion complementary to the subsequent excision of this sugar by glucosidases. Pulse-chase studies indicate that, after the transfer to protein, the removal of all three glucose residues as well as of the first mannose takes place in the endoplasmic reticulum and three additional mannoses are excised in the Golgi complex, because in the presence of an inhibitor of intracellular transport, carbonyl cyanide m-chlorophenylhydrazone (CCCP), there is a pronounced accumulation of protein-linked Man8GlcNAc2. Studies with metabolic inhibitors (CCCP, antimycin, N2) indicate that, under conditions of energy depletion, glucosylation of oligosaccharide-lipid is selectively impaired

Gulose as a constituent of a glycoprotein.

PubMed

Mengele, R; Sumper, M

1992-02-17

The aldohexose gulose was identified as a constituent of a hydroxyproline-rich glycopeptide derived from the glycoprotein SSG 185. This glycoprotein is part of the extracellular matrix of the green alga Volvox carteri. The gulose residue occupies a terminal position in the corresponding saccharide.

Ultrasensitive Electrochemical Detection of Glycoprotein Based on Boronate Affinity Sandwich Assay and Signal Amplification with Functionalized SiO2@Au Nanocomposites.

PubMed

You, Min; Yang, Shuai; Tang, Wanxin; Zhang, Fan; He, Pin-Gang

2017-04-26

Herein we propose a multiple signal amplification strategy designed for ultrasensitive electrochemical detection of glycoproteins. This approach introduces a new type of boronate-affinity sandwich assay (BASA), which was fabricated by using gold nanoparticles combined with reduced graphene oxide (AuNPs-GO) to modify sensing surface for accelerating electron transfer, the composite of molecularly imprinted polymer (MIP) including 4-vinylphenylboronic acid (VPBA) for specific capturing glycoproteins, and SiO 2 nanoparticles carried gold nanoparticles (SiO 2 @Au) labeled with 6-ferrocenylhexanethiol (FcHT) and 4-mercaptophenylboronic acid (MPBA) (SiO 2 @Au/FcHT/MPBA) as tracing tag for binding glycoprotein and generating electrochemical signal. As a sandwich-type sensing, the SiO 2 @Au/FcHT/MPBA was captured by glycoprotein on the surface of imprinting film for further electrochemical detection in 0.1 M PBS (pH 7.4). Using horseradish peroxidase (HRP) as a model glycoprotein, the proposed approach exhibited a wide linear range from 1 pg/mL to 100 ng/mL, with a low detection limit of 0.57 pg/mL. To the best of our knowledge, this is first report of a multiple signal amplification approach based on boronate-affinity molecularly imprinted polymer and SiO 2 @Au/FcHT/MPBA, exhibiting greatly enhanced sensitivity for glycoprotein detection. Furthermore, the newly constructed BASA based glycoprotein sensor demonstrated HRP detection in real sample, such as human serum, suggesting its promising prospects in clinical diagnostics.

116 kDa glycoprotein isolated from Ulmus davidiana Nakai (UDN) inhibits glucose/glucose oxidase (G/GO)-induced apoptosis in BNL CL.2 cells.

PubMed

Ko, Jeong-Hyeon; Lee, Sei-Jung; Lim, Kye-Taek

2005-09-14

Ulmus davidiana Nakai (UDN) has been used in folk medicine for its anti-inflammatory activity. In the present study, we investigated the antiapoptotic effect of UDN glycoprotein in glucose/glucose oxidase (G/GO)-induced BNL CL.2 cells. To evaluate the antiapoptotic effect of UDN glycoprotein, experiments were carried out using Western blot analysis for nuclear factor-kappa B (NF-kappaB), caspase-3, and poly(ADP-ribose) polymerase (PARP). We also examined nitric oxide (NO) production and nuclear staining. When BNL CL.2 cells were treated with G/GO (50 mU/ml), viability of the cells was 54.1%. However, the number of living cells after the addition of UDN glycoprotein in the presence of G/GO increased. UDN glycoprotein protected from cell damage caused by G/GO. Interestingly, UDN glycoprotein decreased NF-kappaB activation and stimulated NO production in G/GO-induced BNL CL.2 cells. In apoptotic parameters, UDN glycoprotein inhibited activations of caspase-3 and PARP cleavage in G/GO-induced BNL CL.2 cells. The results of nuclear staining indicated that UDN glycoprotein (50 microg/ml) has a protective ability from apoptotic cell death caused G/GO (50 mU/ml). In conclusion, UDN glycoprotein has a protective effect on apoptosis induced by G/GO through the inhibition of NF-kappaB, caspase-3, and PARP activity, and the stimulation of NO production in BNL CL.2 cells.

Quantitative Characterization of Shear-Induced Platelet Receptor Shedding: Glycoprotein Ibα, Glycoprotein VI, and Glycoprotein IIb/IIIa.

PubMed

Chen, Zengsheng; Koenig, Steven C; Slaughter, Mark S; Griffith, Bartley P; Wu, Zhongjun J

2017-11-07

The structural integrity of platelet receptors is essential for platelets to play the normal hemostatic function. The high non-physiologic shear stress (NPSS) commonly exists in blood-contacting medical devices and has been shown to cause platelet receptor shedding. The loss of platelet receptors may impair the normal hemostatic function of platelets. The aim of this study was to quantify NPSS-induced shedding of three key receptors on the platelet surface. Human blood was subjected to the matrix of well-defined shear stresses and exposure times, generated by using a custom-designed blood-shearing device. The expression of three key platelet receptors, glycoprotein (GP) Ibα, GPVI, and GPIIb/IIIa, in sheared blood was quantified using flow cytometry. The quantitative relationship between the loss of each of the three receptors on the platelet surface and shear condition (shear stress level and exposure time) was explored. It was found that these relationships followed well the power law functional form. The coefficients of the power law models for the shear-induced shedding of these platelet receptors were derived with coefficients of determination (R) of 0.77, 0.73, and 0.78, respectively. The power law models with these coefficients may be potentially used to predict the shear-induced platelet receptor shedding of human blood.

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity

PubMed Central

Smits, Guillaume; Campillo, Mercedes; Govaerts, Cédric; Janssens, Véronique; Richter, Christine; Vassart, Gilbert; Pardo, Leonardo; Costagliola, Sabine

2003-01-01

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin-like G protein-coupled receptors with a large extracellular N-terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine-rich repeats (LRRs). The LRRs form a succession of β-strands and α-helices organized into a horseshoe-shaped structure. It has been proposed that glycoprotein hormones interact with residues of the β-strands making the concave surface of the horseshoe. Gain-of-function homology scanning of the β-strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild-type LH/CGr. Molecular dynamics simulations and additional site-directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family. PMID:12773385

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

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

Analgesic effects of glycoproteins from Panax ginseng root in mice.

PubMed

Wang, Ying; Chen, Yinghong; Xu, Hong; Luo, Haoming; Jiang, Ruizhi

2013-07-30

The root of Panax ginseng C.A. Mey has various beneficial pharmacological effects. The present study aimed to evaluate the analgesic activities of glycoproteins from the root of Panax ginseng C.A. Mey in mice. Glycoproteins were isolated and purified from the root of Panax ginseng C.A. Mey. Physicochemical properties and molecular mass were determined by chemical assay and HPLC. Acetic acid-induced writhing and hot-plate tests were employed to study the analgesic effect of glycoproteins and compared with that of aspirin or morphine. The locomotor activity was tested in mice by using actophometer. Four glycoproteins were obtained. The glycoproteins which protein content was the highest (73.04%) displayed dose-dependent analgesic effect. In writhing test, the glycoproteins significantly inhibited writhes (P<0.001) at the dose of 20 mg/kg by intraperitoneal injection. In hot-plate test, only at the dose of 20 mg/kg prolong the hot-plate latency (P<0.05, at 30 min). In the locomotor activity test, the glycoproteins were significant decrease of motility counts at the dose of 20 and 40 mg/kg. These findings collectively indicate that the glycoproteins from the root of Panax ginseng C.A. Mey exhibited significant analgesic activities and the proteins were the active site, providing evidence for its pharmacal use. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

N-linked (N-) glycoproteomics of urinary exosomes. [Corrected].

PubMed

Saraswat, Mayank; Joenväära, Sakari; Musante, Luca; Peltoniemi, Hannu; Holthofer, Harry; Renkonen, Risto

2015-02-01

Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes. © 2015 by The

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

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2005-08-09

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2010-11-16

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

2009-07-14

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2006-10-31

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-08-28

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-07-03

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2010-11-02

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-05-15

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

2007-02-27

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Glycoprotein synthesis

DOEpatents

Shultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2007-04-03

Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

Protein and glycoprotein content of lymphocystis disease virus (LCDV).

PubMed

García-Rosado, Esther; Castro, Dolores; Cano, Irene; Alonso, M Carmen; Pérez-Prieto, Sara I; Borrego, Juan J

2004-06-01

The polypeptide and glycoprotein composition of eight strains of the fish-pathogenic lymphocystis disease virus (LCDV) isolated from gilt-head seabream (Sparus aurata), blackspot seabream (Pagellus bogaraveo), and sole (Solea senegalensis) were determined. The protein electrophoretic patterns of all LCDV isolates were quite similar regardless of the host fish, showing two major proteins (79.9 and 55.6 kDa) and a variable number of minor proteins. Three groups of LCDV isolates were distinguished according to the number and molecular masses of the minor proteins. Eight glycoproteins were detected inside viral particles of LCDV 2, LCDV 3 and LCDV 5 isolates, but only seven glycoproteins were found inside viral particles of LCDV 1, LCDV 4, LCDV 6, LCDV 7, and LCDV 11 isolates and the reference virus ATCC VR 342 by using five lectins. LCDV glycoproteins were mainly composed of mannose and sialic acid. These glycoproteins could be part of an external viral envelope probably derived from the host cell membrane.

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

PubMed Central

Li, Fang; Ryu, Byoung Y.; Krueger, Robin L.; Heldt, Scott A.

2012-01-01

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 106 transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins. PMID:22013043

Trypanosoma cruzi Calreticulin Is a Lectin That Binds Monoglucosylated Oligosaccharides but Not Protein Moieties of Glycoproteins

PubMed Central

Labriola, Carlos; Cazzulo, Juan J.; Parodi, Armando J.

1999-01-01

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse–chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin–cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-β-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin–cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages. PMID:10233151

Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins.

PubMed

Labriola, C; Cazzulo, J J; Parodi, A J

1999-05-01

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

Trypanosoma cruzi serinecarboxipeptidase is a sulfated glycoprotein and a minor antigen in human Chagas disease infection.

PubMed

Soprano, Luciana L; Parente, Juliana E; Landoni, Malena; Couto, Alicia S; Duschak, Vilma G

2018-04-01

In this work, the presence of sulfated N-glycans was studied in a high-mannose-type glycoprotein of Trypanosoma cruzi with serinecarboxipeptidase (TcSCP) activity. The immune cross-reactivity between purified SCP and Cruzipain (Cz) was evidenced using rabbit sera specific for both glycoproteins. Taking advantage that SCP co-purifies with Cz from Concanavalin-A affinity columns, the Cz-SCP mixture was desulfated, ascribing the cross-reactivity to the presence of sulfate groups in both molecules. Therefore, knowing that Cz is a sulfated glycoprotein, with antigenic sulfated epitopes (sulfotopes), SCP was excised from SDS-PAGE and the N-glycosydic chains were analyzed by UV-MALDI-TOF-MS, confirming the presence of short-sulfated high-mannose-type oligosaccharidic chains. Besides, the presence of sulfotopes was analyzed in lysates of the different parasite stages demonstrating that a band with apparent molecular weight similar to SCP was highly recognized in trypomastigotes. In addition, SCP was confronted with sera of infected people with different degrees of cardiac dysfunction. Although most sera recognized it in different groups, no statistical association was found between sera antibodies specific for SCP and the severity of the disease. In summary, our findings demonstrate (1) the presence of sulfate groups in the N-glycosidic short chains of native TcSCP, (2) the existence of immune cross-reactivity between Cz and SCP, purified from epimastigotes, (3) the presence of common sulfotopes between both parasite glycoproteins, and (4) the enhanced presence of sulfotopes in trypomastigotes, probably involved in parasite-host relationship and/or infection. Interestingly, we show for the first time that SCP is a minor antigen recognized by most of chronic Chagas disease patient's sera.

The RING domain and the L79 residue of Z protein are involved in both the rescue of nucleocapsids and the incorporation of glycoproteins into infectious chimeric arenavirus-like particles.

PubMed

Casabona, Juan Cruz; Levingston Macleod, Jesica M; Loureiro, Maria Eugenia; Gomez, Guillermo A; Lopez, Nora

2009-07-01

Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome that encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L), and a RING finger protein (Z), which is the driving force of arenavirus budding. We have established a plasmid-based system which allowed the successful packaging of TacV-like nucleocapsids along with Z and GP of JunV into infectious virus-like particles (VLPs). By coexpressing different combinations of the system components, followed by biochemical analysis of the VLPs, the requirements for the assembly of both N and GP into particles were defined. We found that coexpression of N with Z protein in the absence of minigenome and other viral proteins was sufficient to recruit N within lipid-enveloped Z-containing VLPs. In addition, whereas GP was not required for the incorporation of N, coexpression of N substantially enhanced the ratio of GP to Z into VLPs. Disruption of the RING structure or mutation of residue L79 to alanine within Z protein, although it had no effect on Z self-budding, severely impaired VLP infectivity. These mutations drastically altered intracellular Z-N interactions and the incorporation of both N and GP into VLPs. Our results support the conclusion that the interaction between Z and N is required for assembly of both the nucleocapsids and the glycoproteins into infectious arenavirus budding particles.

Chicken galectin-1B inhibits Newcastle disease virus adsorption and replication through binding to hemagglutinin-neuraminidase (HN) glycoprotein.

PubMed

Sun, Junfeng; Han, Zongxi; Qi, Tianming; Zhao, Ran; Liu, Shengwang

2017-12-08

Galectin-1 is an important immunoregulatory factor and can mediate the host-pathogen interaction via binding glycans on the surface of various viruses. We previously reported that avian respiratory viruses, including lentogenic Newcastle disease virus (NDV), can induce up-regulation of chicken galectin (CG)-1B in the primary target organ. In this study, we investigated whether CG-1B participated in the infectious process of NDV in chickens. We demonstrated that velogenic NDV induced up-regulation of CG-1B in target organs. We also found that CG-1B directly bound to NDV virions and inhibited their hemagglutination activity in vitro We confirmed that CG-1B interacted with NDV hemagglutinin-neuraminidase (HN) glycoprotein, in which the specific G4 N -glycans significantly contributed to the interaction between CG-1B and HN glycoprotein. The presence of extracellular CG-1B, rather than the internalization process, inhibited adsorption of NDV. The interaction between intracellular CG-1B and NDV HN glycoproteins inhibited cell-surface expression of HN glycoprotein and reduced the titer of progeny virus in NDV-infected DF-1 cells. Significantly, the replication of parental and HN glycosylation mutant viruses in CG-1B knockdown and overexpression cells demonstrated that the replication of NDV was correlated with the expression of CG-1B in a specific glycan-dependent manner. Collectively, our results indicate that CG-1B has anti-NDV activity by binding to N -glycans on HN glycoprotein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Similarities and Differences of Blood N-Glycoproteins in Five Solid Carcinomas at Localized Clinical Stage Analyzed by SWATH-MS.

PubMed

Sajic, Tatjana; Liu, Yansheng; Arvaniti, Eirini; Surinova, Silvia; Williams, Evan G; Schiess, Ralph; Hüttenhain, Ruth; Sethi, Atul; Pan, Sheng; Brentnall, Teresa A; Chen, Ru; Blattmann, Peter; Friedrich, Betty; Niméus, Emma; Malander, Susanne; Omlin, Aurelius; Gillessen, Silke; Claassen, Manfred; Aebersold, Ruedi

2018-05-29

Cancer is mostly incurable when diagnosed at a metastatic stage, making its early detection via blood proteins of immense clinical interest. Proteomic changes in tumor tissue may lead to changes detectable in the protein composition of circulating blood plasma. Using a proteomic workflow combining N-glycosite enrichment and SWATH mass spectrometry, we generate a data resource of 284 blood samples derived from patients with different types of localized-stage carcinomas and from matched controls. We observe whether the changes in the patient's plasma are specific to a particular carcinoma or represent a generic signature of proteins modified uniformly in a common, systemic response to many cancers. A quantitative comparison of the resulting N-glycosite profiles discovers that proteins related to blood platelets are common to several cancers (e.g., THBS1), whereas others are highly cancer-type specific. Available proteomics data, including a SWATH library to study N-glycoproteins, will facilitate follow-up biomarker research into early cancer detection. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Immunomodulatory Effects of Nontoxic Glycoprotein Fraction Isolated from Rice Bran.

PubMed

Park, Ho-Young; Yu, A-Reum; Hong, Hee-Do; Kim, Ha Hyung; Lee, Kwang-Won; Choi, Hee-Don

2016-05-01

Rice bran, a by-product of brown rice milling, is a rich source of dietary fiber and protein, and its usage as a functional food is expected to increase. In this study, immunomodulatory effects of glycoprotein obtained from rice bran were studied in normal mice and mouse models of cyclophosphamide-induced immunosuppression. We prepared glycoprotein from rice bran by using ammonium precipitation and anion chromatography techniques. Different doses of glycoprotein from rice bran (10, 25, and 50 mg/kg) were administered orally for 28 days. On day 21, cyclophosphamide at a dose of 100 mg/kg was administered intraperitoneally. Glycoprotein from rice bran showed a significant dose-dependent restoration of the spleen index and white blood cell count in the immunocompromised mice. Glycoprotein from rice bran affected the immunomodulatory function by inducing the proliferation of splenic lymphocytes, which produce potential T and B cells. Moreover, it prevented cyclophosphamide-induced damage of Th1-type immunomodulatory function through enhanced secretion of Th1-type cytokines (interferon-γ and interleukin-12). These results indicate that glycoprotein from rice bran significantly recovered cyclophosphamide-induced immunosuppression. Based on these data, it was concluded that glycoprotein from rice bran is a potent immunomodulator and can be developed to recover the immunity of immunocompromised individuals. Georg Thieme Verlag KG Stuttgart · New York.

Detection of glycoproteins in the Acanthamoeba plasma membrane

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

Paatero, G.I.L.; Gahmberg, C.G.

1988-11-01

In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presencemore » of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.« less

The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana.

PubMed

Hancock, C Nathan; Kent, Lia; McClure, Bruce A

2005-09-01

S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia x SI Nicotiana alata (S105S105 or SC10SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors.

21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5440 Beta-2-glycoprotein III immunological test system. (a) Identification. A beta-2-glycoprotein III... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Beta-2-glycoprotein III immunological test system...

21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5430 Beta-2-glycoprotein I immunological test system. (a) Identification. A beta-2-glycoprotein I... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Beta-2-glycoprotein I immunological test system...

Glycoprotein Enrichment Analytical Techniques: Advantages and Disadvantages.

PubMed

Zhu, R; Zacharias, L; Wooding, K M; Peng, W; Mechref, Y

2017-01-01

Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were developed in recent years. Enrichment methods were used to improve the sensitivity of detection, while HPLC and mass spectrometry methods were developed to facilitate the separation of glycopeptides/proteins and enhance detection, respectively. Fragmentation techniques applied in modern mass spectrometers allow the structural interpretation of glycopeptides/proteins, while automated software tools started replacing manual processing to improve the reliability and throughput of the analysis. In this chapter, the current methodologies of glycoprotein analysis were discussed. Multiple analytical techniques are compared, and advantages and disadvantages of each technique are highlighted. © 2017 Elsevier Inc. All rights reserved.

Glycoprotein import: a common feature of complex plastids?

PubMed

Peschke, Madeleine; Hempel, Franziska

2013-10-01

Complex plastids evolved by secondary endosymbiosis and are, in contrast to primary plastids, surrounded by 3 or 4 envelope membranes. Recently, we provided evidence that in diatoms proteins exist that get N-glycosylated during transport across the outermost membrane of the complex plastid. This gives rise to unique questions on the transport mechanisms of these bulky proteins, which get transported across up to 3 further membranes into the plastid stroma. Here we discuss our results in an evolutionary context and speculate about the existence of plastidal glycoproteins in other organisms with complex plastids.

Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein.

PubMed

Li, J; Quilty, J; Popov, M; Reithmeier, R A

2000-07-01

The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn(642) in the fourth extracellular loop of this polytopic membrane protein, while other isoforms (AE2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular loop. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the preceding extracellular loop in an AE1 glycosylation mutant (N555) resulted in processing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface. Pulse-chase experiments showed that the N555 mutant was efficiently converted from a high-mannose to a complex oligosaccharide with a half-time of approx. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane. The turnover of the complex form of the N555 mutant occurred with a half-life of approx. 15 h. The results show that the oligosaccharide attached to the endogenous site in extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 cells, while the oligosaccharide attached to the preceding loop is converted into the complex form.

Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity.

PubMed Central

Johnson, P R; Olmsted, R A; Prince, G A; Murphy, B R; Alling, D W; Walsh, E E; Collins, P L

1987-01-01

The degree of antigenic relatedness between human respiratory syncytial virus (RSV) subgroups A and B was estimated from antibody responses induced in cotton rats by respiratory tract infection with RSV. Glycoprotein-specific enzyme-linked immunosorbent assays of antibody responses induced by RSV infection demonstrated that the F glycoproteins of subgroups A and B were antigenically closely related (relatedness, R approximately 50%), whereas the G glycoproteins were only distantly related (R approximately 5%). Intermediate levels of antigenic relatedness (R approximately 25%) were seen in neutralizing antibodies from cotton rats infected with RSV of the two subgroups. Immunity against the F glycoprotein of subgroup A, induced by vaccinia-A2-F, conferred a high level of protection which was of comparable magnitude against challenge by RSV of either subgroup. In comparison, immunity against the G glycoprotein of subgroup A, induced by vaccinia-A2-G, conferred less complete, but significant, protection. Importantly, in vaccinia-A2-G-immunized animals, suppression of homologous challenge virus replication was significantly greater (13-fold) than that observed for the heterologous virus. PMID:3305988

Glycoproteins Enrichment and LC-MS/MS Glycoproteomics in Central Nervous System Applications.

PubMed

Zhu, Rui; Song, Ehwang; Hussein, Ahmed; Kobeissy, Firas H; Mechref, Yehia

2017-01-01

Proteins and glycoproteins play important biological roles in central nervous systems (CNS). Qualitative and quantitative evaluation of proteins and glycoproteins expression in CNS is critical to reveal the inherent biomolecular mechanism of CNS diseases. This chapter describes proteomic and glycoproteomic approaches based on liquid chromatography/tandem mass spectrometry (LC-MS or LC-MS/MS) for the qualitative and quantitative assessment of proteins and glycoproteins expressed in CNS. Proteins and glycoproteins, extracted by a mass spectrometry friendly surfactant from CNS samples, were subjected to enzymatic (tryptic) digestion and three down-stream analyses: (1) a nano LC system coupled with a high-resolution MS instrument to achieve qualitative proteomic profile, (2) a nano LC system combined with a triple quadrupole MS to quantify identified proteins, and (3) glycoprotein enrichment prior to LC-MS/MS analysis. Enrichment techniques can be applied to improve coverage of low abundant glycopeptides/glycoproteins. An example described in this chapter is hydrophilic interaction liquid chromatographic (HILIC) enrichment to capture glycopeptides, allowing efficient removal of peptides. The combination of three LC-MS/MS-based approaches is capable of the investigation of large-scale proteins and glycoproteins from CNS with an in-depth coverage, thus offering a full view of proteins and glycoproteins changes in CNS.

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

PubMed

Mengele, R; Sumper, M

1992-04-25

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.

Purification, physicochemical characterization, and immunohistochemical localization of a major 11.7 S glycoprotein from the jelly coats of the anuran Lepidobatrachus laevis.

PubMed

Carroll, E J; Wei, S H; Nagel, G M

1991-02-01

Embryos of the frog Lepidobatrachus laevis are encased by a fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). From preparations of total jelly solubilized from cleavage-stage embryos by a solution of alkaline beta-mercaptoethanol we have purified one jelly coat glycoprotein to homogeneity via FPLC gel permeation chromatography on Superose 6H. The purified glycoprotein was 94% protein and 6% carbohydrate, had an s0(20),w of 11.7 S, with a molecular weight of 245,000 measured by sedimentation equilibrium and 263,000 by gel permeation chromatography. SDS-PAGE revealed that the glycoprotein is composed of a single subunit near 29,700 molecular weight; thus we propose that eight of these subunits comprise the native molecule. Amino acid analysis of the glycoprotein indicated a high content of Glx + Asx (32.4 mole%), a low content of basic amino acids (Arg + Lys = 12.2 mole%), and a single cysteine residue per subunit. The N-terminal amino acid was threonine and the sequence of the first twenty amino acids was determined. Monospecific antisera to the glycoprotein were prepared in rabbits and were used to immunohistochemically localize the glycoprotein throughout the matrix of both jelly layers. Antiserum against the glycoprotein had virtually no effect on the fertilizability of jellied eggs in vitro; thus we hypothesize that the glycoprotein fulfills a structural role in both jelly layers.

Paramyxovirus Glycoproteins and the Membrane Fusion Process.

PubMed

Aguilar, Hector C; Henderson, Bryce A; Zamora, J Lizbeth; Johnston, Gunner P

2016-09-01

The family Paramyxoviridae includes many viruses that significantly affect human and animal health. An essential step in the paramyxovirus life cycle is viral entry into host cells, mediated by virus-cell membrane fusion. Upon viral entry, infection results in expression of the paramyxoviral glycoproteins on the infected cell surface. This can lead to cell-cell fusion (syncytia formation), often linked to pathogenesis. Thus membrane fusion is essential for both viral entry and cell-cell fusion and an attractive target for therapeutic development. While there are important differences between viral-cell and cell-cell membrane fusion, many aspects are conserved. The paramyxoviruses generally utilize two envelope glycoproteins to orchestrate membrane fusion. Here, we discuss the roles of these glycoproteins in distinct steps of the membrane fusion process. These findings can offer insights into evolutionary relationships among Paramyxoviridae genera and offer future targets for prophylactic and therapeutic development.

Paramyxovirus Glycoproteins and the Membrane Fusion Process

PubMed Central

Aguilar, Hector C.; Henderson, Bryce A.; Zamora, J. Lizbeth; Johnston, Gunner P.

2016-01-01

The family Paramyxoviridae includes many viruses that significantly affect human and animal health. An essential step in the paramyxovirus life cycle is viral entry into host cells, mediated by virus-cell membrane fusion. Upon viral entry, infection results in expression of the paramyxoviral glycoproteins on the infected cell surface. This can lead to cell-cell fusion (syncytia formation), often linked to pathogenesis. Thus membrane fusion is essential for both viral entry and cell-cell fusion and an attractive target for therapeutic development. While there are important differences between viral-cell and cell-cell membrane fusion, many aspects are conserved. The paramyxoviruses generally utilize two envelope glycoproteins to orchestrate membrane fusion. Here, we discuss the roles of these glycoproteins in distinct steps of the membrane fusion process. These findings can offer insights into evolutionary relationships among Paramyxoviridae genera and offer future targets for prophylactic and therapeutic development. PMID:28138419

Biosynthesis and maturation of cellular membrane glycoproteins.

PubMed

Hunt, L A

1979-01-01

The biosynthesis and the processing of asparagine-linked oligosaccharides of cellular membrane glycoproteins were examined in monolayer cultures of BHK21 cells and human diploid fibroblasts after pulse- and pulse-chase labeling with [2-3H]mannose. After pronase digestion, radiolabeled glycopeptides were characterized by high-resolution gel filtration, with or without additional digestion with various exoglycosidases and endoglycosidases. Pulse-labeled glycoproteins contained a relatively homogenous population of neutral oligosaccharides (major species: Man9GlcNAc2ASN). The vast majority of these asparagine-linked oligosaccharides was smaller than the major fraction of lipid-linked oligosaccharides from the cell and was apparently devoid of terminal glucose. After pulse-chase or long labeling periods, a significant fraction of the large oligomannosyl cores was processed by removal of mannose units and addition of branch sugars (NeuNAc-Gal-GlcNAc), resulting in complex acidic structures containing three and possibly five mannoses. In addition, some of the large oligomannosyl cores were processed by the removal of only several mannoses, resulting in a mixture of neutral structures with 5-9 mannoses. This oligomannosyl core heterogeneity in both neutral and acidic oligosaccharides linked to asparagine in cellular membrane glycoproteins was analogous to the heterogeneity reported for the oligosaccharides of avian RNA tumor virus glycoproteins (Hunt LA, Wright SE, Etchison JR, Summers DF: J Virol 29:336, 1979).

A misfolded protein conformation is not a sufficient condition for in vivo glucosylation by the UDP-Glc:glycoprotein glucosyltransferase.

PubMed

Fernández, F; D'Alessio, C; Fanchiotti, S; Parodi, A J

1998-10-15

A key element in the quality control of glycoprotein folding is the UDP-Glc:glycoprotein glucosyltransferase (GT), which in cell-free assays exclusively glucosylates misfolded glycoproteins. In order to test if such a protein conformation is a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT, a Schizosaccharomyces pombe double mutant (gls2/alg6) was constructed. With this mutant, Man9GlcNAc2 is transferred to proteins and no removal of glucose units added by GT occurs as it lacks glucosidase II. The same proportion of glucosylated (Glc1Man9GlcNAc2) and unglucosylated (Man9GlcNAc2 and Man8GlcNAc2) endoplasmic reticulum (ER)-specific compounds was produced when cells were pre-incubated for 10, 20 or 30 min and further incubated with [14C]glucose for 10 min at 28 degrees C with or without 5 mM dithiothreitol (DTT), thus indicating not only that DTT did not affect protein glucosylation but also that no increased glucosylation of glycoproteins occurred in the presence of the drug. Monitoring Golgi-specific modifications of oligosaccharides after pulse-chase experiments performed in the presence or absence of 5 mM DTT showed that exit of the bulk of glycoproteins synthesized from the ER and thence their proper folding had been prevented by the drug. Cells pulse-chase labeled at 37 degrees C in the absence of DTT also yielded glucosylated and unglucosylated protein-linked oligosaccharides without Golgi-specific modifications. It was concluded that a misfolded protein conformation is not a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT.

Mutations in the conserved carboxy-terminal hydrophobic region of glycoprotein gB affect infectivity of herpes simplex virus.

PubMed

Wanas, E; Efler, S; Ghosh, K; Ghosh, H P

1999-12-01

Glycoprotein gB is the most highly conserved glycoprotein in the herpesvirus family and plays a critical role in virus entry and fusion. Glycoprotein gB of herpes simplex virus type 1 contains a hydrophobic stretch of 69 aa near the carboxy terminus that is essential for its biological activity. To determine the role(s) of specific amino acids in the carboxy-terminal hydrophobic region, a number of amino acids were mutagenized that are highly conserved in this region within the gB homologues of the family HERPESVIRIDAE: Three conserved residues in the membrane anchor domain, namely A786, A790 and A791, as well as amino acids G743, G746, G766, G770 and P774, that are non-variant in Herpesviridae, were mutagenized. The ability of the mutant proteins to rescue the infectivity of the gB-null virus, K082, in trans was measured by a complementation assay. All of the mutant proteins formed dimers and were incorporated in virion particles produced in the complementation assay. Mutants G746N, G766N, F770S and P774L showed negligible complementation of K082, whereas mutant G743R showed a reduced activity. Virion particles containing these four mutant glycoproteins also showed a markedly reduced rate of entry compared to the wild-type. The results suggest that non-variant residues in the carboxy-terminal hydrophobic region of the gB protein may be important in virus infectivity.

Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

PubMed

Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

2008-01-01

Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

26 CFR 1.103(n)-2T - Private activity bond defined (temporary).

Code of Federal Regulations, 2010 CFR

2010-04-01

...(n)-2T Section 1.103(n)-2T Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY....103(n)-2T Private activity bond defined (temporary). Q-1: What is the definition of the term “private activity bond”? A-1: In general, for purposes of §§ 1.103(n)-1T through 1.103(n)-6T, the term “private...

Role of Carbohydrate in Glycoprotein Traffic and Secretion

DTIC Science & Technology

1988-01-01

synthesized in normal amounts but accumu- lated intracellularly, with transport to the cell surface being greatly de - layed. Glycoprotein E2 isolated from...UNcLA ,F E 2 Role of Carbohydrate in Glycoprotein Traffic and Secretion JAMES B. PARENT I. Introduction I!. Evidence for Intracellular Transport Signals...Ill. Oligosaccharide Biosynthesis IV. Role of Carbohydrate in Protein Solubility. Structure, and Stability V. Evidence for Carbohydrate Transport

Metabolic Flux Increases Glycoprotein Sialylation: Implications for Cell Adhesion and Cancer Metastasis*

PubMed Central

Almaraz, Ruben T.; Tian, Yuan; Bhattarcharya, Rahul; Tan, Elaine; Chen, Shih-Hsun; Dallas, Matthew R.; Chen, Li; Zhang, Zhen; Zhang, Hui; Konstantopoulos, Konstantinos; Yarema, Kevin J.

2012-01-01

This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ∼31% showed no change in sialylation, ∼29% exhibited a modest increase, whereas ∼40% experienced an increase of greater than twofold. Increased sialylation of specific glycoproteins resulted in changes to the adhesive properties of SW1990 pancreatic cancer cells (e.g. increased CD44-mediated adhesion to selectins under physiological flow and enhanced integrin-mediated cell mobility on collagen and fibronectin). These results indicate that cancer cells can become more aggressively malignant by controlling the sialylation of proteins implicated in metastatic transformation via metabolic flux. PMID:22457533

Amorphous titania modified with boric acid for selective capture of glycoproteins.

PubMed

Jin, Shanxia; Liu, Liping; Zhou, Ping

2018-05-22

Amorphous titania was modified with boric acid, and the resulting material was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray powder diffraction and X-ray photoelectron spectrometry. The new material, in contrast to conventional boronate affinity materials containing boronic acid ligands, bears boric acid groups. It is shown to exhibit high specificity for glycoproteins, and this was applied to design a method for solid phase extraction of glycoproteins as shown for ribonuclease B, horse radish peroxidase and ovalbumin. Glycoproteins were captured under slightly alkaline environment and released in acidic solutions. The glycoproteins extracted were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The binding capacities for ribonuclease B, horse radish peroxidase and ovalbumin typically are 9.3, 26.0 and 53.0 mg ∙ g -1 , respectively. The method was successfully applied to the selective enrichment of ovalbumin from egg white. Graphical abstract Schematic presentation of the capture of glycoproteins by amorphous titania modified with boric acid.

Structure and polymorphism of the mouse myelin/oligodendrocyte glycoprotein gene

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

Daubas, P.; Pham-Dinh, D.; Dautigny, A.

1994-09-01

The authors have isolated and characterized genomic clones containing the mouse myelin/oligodendrocyte glycoprotein (MOG) gene. It spans a region of 12.5 kb and consists of eight exons. Its exon-intron structure differs from that of classical MHC-class I genes, with which it is linked in the mouse genome. Nucleotide sequencing of the 5{prime} flanking region revelas that it contains several putative protein-binding sites, some of them in common with other myelin gene promoters. One intragenic polymorphism has been identified: it consists of a GA repeat, defining at least three alleles in mouse inbred strains, and is easily detectable using the polymerasemore » chain reaction method.« less

Dissection of the Antibody Response against Herpes Simplex Virus Glycoproteins in Naturally Infected Humans

PubMed Central

Huang, Zhen-Yu; Whitbeck, J. Charles; Ponce de Leon, Manuel; Lou, Huan; Wald, Anna; Krummenacher, Claude; Eisenberg, Roselyn J.; Cohen, Gary H.

2014-01-01

ABSTRACT Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally

Tuning orb spider glycoprotein glue performance to habitat humidity.

PubMed

Opell, Brent D; Jain, Dharamdeep; Dhinojwala, Ali; Blackledge, Todd A

2018-03-26

Orb-weaving spiders use adhesive threads to delay the escape of insects from their webs until the spiders can locate and subdue the insects. These viscous threads are spun as paired flagelliform axial fibers coated by a cylinder of solution derived from the aggregate glands. As low molecular mass compounds (LMMCs) in the aggregate solution attract atmospheric moisture, the enlarging cylinder becomes unstable and divides into droplets. Within each droplet an adhesive glycoprotein core condenses. The plasticity and axial line extensibility of the glycoproteins are maintained by hygroscopic LMMCs. These compounds cause droplet volume to track changes in humidity and glycoprotein viscosity to vary approximately 1000-fold over the course of a day. Natural selection has tuned the performance of glycoprotein cores to the humidity of a species' foraging environment by altering the composition of its LMMCs. Thus, species from low-humidity habits have more hygroscopic threads than those from humid forests. However, at their respective foraging humidities, these species' glycoproteins have remarkably similar viscosities, ensuring optimal droplet adhesion by balancing glycoprotein adhesion and cohesion. Optimal viscosity is also essential for integrating the adhesion force of multiple droplets. As force is transferred to a thread's support line, extending droplets draw it into a parabolic configuration, implementing a suspension bridge mechanism that sums the adhesive force generated over the thread span. Thus, viscous capture threads extend an orb spider's phenotype as a highly integrated complex of large proteins and small molecules that function as a self-assembling, highly tuned, environmentally responsive, adhesive biomaterial. Understanding the synergistic role of chemistry and design in spider adhesives, particularly the ability to stick in wet conditions, provides insight in designing synthetic adhesives for biomedical applications. © 2018. Published by The Company of

Effect of human alpha 2HS glycoprotein on mouse macrophage function.

PubMed Central

Lewis, J G; André, C M

1980-01-01

alpha 2HS glycoprotein was isolated from normal adult serum. The ability of alpha 2HS glycoprotein to promote the endocytosis of radiolabelled DNA and radiolabelled latex particles by mouse macrophages was investigated. The results using both radiolabelled latex particles and radiolabelled DNA show that alpha 2HS glycoprotein enhances the ability of mouse macrophages to take up these radiolabelled substrates as compared to control cells. Images Figure 1 Figure 2 PMID:7439929

Adipokine zinc-α2-glycoprotein regulated by growth hormone and linked to insulin sensitivity.

PubMed

Balaz, Miroslav; Ukropcova, Barbara; Kurdiova, Timea; Gajdosechova, Lucia; Vlcek, Miroslav; Janakova, Zuzana; Fedeles, Jozef; Pura, Mikulas; Gasperikova, Daniela; Smith, Steven R; Tkacova, Ruzena; Klimes, Iwar; Payer, Juraj; Wolfrum, Christian; Ukropec, Jozef

2015-02-01

Hypertrophic obesity is associated with impaired insulin sensitivity and lipid-mobilizing activity of zinc-α2-glycoprotein. Adipose tissue (AT) of growth hormone (GH) -deficient patients is characterized by extreme adipocyte hypertrophy due to defects in AT lipid metabolism. It was hypothesized that zinc-α2-glycoprotein is regulated by GH and mediates some of its beneficial effects in AT. AT from patients with GH deficiency and individuals with obesity-related GH deficit was obtained before and after 5-year and 24-month GH supplementation therapy. GH action was tested in primary human adipocytes. Relationships of GH and zinc-α2-glycoprotein with adipocyte size and insulin sensitivity were evaluated in nondiabetic patients with noncancerous cachexia and hypertrophic obesity. AT in GH-deficient adults displayed a substantial reduction of zinc-α2-glycoprotein. GH therapy normalized AT zinc-α2-glycoprotein. Obesity-related relative GH deficit was associated with almost 80% reduction of zinc-α2-glycoprotein mRNA in AT. GH increased zinc-α2-glycoprotein mRNA in both AT of obese men and primary human adipocytes. Interdependence of GH and zinc-α2-glycoprotein in regulating AT morphology and metabolic phenotype was evident from their relationship with adipocyte size and AT-specific and whole-body insulin sensitivity. The results demonstrate that GH is involved in regulation of AT zinc-α2-glycoprotein; however, the molecular mechanism linking GH and zinc-α2-glycoprotein in AT is yet unknown. © 2014 The Obesity Society.

Glycoprotein expression by adenomatous polyps of the colon

NASA Astrophysics Data System (ADS)

Roney, Celeste A.; Xie, Jianwu; Xu, Biying; Jabour, Paul; Griffiths, Gary; Summers, Ronald M.

2008-03-01

Colon cancer is the second leading cause of cancer related deaths in the United States. Specificity in diagnostic imaging for detecting colorectal adenomas, which have a propensity towards malignancy, is desired. Adenomatous polyp specimens of the colon were obtained from the mouse model of colorectal cancer called adenomatous polyposis coli-multiple intestinal neoplasia (APC Min). Histological evaluation, by the legume protein Ulex europaeus agglutinin I (UEA-1), determined expression of the glycoprotein α-L-fucose. FITC-labelled UEA-1 confirmed overexpression of the glycoprotein by the polyps on fluorescence microscopy in 17/17 cases, of which 13/17 included paraffin-fixed mouse polyp specimens. In addition, FITC-UEA-1 ex vivo multispectral optical imaging of 4/17 colonic specimens displayed over-expression of the glycoprotein by the polyps, as compared to non-neoplastic mucosa. Here, we report the surface expression of α-L-fucosyl terminal residues by neoplastic mucosal cells of APC specimens of the mouse. Glycoprotein expression was validated by the carbohydrate binding protein UEA-1. Future applications of this method are the development of agents used to diagnose cancers by biomedical imaging modalities, including computed tomographic colonography (CTC). UEA-1 targeting to colonic adenomas may provide a new avenue for the diagnosis of colorectal carcinoma by CT imaging.

Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

PubMed Central

Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

2013-01-01

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and

The Neutralizing Linear Epitope of Human Herpesvirus 6A Glycoprotein B Does Not Affect Virus Infectivity.

PubMed

Wakata, Aika; Kanemoto, Satoshi; Tang, Huamin; Kawabata, Akiko; Nishimura, Mitsuhiro; Jasirwan, Chyntia; Mahmoud, Nora Fahmy; Mori, Yasuko

2018-03-01

Human herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity. IMPORTANCE Glycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents

Glycoprotein interactions in paramyxovirus fusion

PubMed Central

Iorio, Ronald M; Melanson, Vanessa R; Mahon, Paul J

2009-01-01

The Paramyxoviridae are enveloped, negative-stranded RNA viruses, some of which recognize sialic acid-containing receptors, while others recognize specific proteinaceous receptors. The major cytopathic effect of paramyxovirus infection is membrane fusion-induced syncytium formation. Paramyxoviruses are unusual in that the receptor-binding and fusion-promoting activities reside on two different spike structures, the attachment and fusion glycoproteins, respectively. For most paramyxoviruses, this distribution of functions requires a mechanism by which the two processes can be linked for the promotion of fusion. This is accomplished by a virus-specific interaction between the two proteins. An increasing body of evidence supports the notion that members of this family of viruses utilize this glycoprotein interaction in different ways in order to mediate the regulation of the fusion protein activation, depending on the type of receptor utilized by the virus. PMID:20161127

Glycoprotein Biochemistry--Some Clinical Aspects of Interest to Biochemistry Students.

ERIC Educational Resources Information Center

Smith, Christopher A.; And Others

1991-01-01

Authors describe some clinical features of glycoprotein biochemistry, including recognition, selected blood glycoproteins, glycated proteins, histochemistry, and cancer. The material presented has largely been taught to medical laboratory students; however, it can be used to teach premedical students and pure biochemistry students. Includes two…

Effects of carrying a backpack in an asymmetrical manner on the asymmetries of the trunk and parameters defining lateral flexion of the spine.

PubMed

Drzał-Grabiec, Justyna; Snela, Sławomir; Rachwał, Maciej; Podgórska, Justyna; Rykała, Justyna

2015-03-01

The aim of this study was to examine changes in the body posture parameters defining asymmetry of the trunk and lateral flexion of the spine in children while carrying a backpack weighing 10% of a child's weight. Carrying a backpack may negatively affect the posture of schoolchildren and contribute to spinal pain. The study involved 162 primary school students ages 11 to 13 years. The parameters describing body posture were assessed with a backpack carried on the right or left shoulder as well as without a load. To assess the predefined parameters, we used the CQ Elektronik System, employing the photogrammetric method. Trunk inclination shifted significantly in the opposite direction to the shoulder the backpack was carried on, and an increase in shoulder asymmetry was also found. We also observed a more pronounced right-side lateral flexion of the spine when the backpack was carried on the right shoulder and an analogous relationship for the left side. The results of this study show that carrying a backpack in an asymmetrical manner negatively affects spine, even if the backpack weight constitutes 10% of the child's weight, which has been previously recommended as a safe load for a child's shoulders. We suggest that the issue of safe backpack weight be reassessed and that students be taught basic ergonomic principles on how to carry loads. Changes to the management pattern of carrying textbooks to and from school also should be considered. © 2014, Human Factors and Ergonomics Society.

Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

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

Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.

2007-09-30

The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed intomore » the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral

Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

PubMed Central

Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Tyagi, Charu; du Toit, Lisa C.

2015-01-01

The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

Vesicular Stomatitis Virus Pseudotyped with Ebola Virus Glycoprotein Serves as a Protective, Noninfectious Vaccine against Ebola Virus Challenge in Mice

PubMed Central

Lennemann, Nicholas J.; Herbert, Andrew S.; Brouillette, Rachel; Rhein, Bethany; Bakken, Russell A.; Perschbacher, Katherine J.; Cooney, Ashley L.; Miller-Hunt, Catherine L.; Ten Eyck, Patrick; Biggins, Julia; Olinger, Gene; Dye, John M.

2017-01-01

ABSTRACT The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success. IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant

The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases.

PubMed

Naidu, Rayapati A; Ingle, Caroline J; Deom, Carl M; Sherwood, John L

2004-02-05

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G(N) (N-terminal) and G(C) (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G(N) and G(C). The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G(C) showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G(N) was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G(C), with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G(N). None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G(C) or G(N). Treatment of the denatured virions with endoglycosidase H and peptide:N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G(C) to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G(N). These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G(C) and possibly high mannose-type on G(N). Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G(C) is likely to be more

Immunogold detection of glycoprotein antigens in sea urchin embryos.

PubMed

Benson, N C; Benson, S C; Wilt, F

1989-01-01

Four developmental stages of sea urchin embryos were labeled with colloidal gold in an attempt to elucidate the intracellular trafficking patterns within the cells that produce the glycoprotein matrix of the embryonic spicule. The primary mesenchyme cells (PMCs) form a syncytium and secrete an organic matrix on which calcium carbonate is laid down to form an endoskeletal spicule. The organic matrix has been isolated and characterized as glycoprotein consisting of four major bands. Polyclonal antibodies to these glycoproteins were used to label embryos from the mesenchyme blastula, early gastrula, late gastrula, and plutei stages of development. The label is concentrated in the Golgi complex and associated vesicles, in secretory vesicles, and in the organic matrix. The density of the labeling increases as development proceeds.

Glycan Reader: Automated Sugar Identification and Simulation Preparation for Carbohydrates and Glycoproteins

PubMed Central

Jo, Sunhwan; Song, Kevin C.; Desaire, Heather; MacKerell, Alexander D.; Im, Wonpil

2011-01-01

Understanding how glycosylation affects protein structure, dynamics, and function is an emerging and challenging problem in biology. As a first step toward glycan modeling in the context of structural glycobiology, we have developed Glycan Reader and integrated it into the CHARMM-GUI, http://www.charmm-gui.org/input/glycan. Glycan Reader greatly simplifies the reading of PDB structure files containing glycans through (i) detection of carbohydrate molecules, (ii) automatic annotation of carbohydrates based on their three-dimensional structures, (iii) recognition of glycosidic linkages between carbohydrates as well as N-/O-glycosidic linkages to proteins, and (iv) generation of inputs for the biomolecular simulation program CHARMM with the proper glycosidic linkage setup. In addition, Glycan Reader is linked to other functional modules in CHARMM-GUI, allowing users to easily generate carbohydrate or glycoprotein molecular simulation systems in solution or membrane environments and visualize the electrostatic potential on glycoprotein surfaces. These tools are useful for studying the impact of glycosylation on protein structure and dynamics. PMID:21815173

Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

PubMed

Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

2015-01-01

Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

[Glycoproteins of mucus of gastric and duodenal wall surface during ulcerogenesis and the impact of fenugreek].

PubMed

Khil'ko, T D; Iakubtsova, I V; Preobrazhens'ka, T D; Ostapchenko, L I

2013-01-01

The comparative evaluation of qualitative and quantitative composition of glycoproteins of gastric and duodenal wall surface layer of protective mucus in the normal, at the modeling of ulcers in rats and at the introduction to animals with ulcerative lesions of fenugreek extract carried out. It was shown in control (normally) the general level of glycosylation of glycoproteins gastric mucus is 1.7 times more than the duodenum. Under acute stress model ulceration in the stomach mucus decrease in hexosamine (1.4 times), galactose (2.2 times), fucose (1.3-fold) and an increase in NANA (3.6 times) observed. Under cysteamine model ulceration in duodenal mucus increase galactose (2.7 times), NANA (2.4 times), fucose (1.8-fold) but significant decrease in the amount of hexosamines 3 times compared to the control occurred. It was proved the protective effect of fenugreek extract to the wall surface mucus of the stomach and duodenum mucosa under conditions modeling ulceration in rats.

cap alpha. -D-Mannopyranosylmethyl-P-nitrophenyltriazene effects on the degradation and biosynthesis of N-linked oligosaccharide chains on. cap alpha. /sub 1/-acid glycoprotein by liver cells

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

Docherty, P.A.; Aronson, N.N. Jr.

1986-05-01

The effects of ..cap alpha..-D-mannopyranosylmethyl-p-nitrophenyltriazene (..cap alpha..-ManMNT) on the degradation and processing of oligosaccharide chains on ..cap alpha../sub 1/-acid glycoprotein (AGP) were studied. Addition of the triazene to a perfused liver blocked the complete degradation of endocytosed N-acetyl (/sup 14/C)glucosamine-labeled asialo-AGP and caused the accumulation of Man/sub 2/GlcNAc/sub 1/ fragments in the lysosome-enriched fraction of the liver homogenate. This compound also reduced the reincorporation of lysosomally-derived (/sup 14/C)GlcNAc into newly secreted glycoproteins. Cultured hepatocytes treated with the inhibitor synthesized and secreted fully-glycosylated AGP. However, the N-linked oligosaccharide chains on AGP secreted by the ..cap alpha..-ManMNT-treated hepatocytes remained sensitive to digestionmore » with endoglycosidase H, were resistant to neuraminidase, and consisted of Man/sub 9-7/GlcNAc/sub 2/ structures as analyzed by high resolution Bio-Gel P-4 chromatography. As measured by their resistance to cleavage by endoglycosidase H, the normal processing of all six carbohydrate chains on AGP to the complex form did not completely resume until nearly 24 h after triazene treatment. Since ManMNT is likely to irreversibly inactivate ..cap alpha..-D-mannosidases, the return of AGP to secretory forms with complex chains after 24 h probably resulted from synthesis of new processing enzymes.« less

Establishment of a fluorescence-based method to evaluate endocytosis of desialylated glycoproteins in vitro.

PubMed

Luo, Cheng; Chen, Song; Xu, Na; Sai, Wen Bo; Zhao, Wei; Li, Ying Chun; Hu, Xiao Jing; Tian, Hong; Gao, Xiang Dong; Yao, Wen Bing

2017-04-01

Insufficient sialylation can result in rapid clearance of therapeutic glycoproteins by intracellular degradation, which is mainly mediated by asialoglycoprotein receptors (ASGPRs) on hepatic cells. In contrast, for glycoproteins, a long half-life is often related to high level of terminal sialic acid. These could be extremely important for insufficient sialylated biomedicines in clinic, and development of therapeutic glycoproteins in laboratory. However, how the desialylated glycoproteins are removed and how to evaluate the ASGPRs mediated endocytosis in vitro needs further investigate. Herein we described an integrative characterization of ASGPRs in vitro to elucidate its endocytosis properties. The endocytosis was determined by a fluorescence-based quantization method. The results showed that the ASGPRs could bind to poorly sialylated glycoproteins including asialofetuin and low sialylated recombinant Factor VIIa with a relatively higher ASGPRs binding affinity, and induce a more rapid endocytosis in vitro. Moreover, the mechanism under the internalization of ASGPRs was also investigated, which was found to depend on clathrin and caveolin. Utilizing the relative fluorescence quantification can be suitable for measurement of insufficient sialylated glycoprotein endocytosis and quality control of therapeutic glycoproteins, which could be useful for the understanding of the development of therapeutic glycoproteins. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

PubMed

Simó, Rafael; Hernández, Cristina; Sáez-López, Cristina; Soldevila, Berta; Puig-Domingo, Manel; Selva, David M

2014-01-01

Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight

Thyroid Hormone Upregulates Zinc-α2-glycoprotein Production in the Liver but Not in Adipose Tissue

PubMed Central

Simó, Rafael; Hernández, Cristina; Sáez-López, Cristina; Soldevila, Berta; Puig-Domingo, Manel; Selva, David M.

2014-01-01

Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight

Serodiagnosis of infectious mononucleosis with a bovine erythrocyte glycoprotein.

PubMed

Fletcher, M A; Klimas, N G; Latif, Z A; Caldwell, K E

1983-09-01

A glycoprotein from bovine erythrocyte membrane was evaluated in two immunoassays as a reagent for the serodiagnosis of infectious mononucleosis (IM). We previously reported that a partially purified preparation of this glycoprotein, when attached to latex beads, agglutinated in the presence of IM heterophile antibody. In the present study, we used a highly purified form of the glycoprotein both as an agglutinating reagent, covalently bound to latex, and in a solid-phase sandwich-type radioimmunoassay (RIA) for IM antibody detection in a larger population of patients. We tested serum samples from college students with symptoms suggestive of IM with the latex reagent (143 samples) and with the RIA (245 samples). Correlation of these two tests, both with each other and with the classical differentially absorbed, agglutination tests for Paul-Bunnell antibody in IM sera, using fresh sheep or horse cells, was excellent (greater than 97% agreement). The new tests also corresponded in most cases with a rapid, unabsorbed preserved horse erythrocyte slide test. However, in this study of 245 samples, both apparent false-positives (5 samples) and apparent false-negatives (3 samples) were observed with this slide test. In conclusion, we found that the bovine glycoprotein as a reagent can facilitate the diagnosis of IM, giving results comparable to those with erythrocyte agglutination tests on differentially absorbed sera. The advantages are ease and speed of performance (latex test), potential for automation (RIA test), stability and uniformity of the glycoprotein reagent (latex and RIA tests), and most importantly, the ability to use unabsorbed sera (latex and RIA tests).

Core Structure of S2 from the Human Coronavirus NL63 Spike Glycoprotein

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

Zheng,Q.; Deng, Y.; Liu, J.

2006-01-01

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARSmore » coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an {alpha}-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 {sup o}C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.« less

Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

PubMed

Rasheed, Muhammad Asif; Ansari, Abdur Rahman; Ihsan, Awais; Navid, Muhammad Tariq; Ur-Rehman, Shahid; Raza, Sohail

2018-03-01

Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses. Copyright © 2018 Elsevier Ltd. All rights reserved.

90K Glycoprotein Promotes Degradation of Mutant β-Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus.

PubMed

Park, So-Yeon; Yoon, Somy; Kim, Hangun; Kim, Kyung Keun

2016-10-01

β-Catenin is a major transducer of the Wnt signaling pathway, which is aberrantly expressed in colorectal and other cancers. Previously, we showed that β-catenin is downregulated by the 90K glycoprotein via ISGylation-dependent degradation. However, the further mechanisms of β-catenin degradation by 90K-mediated ISGylation pathway were not investigated. This study aimed to identify the β-catenin domain responsible for the action of 90K and to compare the mechanism of 90K on β-catenin degradation with phosphorylation-dependent ubiquitinational degradation of β-catenin. The deletion mutants of β-catenin lacking N- or C-terminal domain or mutating the N-terminal lysine or nonlysine residue were employed to delineate the characteristics of β-catenin degradation by 90K-mediated ISGylation pathway. 90K induced Herc5 and ISG15 expression and reduced β-catenin levels in HeLa and CSC221 cells. The N-terminus of β-catenin is required for 90K-induced β-catenin degradation, but the N-terminus of β-catenin is not essential for interaction with Herc5. However, substituting lysine residues in the N-terminus of β-catenin with arginine or deleting serine or threonine residue containing domains from the N-terminus does not affect 90K-induced β-catenin degradation, indicating that the N-terminal 86 amino acids of β-catenin are crucial for 90K-mediated ISGylation/degradation of β-catenin in which the responsible lysine or nonlysine residues were not identified. Our present results highlight the action of 90K on promoting degradation of mutant β-catenin lacking the phosphorylation sites in the N-terminus. It provides further insights into the discrete pathway downregulating the stabilized β-catenin via acquiring mutations at the serine/threonine residues in the N-terminus. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

CHAPTER 7: Glycoprotein Enrichment Analytical Techniques: Advantages and Disadvantages

PubMed Central

Zhu, Rui; Zacharias, Lauren; Wooding, Kerry M.; Peng, Wenjing; Mechref, Yehia

2017-01-01

Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were developed in recent years. Enrichment methods were used to improve the sensitivity of detection while HPLC and mass spectrometry methods were developed to facilitate the separation of glycopeptides/proteins and enhance detection, respectively. Fragmentation techniques applied in modern mass spectrometers allow the structural interpretation of glycopeptides/proteins while automated software tools started replacing manual processing to improve the reliability and throughout of the analysis. In this chapter, the current methodologies of glycoprotein analysis were discussed. Multiple analytical techniques are compared, and advantages and disadvantages of each technique are highlighted. PMID:28109440

21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in the... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in the... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in the... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in the... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

PubMed Central

Lech, Patrycja J.; Tobin, Gregory J.; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D.; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P.; Russell, Stephen J.; Nara, Peter L.

2013-01-01

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

HIV-1 envelope glycoprotein

DOEpatents

Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

2017-08-22

The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

Mechanisms of ammonia and ammonium transport by rhesus-associated glycoproteins

PubMed Central

Caner, Tolga; Abdulnour-Nakhoul, Solange; Brown, Karen; Islam, M. Toriqul; Hamm, L. Lee

2015-01-01

In this study we characterized ammonia and ammonium (NH3/NH4+) transport by the rhesus-associated (Rh) glycoproteins RhAG, Rhbg, and Rhcg expressed in Xenopus oocytes. We used ion-selective microelectrodes and two-electrode voltage clamp to measure changes in intracellular pH, surface pH, and whole cell currents induced by NH3/NH4+ and methyl amine/ammonium (MA/MA+). These measurements allowed us to define signal-specific signatures to distinguish NH3 from NH4+ transport and to determine how transport of NH3 and NH4+ differs among RhAG, Rhbg, and Rhcg. Our data indicate that expression of Rh glycoproteins in oocytes generally enhanced NH3/NH4+ transport and that cellular changes induced by transport of MA/MA+ by Rh proteins were different from those induced by transport of NH3/NH4+. Our results support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4+ and neutral NH3 species; 2) transport of NH4+ is electrogenic; 3) like Rhbg, RhAG transport of NH4+ masks NH3 transport; and 4) Rhcg is likely to be a predominantly NH3 transporter, with no evidence of enhanced NH4+ transport by this transporter. The dual role of Rh proteins as NH3 and NH4+ transporters is a unique property and may be critical in understanding how transepithelial secretion of NH3/NH4+ occurs in the renal collecting duct. PMID:26354748

QUANTITATIVE MASS SPECTROMETRIC ANALYSIS OF GLYCOPROTEINS COMBINED WITH ENRICHMENT METHODS

PubMed Central

Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

2015-01-01

Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:148–165, 2015. PMID:24889823

Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium

PubMed Central

Blaschke, A. P.; Toze, S.; Sidhu, J. P. S.; Ahmed, W.; van Driezum, I. H.; Sommer, R.; Kirschner, A. K. T.; Cervero-Aragó, S.; Farnleitner, A. H.; Pang, L.

2015-01-01

Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log10 reduction of biotin-coated CPMs, and a 2.6-log10 reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log10 reduction, while the uncoated CPMs displayed a 3.7-log10 reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water. PMID:25888174

Genetics Home Reference: glycoprotein VI deficiency

MedlinePlus

... protein called glycoprotein VI (GPVI). This protein is embedded in the outer membrane of blood cell fragments ... erythematosus (SLE). Autoimmune disorders occur when the immune system malfunctions and attacks the body's own cells and ...

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.

PubMed

Wilke, Sonja; Krausze, Joern; Gossen, Manfred; Groebe, Lothar; Jäger, Volker; Gherardi, Ermanno; van den Heuvel, Joop; Büssow, Konrad

2010-06-01

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals

PubMed Central

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-01-01

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans. PMID:27094903

The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals.

PubMed

Shanmuganatham, Karthik K; Jones, Jeremy C; Marathe, Bindumadhav M; Feeroz, Mohammed M; Jones-Engel, Lisa; Walker, David; Turner, Jasmine; Rabiul Alam, S M; Kamrul Hasan, M; Akhtar, Sharmin; Seiler, Patrick; McKenzie, Pamela; Krauss, Scott; Webby, Richard J; Webster, Robert G

2016-04-20

H9N2 avian influenza viruses are continuously monitored by the World Health Organization because they are endemic; they continually reassort with H5N1, H7N9 and H10N8 viruses; and they periodically cause human infections. We characterized H9N2 influenza viruses carrying internal genes from highly pathogenic H7N3 viruses, which were isolated from chickens or quail from live-bird markets in Bangladesh between 2010 and 2013. All of the H9N2 viruses used in this study carried mammalian host-specific mutations. We studied their replication kinetics in normal human bronchoepithelial cells and swine tracheal and lung explants, which exhibit many features of the mammalian airway epithelium and serve as a mammalian host model. All H9N2 viruses replicated to moderate-to-high titers in the normal human bronchoepithelial cells and swine lung explants, but replication was limited in the swine tracheal explants. In Balb/c mice, the H9N2 viruses were nonlethal, replicated to moderately high titers and the infection was confined to the lungs. In the ferret model of human influenza infection and transmission, H9N2 viruses possessing the Q226L substitution in hemagglutinin replicated well without clinical signs and spread via direct contact but not by aerosol. None of the H9N2 viruses tested were resistant to the neuraminidase inhibitors. Our study shows that the Bangladeshi H9N2 viruses have the potential to infect humans and highlights the importance of monitoring and characterizing this influenza subtype to better understand the potential risk these viruses pose to humans.

The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

PubMed

Voigt, Jürgen; Frank, Ronald; Wöstemeyer, Johannes

2009-02-01

Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.

The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.

PubMed

Feldmann, H; Volchkov, V E; Volchkova, V A; Klenk, H D

1999-01-01

Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.

Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

PubMed

Bender, Ruben R; Muth, Anke; Schneider, Irene C; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J

2016-06-01

Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

NASA Technical Reports Server (NTRS)

Wang, W.; Takezawa, D.; Poovaiah, B. W.

1996-01-01

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

Natural Products based P-glycoprotein Activators for Improved β-amyloid Clearance in Alzheimer's Disease: An in silico Approach.

PubMed

Shinde, Pravin; Vidyasagar, Nikhil; Dhulap, Sivakami; Dhulap, Abhijeet; Hirwani, Raj

2015-01-01

Alzheimer's disease is an age related disorder and is defined to be progressive, irreversible neurodegenerative disease. The potential targets which are associated with the Alzheimer's disease are cholinesterases, N-methyl-D-aspartate receptor, Beta secretase 1, Pregnane X receptor (PXR) and P-glycoprotein (Pgp). P-glycoprotein is a member of the ATP binding cassette (ABC) transporter family, which is an important integral of the blood-brain, blood-cerebrospinal fluid and the blood-testis barrier. Reports from the literature provide evidences that the up-regulation of the efflux pump is liable for a decrease in β -amyloid intracellular accumulation and is an important hallmark in Alzheimer's disease (AD). Thus, targeting β-amyloid clearance by stimulating Pgp could be a useful strategy to prevent Alzheimer's advancement. Currently available drugs provide limited effectiveness and do not assure to cure Alzheimer's disease completely. On the other hand, the current research is now directed towards the development of synthetic or natural based therapeutics which can delay the onset or progression of Alzheimer's disease. Since ancient time medicinal plants such as Withania somnifera, Bacopa monieri, Nerium indicum have been used to prevent neurological disorders including Alzheimer's disease. Till today around 125 Indian medicinal plants have been screened on the basis of ethnopharmacology for their activity against neurological disorders. In this paper, we report bioactives from natural sources which show binding affinity towards the Pgp receptor using ligand based pharmacophore development, virtual screening, molecular docking and molecular dynamics simulation studies for the bioactives possessing acceptable ADME properties. These bioactives can thus be useful to treat Alzheimer's disease.

Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

PubMed

Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

2016-07-01

Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection.

P-glycoprotein in autoimmune rheumatic diseases.

PubMed

García-Carrasco, M; Mendoza-Pinto, C; Macias Díaz, S; Vera-Recabarren, M; Vázquez de Lara, L; Méndez Martínez, S; Soto-Santillán, P; González-Ramírez, R; Ruiz-Arguelles, A

2015-07-01

P-glycoprotein (Pgp) is a transmembrane protein of 170 kD encoded by the multidrug resistance 1 (MDR-1) gene, localized on chromosome 7. More than 50 polymorphisms of the MDR-1 gene have been described; a subset of these has been shown to play a pathophysiological role in the development of inflammatory bowel disease, femoral head osteonecrosis induced by steroids, lung cancer and renal epithelial tumors. Polymorphisms that have a protective effect on the development of conditions such as Parkinson disease have also been identified. P-glycoprotein belongs to the adenosine triphosphate binding cassette transporter superfamily and its structure comprises a chain of approximately 1280 aminoacid residues with an N-C terminal structure, arranged as 2 homologous halves, each of which has 6 transmembrane segments, with a total of 12 segments with 2 cytoplasmic nucleotide binding domains. Many cytokines like interleukin 2 and tumor necrosis factor alpha increase Pgp expression and activity. Pgp functions as an efflux pump for a variety of toxins in order to protect particular organs and tissues as the central nervous system. Pgp transports a variety of substrates including glucocorticoids while other drugs such as tacrolimus and cyclosporine A act as modulators of this protein. The most widely used method to measure Pgp activity is flow cytometry using naturally fluorescent substrates such as anthracyclines or rhodamine 123. The study of drug resistance and its association to Pgp began with the study of resistance to chemotherapy in the treatment of cancer and antiretroviral therapy for human immunodeficiency virus; however, the role of Pgp in the treatment of systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis has been a focus of study lately and has emerged as an important mechanism by which treatment failure occurs. The present review analyzes the role of Pgp in these autoimmune diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever

PubMed Central

Golden, Joseph W.; Maes, Piet; Kwilas, Steven A.; Ballantyne, John

2016-01-01

ABSTRACT Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT50), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. IMPORTANCE Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can

In-Depth N-Glycosylation Reveals Species-Specific Modifications and Functions of the Royal Jelly Protein from Western (Apis mellifera) and Eastern Honeybees (Apis cerana).

PubMed

Feng, Mao; Fang, Yu; Han, Bin; Xu, Xiang; Fan, Pei; Hao, Yue; Qi, Yuping; Hu, Han; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

2015-12-04

Royal jelly (RJ), secreted by honeybee workers, plays diverse roles as nutrients and defense agents for honeybee biology and human health. Despite being reported to be glycoproteins, the glycosylation characterization and functionality of RJ proteins in different honeybee species are largely unknown. An in-depth N-glycoproteome analysis and functional assay of RJ produced by Apis mellifera lingustica (Aml) and Apis cerana cerana (Acc) were conducted. RJ produced by Aml yielded 80 nonredundant N-glycoproteins carrying 190 glycosites, of which 23 novel proteins harboring 35 glycosites were identified. For Acc, all 43 proteins glycosylated at 138 glycosites were reported for the first time. Proteins with distinct N-glycoproteomic characteristics in terms of glycoprotein species, number of N-glycosylated sites, glycosylation motif, abundance level of glycoproteins, and N-glycosites were observed in this two RJ samples. The fact that the low inhibitory efficiency of N-glycosylated major royal jelly protein 2 (MRJP2) against Paenibacillus larvae (P. larvae) and the absence of antibacterial related glycosylated apidaecin, hymenoptaecin, and peritrophic matrix in the Aml RJ compared to Acc reveal the mechanism for why the Aml larvae are susceptible to P. larvae, the causative agent of a fatal brood disease (American foulbrood, AFB). The observed antihypertension activity of N-glycosylated MRJP1 in two RJ samples and a stronger activity found in Acc than in Aml reveal that specific RJ protein and modification are potentially useful for the treatment of hypertensive disease for humans. Our data gain novel understanding that the western and eastern bees have evolved species-specific strategies of glycosylation to fine-tune protein activity for optimizing molecular function as nutrients and immune agents for the good of honeybee and influence on the health promoting activity for human as well. This serves as a valuable resource for the targeted probing of the biological

Metabolism of Glycoproteins in Turpentine Granuloma*

PubMed Central

Prodi, G.; Pane, G.; Romeo, G.

1970-01-01

The local synthesis of sialic acid and sialic acid containing glycoproteins in granuloma experimentally produced with turpentine has been investigated by incubating them in vitro with 14C glucosamine. The content and activity of chromatographically isolated sialic acid of water soluble and water insoluble fractions of tissue incubated at different times after injection of turpentine was determined. A local synthesis of sialic acid and its incorporation both in the soluble and insoluble fractions were found, with a time depending slope. Chromatography on DEAE Sephadex of glycoproteins obtained from water soluble fraction showed that radioactivity was present in 2 peaks. After papain digestion of the insoluble fraction, the sialic acid containing material could be separated into 2 groups of radioactive glycopeptides on DEAE Sephadex. The data demonstrates that granuloma can synthestize in vitro a considerable variety of glycoproteic materials. PMID:5491911

Plasmin-Cleaved β-2-Glycoprotein 1 Is an Inhibitor of Angiogenesis

PubMed Central

Sakai, Taro; Balasubramanian, Krishnakumar; Maiti, Sourindra; Halder, Jyotsna B.; Schroit, Alan J.

2007-01-01

β-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved β-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance. PMID:17872974

A sea lamprey glycoprotein hormone receptor similar with gnathostome thyrotropin hormone receptor.

PubMed

Freamat, Mihael; Sower, Stacia A

2008-10-01

The specificity of the vertebrate hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes is explained by the evolutionary refinement of the specificity of expression and selectivity of interaction between the glycoprotein hormones GpH (FSH, LH, and TSH) and their cognate receptors GpH-R (FSH-R, LH-R, and TSH-R). These two finely tuned signaling pathways evolved by gene duplication and functional divergence from an ancestral GpH/GpH-R pair. Comparative analysis of the protochordate and gnathostome endocrine systems suggests that this process took place prior or concomitantly with the emergence of the gnathostome lineage. Here, we report identification and characterization of a novel glycoprotein hormone receptor (lGpH-R II) in the Agnathan sea lamprey. This 781 residue protein was found approximately 43% identical with mammalian TSH-R and FSH-R representative sequences, and similarly with these two classes of mammalian receptors it is assembled from ten exons. A synthetic ligand containing the lamprey glycoprotein hormone beta-chain tethered upstream of a mammalian alpha-chain activated the lGpH-R II expressed in COS-7 cells but in a lesser extent than lGpH-R I. Molecular phylogenetic analysis of vertebrate GpH-R protein sequences suggests a closer relationship between lGpH-R II and gnathostome thyrotropin receptors. Overall, the presence and characteristics of the lamprey glycoprotein hormone receptors suggest existence of a primitive functionally overlapping glycoprotein hormone/glycoprotein hormone receptor system in this animal.

Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay.

PubMed

Urbanowicz, Richard A; McClure, C Patrick; King, Barnabas; Mason, Christopher P; Ball, Jonathan K; Tarr, Alexander W

2016-09-01

Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, preclinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from quasispecies in individual patients were discovered to behave very differently in this entry model. Empirical optimization of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterized as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) was also sensitive to the amount and ratio of plasmids used, and that protocols for optimal production of these pseudoviruses are dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilizing pseudoviruses to conduct empirical optimization of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping.

Vaccinia Virus Recombinant Expressing Herpes Simplex Virus Type 1 Glycoprotein D Prevents Latent Herpes in Mice

NASA Astrophysics Data System (ADS)

Cremer, Kenneth J.; Mackett, Michael; Wohlenberg, Charles; Notkins, Abner Louis; Moss, Bernard

1985-05-01

In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

Identification of Roles for Peptide: N-Glycanase and Endo-β-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells

PubMed Central

Chantret, Isabelle; Fasseu, Magali; Zaoui, Karim; Le Bizec, Christiane; Sadou Yayé, Hassane; Dupré, Thierry; Moore, Stuart E. H.

2010-01-01

Background During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo β-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells. Methods/Principal Findings During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Δ deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man8GlcNAc2, corresponding to ∼70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc1Man9GlcNAc2 and Man9GlcNAc2 that corresponds to ∼30% of total fOS. Conclusions/Significance As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either

Evidence that maturation of the N-linked glycans of the respiratory syncytial virus (RSV) glycoproteins is required for virus-mediated cell fusion: The effect of {alpha}-mannosidase inhibitors on RSV infectivity

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

McDonald, Terence P.; Jeffree, Chris E.; Li, Ping

2006-07-05

Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the {alpha}-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affectedmore » by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity.« less

Release of chromaffin granule glycoproteins and proteoglycans from potassium-stimulated PC12 pheochromocytoma cells.

PubMed

Salton, S R; Margolis, R U; Margolis, R K

1983-10-01

Cultured PC12 pheochromocytoma cells were labeled with [3H]glucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM). The released complex carbohydrates include chromogranins, dopamine beta-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(beta 1 leads to 3)N-acetylgalactosamine, as well as several mono- and disialyl O-glycosidically-linked oligosaccharides, and the tetrasaccharide AcNeu(alpha 2 leads to 3)Gal(beta 1 leads to 3)[AcNeu(alpha 2 leads to 6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23-68%), heparan sulfate (16-23%), and glycoprotein oligosaccharides (16-54%), which are of the tri- and tetraantennary and O-glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

PubMed Central

Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

2015-01-01

Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

Expression from cloned DNA of biologically active glycoprotein C of herpes simplex virus type 1 in mammalian cells.

PubMed

Ghosh-Choudhury, N; Butcher, M; Ghosh, H P

1990-03-01

A DNA fragment of the herpes simplex virus type 1 genome encoding glycoprotein C (gC-1) has been cloned into different eukaryotic expression vectors for transient and stable expression of the glycoprotein in a number of cell lines. All of these expression vectors use a non-HSV promoter, such as the adenovirus major late promoter or murine leukemia virus long terminal repeat promoter to express gC-1 in COS and CHO cells or 3T3 cells. The gC-1 protein synthesized was fully glycosylated with both N- and O-linked oligosaccharides. Synthesis of the mature 120K gC-1 glycoprotein involved partially glycosylated 100K and 105K proteins and the non-glycosylated 70K protein as intermediate molecules. Immunofluorescence studies showed that the expressed gC-1 was localized intracellularly in the nuclear envelope as well as on the cell surface. The expressed gC-1 was biologically active and could act as a receptor for the complement component C3b in the absence of other HSV proteins.

Shoot inversion inhibition of stem elongation in Pharbitis nil: a possible role for ethylene-induced glycoprotein and lignin

NASA Technical Reports Server (NTRS)

Prasad, T. K.; Cline, M. G.

1987-01-01

Inversion of the upper shoot of Pharbitis nil results in the inhibition of elongation in the inverted stem. The objective of the present study was to determine how shoot inversion-induced gravity stress inhibited elongation and to elucidate the possible role of ethylene-induced glycoprotein and lignin in this process. Determinations of hydroxyproline, peroxidase, phenylalanine ammonia-lyase (PAL), phenol, and lignin content/activity were carried out by appropriate spectrophotometric methods. It was found that inversion and Ethrel treatments of upright shoots caused significant increases in hydroxyproline content, peroxidase, and PAL activity in 12 hours and in phenol and lignin contents in 24 hours. All of these increases except for that of cytoplasmic peroxidase activity were partially reversed by AgNO3, the ethylene action inhibitor. It is concluded that possible cross-linking associated with the accumulation of the ethylene-induced hydroxyproline-rich glycoprotein and lignin may be responsible for the later stages of cessation of elongation in the inverted Pharbitis shoot.

21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...

Hepatitis C Patient-Derived Glycoproteins Exhibit Marked Differences in Susceptibility to Serum Neutralizing Antibodies: Genetic Subtype Defines Antigenic but Not Neutralization Serotype▿

PubMed Central

Tarr, Alexander W.; Urbanowicz, Richard A.; Hamed, Mohamed R.; Albecka, Anna; McClure, C. Patrick; Brown, Richard J. P.; Irving, William L.; Dubuisson, Jean; Ball, Jonathan K.

2011-01-01

Neutralizing antibodies have a role in controlling hepatitis C virus (HCV) infection. A successful vaccine will need to elicit potently neutralizing antibodies that are capable of preventing the infection of genetically diverse viral isolates. However, the specificity of the neutralizing antibody response in natural HCV infection still is poorly understood. To address this, we examined the reactivity of polyclonal antibodies isolated from chronic HCV infection to the diverse patient-isolated HCV envelope glycoproteins E1 and E2 (E1E2), and we also examined the potential to neutralize the entry of pseudoparticles bearing these diverse E1E2 proteins. The genetic type of the infection was found to determine the pattern of the antibody recognition of these E1E2 proteins, with the greatest reactivity to homologous E1E2 proteins. This relationship was strongest when the component of the antibody response directed only to linear epitopes was analyzed. In contrast, the neutralization serotype did not correlate with genotype. Instead, serum-derived antibodies displayed a range of neutralization breadth and potency, while different E1E2 glycoproteins displayed different sensitivities to neutralization, such that these could be divided broadly into neutralization-sensitive and -resistant phenotypes. An important additional observation was that entry mediated by some E1E2 proteins was enhanced in the presence of some of the polyclonal antibody fractions isolated during chronic infection. These data highlight the need to use diverse E1E2 isolates, which represent extremes of neutralization sensitivity, when screening antibodies for therapeutic potential and for testing antibodies generated following immunization as part of vaccine development. PMID:21325403

Increased Expression of P-Glycoprotein Is Associated With Chlorpyrifos Resistance in the German Cockroach (Blattodea: Blattellidae).

PubMed

Hou, Weiyuan; Jiang, Chu; Zhou, Xiaojie; Qian, Kun; Wang, Lei; Shen, Yanhui; Zhao, Yan

2016-12-01

A principal method for control of the German cockroach, Blattella germanica (L.), is the broad-spectrum organophosphorus insecticide, chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate); however, extensive and repeated application has resulted in the development of resistance to chlorpyrifos in this insect. Evidence suggests that ATP-binding cassette protein transporters, including P-glycoprotein, are involved in insecticide resistance. However, little is known of the role of P-glycoprotein in insecticide resistance in the German cockroach. Here, we developed a chlorpyrifos-resistant strain of German cockroach and investigated the relationship between P-glycoprotein and chlorpyrifos resistance using toxicity assays; inhibition studies with two P-glycoprotein inhibitors, verapamil and quinine; P-glycoprotein-ATPase activity assays; and western blotting analysis. After 23 generations of selection from susceptible strain cockroaches, we obtained animals with high resistance to chlorpyrifos. When P-glycoprotein-ATPase activity was inhibited by verapamil and quinine, we observed enhanced susceptibility to chlorpyrifos in both control and chlorpyrifos-resistant cockroaches. No significant alterations of P-glycoprotein expression or ATPase activity were observed in cockroaches acutely exposed to LD50 doses of chlorpyrifos for 24 h, while P-glycoprotein expression and ATPase activity were clearly elevated in the chlorpyrifos-resistant cockroach strain. Thus, we conclude that P-glycoprotein is associated with chlorpyrifos resistance in the German cockroach and that elevated levels of P-glycoprotein expression and ATPase activity may be an important mechanism of chlorpyrifos resistance in the German cockroach. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase.

PubMed

Kukushkin, Nikolay V; Alonzi, Dominic S; Dwek, Raymond A; Butters, Terry D

2011-08-15

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase. © The Authors Journal compilation © 2011 Biochemical Society

Spatial localization of the Ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography.

PubMed

Tran, Erin E H; Simmons, James A; Bartesaghi, Alberto; Shoemaker, Charles J; Nelson, Elizabeth; White, Judith M; Subramaniam, Sriram

2014-09-01

The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Dietary Hizikia fusiformis glycoprotein-induced IGF-I and IGFBP-3 associated to somatic growth, polyunsaturated fatty acid metabolism, and immunity in juvenile olive flounder Paralichthys olivaceus.

PubMed

Choi, Youn Hee; Kim, Kang-Woong; Han, Hyon-Sob; Nam, Taek Jeong; Lee, Bong-Joo

2014-01-01

This study was aimed to examine the effect of dietary glycoprotein extracted from the sea mustard Hizikia fusiformis (Phaeophyceae: Sargassaceae) as a dietary supplement on growth performance in association with somatotropin level, proximate compositions, and immunity in juvenile olive flounder Paralichthys olivaceus. Water-ethanol extracted glycoprotein from H. fusiformis was supplemented to three fishmeal-based diets at the concentration of 0, 5, and 10gkg(-1) diet (designated as H0, H5, and H10, respectively). After a 12week-long feeding trial, growth performance and biochemical responses were analyzed including proximate composition, and whole body amino acids and fatty acids. We also measured plasma insulin like growth factor (IGF), IGF-binding protein (IGFBP) and interleukin (IL). The fish fed H5 showed the greatest weight gain among the dietary treatments. In parallel with the growth, the fish fed the diets containing H. fusiformis glycoprotein showed an increased plasma IGF-I activity and increased expression of 43-kDa IGFBP-3 compared to that in the control, whereas an opposite trend was observed for 34-kDa IGFBP-1. Although no differences were found in the level of whole body linoleic acid (C18:2n-6) and linolenic acid (C18:3n-3) among treatments, increases in arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were observed in fish fed H5 compared to control. IL-2 and -6 levels increased significantly in fish fed H10 compared to those in the control indicating increased immunity. These results suggest that supplementation of H. fusiformis glycoprotein in fish diet may be beneficial for fish growth and immunity in juvenile olive flounder. © 2013.

Interaction of forskolin with the P-glycoprotein multidrug transporter

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

Ming s, D.I.; Seamon, K.B.; Speicher, L.A.

1991-08-27

Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug reing ance (MDR) phenotype. Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells. Two photoactive derivatives of forskolin have been synthesized, 7-O-((2-(3-(4-azido-3-({sup 125}I)iodophenyl)propionamido)ethyl)carbamyl)forskolin, {sup 125}I-6-AIPP-Fsk, and 6-O-((2-(3-(4-azido-3-({sup 125}I)iodophenyl)propionamido)ethyl)carbamyl)forskolin, {sup 125}I-6-AIPP-Fsk, which exhibit specificity for labelingmore » the glucose transporter and aing lyl cyclase, respectively. Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin. The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR. The ability of forskolin photolabels to specifically label the transporter, the adenylyl cyclase, and the P-glycoprotein suggests that these proteins may share a common biing g domain for forskolin analogues.« less

Marine Natural Products with P-Glycoprotein Inhibitor Properties

PubMed Central

Lopez, Dioxelis; Martinez-Luis, Sergio

2014-01-01

P-glycoprotein (P-gp) is a protein belonging to the ATP-binding cassette (ABC) transporters superfamily that has clinical relevance due to its role in drug metabolism and multi-drug resistance (MDR) in several human pathogens and diseases. P-gp is a major cause of drug resistance in cancer, parasitic diseases, epilepsy and other disorders. This review article aims to summarize the research findings on the marine natural products with P-glycoprotein inhibitor properties. Natural compounds that modulate P-gp offer great possibilities for semi-synthetic modification to create new drugs and are valuable research tools to understand the function of complex ABC transporters. PMID:24451193

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

A Diverse Panel of Hepatitis C Virus Glycoproteins for Use in Vaccine Research Reveals Extremes of Monoclonal Antibody Neutralization Resistance.

PubMed

Urbanowicz, Richard A; McClure, C Patrick; Brown, Richard J P; Tsoleridis, Theocharis; Persson, Mats A A; Krey, Thomas; Irving, William L; Ball, Jonathan K; Tarr, Alexander W

2015-12-23

Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies. Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and

Instabilities of Current Carrying Torus

NASA Astrophysics Data System (ADS)

Liu, Wenjuan; Qiu, J.

2010-05-01

We investigate the initial equilibrium and stability conditions for an uniform current-carrying plasma ring with a non-trivial toroidal magnetic field Bt. Realistic parameters comparable to observations are used to describe the magnetic field inside and outside the torus. The external poloidal magnetic field is assumed to fall off as a power function with decay index n (n = - d log (Bex) /d log(h)). The parameter space is explored to find all initial equilibrium solutions, at which perturbation is introduced. It is shown that with non-trivial toroidal field, the current ring attains equilibrium with a weaker external field. It is also shown that the torus attains equilibrium at higher altitude when the external field decays more rapidly (greater n) or the ratio of the toroidal flux in the torus to the external field increases. We further study stabilities of the torus at equilibrium by defining a critical decay index ncr (Kliem and Török 2006). A sufficiently strong toroidal field can completely suppress the torus instability due to the current hoop force. With a weak toroidal field, similar to the case of Bt=0, the instability occurs when the external magnetic field declines rapidly with height when the field decay index n>ncr. For realistic sets of parameters, the equilibrium height is within 10 solar radii, and the effective ncr is in the range of 1.0-1.6. The critical decay index increases when the ratio of the toroidal flux to the external field decreases. This work is supported by NSF CAREER grant ATM-0748428.

Glycoproteins functionalized natural and synthetic polymers for prospective biomedical applications: A review.

PubMed

Tabasum, Shazia; Noreen, Aqdas; Kanwal, Arooj; Zuber, Mohammad; Anjum, Muhammad Naveed; Zia, Khalid Mahmood

2017-05-01

Glycoproteins have multidimensional properties such as biodegradability, biocompatibility, non-toxicity, antimicrobial and adsorption properties; therefore, they have wide range of applications. They are blended with different polymers such as chitosan, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone (PVP), polycaprolactone (PCL), heparin, polystyrene fluorescent nanoparticles (PS-NPs) and carboxyl pullulan (PC) to improve their properties like thermal stability, mechanical properties, resistance to pH, chemical stability and toughness. Considering the versatile charateristics of glycoprotein based polymers, this review sheds light on synthesis and characterization of blends and composites of glycoproteins, with natural and synthetic polymers and their potential applications in biomedical field such as drug delivery system, insulin delivery, antimicrobial wound dressing uses, targeting of cancer cells, development of anticancer vaccines, development of new biopolymers, glycoproteome research, food product and detection of dengue glycoproteins. All the technical scientific issues have been addressed; highlighting the recent advancement. Copyright © 2017 Elsevier B.V. All rights reserved.

[C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

PubMed

Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

2017-01-01

The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

NASA Astrophysics Data System (ADS)

Smeekens, Johanna M.; Chen, Weixuan; Wu, Ronghu

2015-04-01

Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide- N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

Defining Putative Glycan Cancer Biomarkers by Mass Spectrometry

PubMed Central

Mechref, Yehia; Hu, Yunli; Garcia, Aldo; Hussein, Ahmed

2013-01-01

Summary For decades, the association between aberrant glycosylation and many types of cancers has been shown. However, defining the changes of glycan structures has not been demonstrated until recently. This has been facilitated by the major advances in mass spectrometry and separation science which allowed the detailed characterization of glycan changes associated with cancer. Mass spectrometry glycomics methods have been successfully employed to compare the glycomic profiles of different human specimen collected from disease-free individuals and patients with cancer. Additionally, comparing the glycomic profiles of glycoproteins purified from specimen collected from disease-free individuals and patients with cancer has also been performed. These types of glycan analyses employing mass spectrometry or liquid-chromatography mass spectrometry allowed the characterization of native, labeled, and permethylated glycans. This review discusses the different glycomic and glycoproteomic methods employed for defining glycans as cancer biomarkers of different organs, including breast, colon, esophagus, liver, lung, ovarian, pancreas and prostate. PMID:23157355

Detection and initial characterization of protein entities consisting of the HIV glycoprotein cytoplasmic C-terminal domain alone.

PubMed

Pfeiffer, Tanya; Ruppert, Thomas; Schaal, Heiner; Bosch, Valerie

2013-06-20

Employing antibodies against the cytoplasmic tail of the HIV-1 glycoprotein (Env-CT), in addition to gp160/gp41, we have identified several novel small Env proteins (<25kD) in HIV-1 transfected and infected cells. Mass spectrometric and mutational analyses show that two mechanisms contribute to their generation. Thus the protein, designated Tr-Env-CT (for truncated Env-CT), consists of the C-terminal 139 amino acids (aa) of Env (aa 718-856) with the N-terminal Q718 modified to pyroglutamic acid. It is likely derived from full-length Env protein by proteolytic processing. A further heterogeneous set of slightly larger proteins, termed Env-CT* species, are rather derived from spliced mRNAs containing only those Env C-terminal residues (aa 719-856) which overlap with the second tat and rev coding exons. They are N-terminally extended in the same reading frame. It is conceivable that essential Env-CT functions may be fulfilled by these novel species rather than by the full-length glycoprotein itself. Copyright © 2013 Elsevier Inc. All rights reserved.

Extraction of physical Schottky parameters using the Lambert function in Ni/AlGaN/GaN HEMT devices with defined conduction phenomena

NASA Astrophysics Data System (ADS)

Latry, O.; Divay, A.; Fadil, D.; Dherbécourt, P.

2017-01-01

Electrical characterization analyses are proposed in this work using the Lambert function on Schottky junctions in GaN wide band gap semiconductor devices for extraction of physical parameters. The Lambert function is used to give an explicit expression of the current in the Schottky junction. This function is applied with defined conduction phenomena, whereas other work presented arbitrary (or undefined) conduction mechanisms in such parameters’ extractions. Based upon AlGaN/GaN HEMT structures, extractions of parameters are undergone in order to provide physical characteristics. This work highlights a new expression of current with defined conduction phenomena in order to quantify the physical properties of Schottky contacts in AlGaN/GaN HEMT transistors. Project supported by the French Department of Defense (DGA).

Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing.

PubMed

Shrivastava-Ranjan, Punya; Flint, Mike; Bergeron, Éric; McElroy, Anita K; Chatterjee, Payel; Albariño, César G; Nichol, Stuart T; Spiropoulou, Christina F

2018-05-01

Ebola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013-2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection in vitro Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD. IMPORTANCE Treatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune

Structural and immunologic correlates of chemically stabilized HIV-1 envelope glycoproteins

PubMed Central

de Val, Natalia; Montefiori, David; Tomaras, Georgia D.; Shen, Xiaoying; Kalyuzhniy, Oleksandr; Sanders, Rogier W.; McCoy, Laura E.; Moore, John P.; Ward, Andrew B.

2018-01-01

Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes*

PubMed Central

Kaulfürst-Soboll, Heidi; Rips, Stephan; Koiwa, Hisashi; Kajiura, Hiroyuki; Fujiyama, Kazuhito; von Schaewen, Antje

2011-01-01

Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry β1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-β1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins. PMID:21478158

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

PubMed

Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

2010-03-01

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. Copyright 2009 Elsevier B.V. All rights reserved.

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system

PubMed Central

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D.; Baba, Hiroko; Ikenaka, Kazuhiro

2017-01-01

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy. PMID:28186137

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system.

PubMed

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D; Baba, Hiroko; Ikenaka, Kazuhiro

2017-02-10

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P 0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy.

Intracellular transport and stability of varicella-zoster virus glycoprotein K

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

Hall, Susan L.; Govero, Jennifer L.; Heineman, Thomas C.

2007-02-20

VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established thatmore » gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.« less

Sweating the small stuff: Glycoproteins in human sweat and their unexplored potential for microbial adhesion.

PubMed

Peterson, Robyn A; Gueniche, Audrey; Adam de Beaumais, Ségolène; Breton, Lionel; Dalko-Csiba, Maria; Packer, Nicolle H

2016-03-01

There is increasing evidence that secretory fluids such as tears, saliva and milk play an important role in protecting the human body from infection via a washing mechanism involving glycan-mediated adhesion of potential pathogens to secretory glycoproteins. Interaction of sweat with bacteria is well established as the cause of sweat-associated malodor. However, the role of sweat glycoproteins in microbial attachment has received little, if any, research interest in the past. In this review, we demonstrate how recent published studies involving high-throughput proteomic analysis have inadvertently, and fortuitously, exposed an abundance of glycoproteins in sweat, many of which have also been identified in other secretory fluids. We bring together research demonstrating microbial adhesion to these secretory glycoproteins in tears, saliva and milk and suggest a similar role of the sweat glycoproteins in mediating microbial attachment to sweat and/or skin. The contribution of glycan-mediated microbial adhesion to sweat glycoproteins, and the associated impact on sweat derived malodor and pathogenic skin infections are unchartered new research areas that we are beginning to explore. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Analytical Pipeline for Discovery and Verification of Glycoproteins from Plasma-Derived Extracellular Vesicles as Breast Cancer Biomarkers.

PubMed

Chen, I-Hsuan; Aguilar, Hillary Andaluz; Paez Paez, J Sebastian; Wu, Xiaofeng; Pan, Li; Wendt, Michael K; Iliuk, Anton B; Zhang, Ying; Tao, W Andy

2018-05-15

Glycoproteins comprise more than half of current FDA-approved protein cancer markers, but the development of new glycoproteins as disease biomarkers has been stagnant. Here we present a pipeline to develop glycoproteins from extracellular vesicles (EVs) through integrating quantitative glycoproteomics with a novel reverse phase glycoprotein array and then apply it to identify novel biomarkers for breast cancer. EV glycoproteomics show promise in circumventing the problems plaguing current serum/plasma glycoproteomics and allowed us to identify hundreds of glycoproteins that have not been identified in blood. We identified 1,453 unique glycopeptides representing 556 glycoproteins in EVs, among which 20 were verified significantly higher in individual breast cancer patients. We further applied a novel glyco-specific reverse phase protein array to quantify a subset of the candidates. Together, this study demonstrates the great potential of this integrated pipeline for biomarker discovery.

Homologous and heterologous antibody responses of mice immunized with purified feline herpesvirus type 1 and canine herpesvirus glycoproteins.

PubMed

Limcumpao, J A; Horimoto, T; Xuan, X N; Tohya, Y; Azetaka, M; Takahashi, E; Mikami, T

1991-06-01

The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.

N-linked glycoprotein analysis using dual-extraction ultrahigh-performance liquid chromatography and electrospray tandem mass spectrometry.

PubMed

Siu, S O; Lam, Maggie P Y; Lau, Edward; Yeung, William S B; Cox, David M; Chu, Ivan K

2010-01-01

Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever.

PubMed

Golden, Joseph W; Maes, Piet; Kwilas, Steven A; Ballantyne, John; Hooper, Jay W

2016-01-20

Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT(50)), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can mitigate the

Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

PubMed Central

Saw, Wan Ting; Eisenberg, Roselyn J.; Cohen, Gary H.

2016-01-01

ABSTRACT Receptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH2/gL2), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD1) and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing the activity of the mutants. Under low-pH conditions or when

Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

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

Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

1986-10-01

A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX;more » gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.« less

Bioactivity of proteins isolated from Lactobacillus plantarum L67 treated with Zanthoxylum piperitum DC glycoprotein.

PubMed

Song, S; Oh, S; Lim, K-T

2015-06-01

Lactobacilli in the human gastrointestinal tract have beneficial effects on the health of their host. To enhance these effects, the bioactivity of lactobacilli can be fortified through exogenous dietary or pharmacological agents, such as glycoproteins. To elucidate the inductive effect of Zanthoxylum piperitum DC (ZPDC) glycoprotein on Lactobacillus plantarum L67, we evaluated the radical-scavenging activity, anti-oxidative enzymes (SOD, GPx and CAT), growth rate, ATPase activity and β-galactosidase activity of this strain. When Lact. plantarum L67 was treated with ZPDC glycoprotein at different concentrations, the intensities of a few SDS-PAGE bands were slightly changed. The amount of a 23 kDa protein was increased upon treatment with increasing concentrations of ZPDC glycoprotein. The results of this study indicate that the radical-scavenging activity for O2(-) and OH¯, but not for the DPPH radical, increased in a concentration-dependent manner after treatment with ZPDC glycoprotein. The activation of anti-oxidative enzymes (SOD, GPx and CAT), growth rate and β-galactosidase activity also increased in a concentration-dependent manner in response to ZPDC glycoprotein treatment, whereas ATPase activity was decreased. In summary, ZPDC glycoprotein stimulated an increase in the bioactivity of Lact. plantarum L67. Significance and impact of the study: This study demonstrated that Lactobacillus plantarum L67 possesses anti-oxidative activity. This strain of lactic bacteria has been known to have various probiotic uses, such as yogurt starters and dietary additional supplements. We found, through this experiment, that the protein has a strong anti-oxidative character, and the activity can be enhanced by treatment with Zanthoxylum piperitum DC (ZPDC) glycoprotein. This study may be application of Lact. plantarum L67 treated by ZPDC glycoprotein in yogurt fermentation. It could be one of the avenues of minimizing yogurt postacidification during storage. In addition

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

PubMed

Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

2015-01-02

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

The effect of P-glycoprotein on methadone hydrochloride flux in equine intestinal mucosa.

PubMed

Linardi, R L; Stokes, A M; Andrews, F M

2013-02-01

Methadone is an effective analgesic opioid that may have a place for the treatment of pain in horses. However, its absorption seems to be impaired by the presence of a transmembrane protein, P-glycoprotein, present in different tissues including the small intestine in other species. This study aims to determine the effect of the P-glycoprotein on methadone flux in the equine intestinal mucosa, as an indicator of in vivo drug absorption. Jejunum tissues from five horses were placed into the Ussing chambers and exposed to methadone solution in the presence or absence of Rhodamine 123 or verapamil. Electrical measurements demonstrated tissue viability for 120 min, and the flux of methadone across the jejunal membrane (mucosal to submucosal direction) was calculated based on the relative drug concentration measured by ELISA. The flux of methadone was significantly higher only in the presence of verapamil. P-glycoprotein was immunolocalized in the apical membrane of the jejunal epithelial cells (enterocytes), mainly located in the tip of the villi compared to cells of the crypts. P-glycoprotein is present in the equine jejunum and may possibly mediate the intestinal transport of methadone. This study suggests that P-glycoprotein may play a role in the poor intestinal absorption of methadone in vivo. © 2012 Blackwell Publishing Ltd.

HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

PubMed

Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

2016-01-01

Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins.

PubMed

Anumula, K R; Du, P

1999-11-15

Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose. Copyright 1999 Academic Press.

[Pregnancy-specific beta-glycoprotein in the serum of women with a complicated early pregnancy].

PubMed

Radikov, N

1989-01-01

The author determined pregnancy specific beta 1-glycoprotein in 109 women with threatened early pregnancy as 32 of the women suffered from abortus imminens with several unsuccessful pregnancies in the past as well as 67 women with abortus incipiens with bleeding ex utero. The author established that 87% of women with abortus imminens and preserved pregnancies had values of beta 1-glycoprotein close to those of normal pregnancy for the respective gestational week. 93% of women with abortus incipiens preserved pregnancies till term, but the specific glycoprotein was with in normal ranges. Spontaneous abortion occurred in 7% of women with low values under the 10th percentile. The present study show that examination of pregnancy specific beta 1-glycoprotein in women with threatened early pregnancy is of prognostic significance for the outcome of pregnancy.

Study on the extraction and purification of glycoprotein from the yellow seahorse, Hippocampus kuda Bleeker

PubMed Central

Su, Yuting; Xu, Yongjian

2015-01-01

The optimum parameters of extraction for glycoprotein from seahorse were examined and determined by Box-Behnken combined with ultrasonic extraction technology. Column chromatography of glycoprotein was used for further purification. The optimal extraction conditions of seahorse glycoprotein were extracting time 4.3 h, salt concentration 0.08 mol/L, extracting temperature 73°C, raw material, and water ratio 1:6. At the optimal conditions, the yield of saccharide reached to 1.123%, and the yield of protein reached to 5.898%. For purifying the crude glycoprotein, the stage renounces of DEAE-52 column chromatography were done, respectively, with 0.05, 0.1, 0.5 mol/L NaHCO3 solution, and further purification was done with Sephadex G-100 column chromatography. Finally, two pieces of seahorse glycoprotein were obtained by the column chromatography, that is, HG-11 and HG-21. The saccharide content was 56.7975% and 39.479%, the protein content was 30.5475% and 51.747%, respectively. PMID:26288722

A recombinant rabies virus carrying GFP between N and P affects viral transcription in vitro.

PubMed

Luo, Jun; Zhao, Jing; Tian, Qin; Mo, Weiyu; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

2016-06-01

Several studies have demonstrated the rabies virus to be a perfect potential vaccine vector to insert foreign genes into the target genome. For this study, a green fluorescent protein (GFP) gene was cloned into the rabies virus (RABV) genome between the N and P gene. CT dinucleotide was inserted as intergenic region. The recombinant high egg passage Flury strain (HEP-Flury) of RABV, carrying GFP (rHEP-NP-GFP), was generated in BHK-21 cells using reverse genetics. According to the viral growth kinetics assay, the addition of GFP between N and P gene has little effect on the viral growth compared to the parental strain HEP-Flury. Quantitative real-time PCR (qPCR) indicated that rHEP-NP-GFP showed different viral gene transcription, especially for G gene, compared to HEP-Flury. The same is true for one other recombinant RABV carrying GFP between G and L gene in NA cells. In addition, parent HEP-Flury showed more expression of innate immune-related molecules in NA cells. Compared to HEP-Flury, Western blotting (WB) indicated that insertion of a foreign gene following N gene enhanced the expression of M and G proteins. According to the qPCR and WB, GFP expression levels of rHEP-NP-GFP were significantly higher than rHEP-GFP. This study indicates HEP-Flury as valid vector to express exogenous genes between N and P.

Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

PubMed Central

Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

2010-01-01

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

Energy carries information

NASA Astrophysics Data System (ADS)

Ilgin, Irfan; Yang, I.-Sheng

2014-08-01

We show that for every qubit of quantum information, there is a well-defined notion of "the amount of energy that carries it," because it is a conserved quantity. This generalizes to larger systems and any conserved quantities: the eigenvalue spectrum of conserved charges has to be preserved while transferring quantum information. It is possible to "apparently" violate these conservations by losing a small fraction of information, but that must invoke a specific process which requires a large scale coherence. We discuss its implication regarding the black hole information paradox.

Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity.

PubMed Central

Dubay, J W; Roberts, S J; Hahn, B H; Hunter, E

1992-01-01

Human immunodeficiency virus type 1 contains a transmembrane glycoprotein with an unusually long cytoplasmic domain. To determine the role of this domain in virus replication, a series of single nucleotide changes that result in the insertion of premature termination codons throughout the cytoplasmic domain has been constructed. These mutations delete from 6 to 192 amino acids from the carboxy terminus of gp41 and do not affect the amino acid sequence of the regulatory proteins encoded by rev and tat. The effects of these mutations on glycoprotein biosynthesis and function as well as on virus infectivity have been examined in the context of a glycoprotein expression vector and the viral genome. All of the mutant glycoproteins were synthesized, processed, and transported to the cell surface in a manner similar to that of the wild-type glycoprotein. With the exception of mutants that remove the membrane anchor domain, all of the mutant glycoproteins retained the ability to cause fusion of CD4-bearing cells. However, deletion of more than 19 amino acids from the C terminus of gp41 blocked the ability of mutant virions to infect cells. This defect in virus infectivity appeared to be due at least in part to a failure of the virus to efficiently incorporate the truncated glycoprotein. Similar data were obtained for mutations in two different env genes and two different target cell lines. These results indicate that the cytoplasmic domain of gp41 plays a critical role during virus assembly and entry in the life cycle of human immunodeficiency virus type 1. Images PMID:1357190

Metabolism of vertebrate amino sugars with N-glycolyl groups: mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid.

PubMed

Banda, Kalyan; Gregg, Christopher J; Chow, Renee; Varki, Nissi M; Varki, Ajit

2012-08-17

Although N-acetyl groups are common in nature, N-glycolyl groups are rare. Mammals express two major sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). Although humans cannot produce Neu5Gc, it is detected in the epithelial lining of hollow organs, endothelial lining of the vasculature, fetal tissues, and carcinomas. This unexpected expression is hypothesized to result via metabolic incorporation of Neu5Gc from mammalian foods. This accumulation has relevance for diseases associated with such nutrients, via interaction with Neu5Gc-specific antibodies. Little is known about how ingested sialic acids in general and Neu5Gc in particular are metabolized in the gastrointestinal tract. We studied the gastrointestinal and systemic fate of Neu5Gc-containing glycoproteins (Neu5Gc-glycoproteins) or free Neu5Gc in the Neu5Gc-free Cmah(-/-) mouse model. Ingested free Neu5Gc showed rapid absorption into the circulation and urinary excretion. In contrast, ingestion of Neu5Gc-glycoproteins led to Neu5Gc incorporation into the small intestinal wall, appearance in circulation at a steady-state level for several hours, and metabolic incorporation into multiple peripheral tissue glycoproteins and glycolipids, thus conclusively proving that Neu5Gc can be metabolically incorporated from food. Feeding Neu5Gc-glycoproteins but not free Neu5Gc mimics the human condition, causing tissue incorporation into human-like sites in Cmah(-/-) fetal and adult tissues, as well as developing tumors. Thus, glycoproteins containing glycosidically linked Neu5Gc are the likely dietary source for human tissue accumulation, and not the free monosaccharide. This human-like model can be used to elucidate specific mechanisms of Neu5Gc delivery from the gut to tissues, as well as general mechanisms of metabolism of ingested sialic acids.

Presynaptic neurones may contribute a unique glycoprotein to the extracellular matrix at the synapse

NASA Astrophysics Data System (ADS)

Caroni, Pico; Carlson, Steven S.; Schweitzer, Erik; Kelly, Regis B.

1985-04-01

As the extracellular matrix at the original site of a neuromuscular junction seems to play a major part in the specificity of synaptic regeneration1-5, considerable attention has been paid to unique molecules localized to this region6-11. Here we describe an extracellular matrix glycoprotein of the elasmobranch electric organ that is localized near the nerve endings. By immunological criteria, it is synthesized in the cell bodies, transported down the axons and is related to a glycoprotein in the synaptic vesicles of the neurones that innervate the electric organ. It is apparently specific for these neurones, as it cannot be detected elsewhere in the nervous system of the fish. Therefore, neurones seem to contribute unique extracellular matrix glycoproteins to the synaptic region. Synaptic vesicles could be involved in transporting these glycoproteins to or from the nerve terminal surface.

Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

PubMed Central

Ploegh, Hidde L.

2012-01-01

The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging. PMID:22457626

Platelet Glycoprotein Ib-IX and Malignancy

DTIC Science & Technology

2010-09-01

provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

Antibodies to the Glycoprotein GP2 Subunit Cross-React between Old and New World Arenaviruses.

PubMed

Amanat, Fatima; Duehr, James; Oestereich, Lisa; Hastie, Kathryn M; Ollmann Saphire, Erica; Krammer, Florian

2018-01-01

Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays. IMPORTANCE Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective

Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

PubMed Central

Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta

2015-01-01

For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

Structure and assembly of desmosome junctions: biosynthesis, processing, and transport of the major protein and glycoprotein components in cultured epithelial cells.

PubMed

Penn, E J; Hobson, C; Rees, D A; Magee, A I

1987-07-01

Extracts of metabolically labeled cultured epithelial cells have been analyzed by immunoprecipitation followed by SDS-PAGE, using antisera to the major high molecular mass proteins and glycoproteins (greater than 100 kD) from desmosomes of bovine muzzle epidermis. For nonstratifying cells (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney), and A431 cells that have lost the ability to stratify through transformation, and a stratifying cell type (primary human keratinocytes) apparently similar polypeptides were immunoprecipitated with our antisera. These comprised three glycoproteins (DGI, DGII, and DGIII) and one major nonglycosylated protein (DPI). DPII, which has already been characterized by others in stratifying tissues, appeared to be absent or present in greatly reduced amounts in the nonstratifying cell types. The desmosome glycoproteins were further characterized in MDCK cells. Pulse-chase studies showed all three DGs were separate translation products. The two major glycoprotein families (DGI and DGII/III) were both found to be synthesized with co-translational addition of 2-4 high mannose cores later processed into complex type chains. However, they became endo-beta-N-acetylglucosaminidase H resistant at different times (DGII/III being slower). None of the DGs were found to have O-linked oligosaccharides unlike bovine muzzle DGI. Transport to the cell surface was rapid for all glycoproteins (60-120 min) as demonstrated by the rate at which they became sensitive to trypsin in intact cells. This also indicated that they were exposed at the outer cell surface. DGII/III, but not DGI, underwent a proteolytic processing step, losing 10 kD of carbohydrate-free peptide, during transport to the cell surface suggesting a possible regulatory mechanism in desmosome assembly.

β-casein nanovehicles for oral delivery of chemotherapeutic Drug combinations overcoming P-glycoprotein-mediated multidrug resistance in human gastric cancer cells.

PubMed

Bar-Zeev, Maya; Assaraf, Yehuda G; Livney, Yoav D

2016-04-26

Multidrug resistance (MDR) is a primary obstacle to curative cancer therapy. We have previously demonstrated that β-casein (β-CN) micelles (β-CM) can serve as nanovehicles for oral delivery and target-activated release of hydrophobic drugs in the stomach. Herein we introduce a novel nanosystem based on β-CM, to orally deliver a synergistic combination of a chemotherapeutic drug (Paclitaxel) and a P-glycoprotein-specific transport inhibitor (Tariquidar) individually encapsulated within β-CM, for overcoming MDR in gastric cancer. Light microscopy, dynamic light scattering and zeta potential analyses revealed solubilization of these drugs by β-CN, suppressing drug crystallization. Spectrophotometry demonstrated high loading capacity and good encapsulation efficiency, whereas spectrofluorometry revealed high affinity of these drugs to β-CN. In vitro cytotoxicity assays exhibited remarkable synergistic efficacy against human MDR gastric carcinoma cells with P-glycoprotein overexpression. Oral delivery of β-CN - based nanovehicles carrying synergistic drug combinations to the stomach constitutes a novel efficacious therapeutic system that may overcome MDR in gastric cancer.

Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein

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

York, Joanne; Agnihothram, Sudhakar S.; Romanowski, Victor

2005-12-20

The G2 fusion subunit of the Junin virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins,more » four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that {alpha}-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever.« less

A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

Glycosylation is a common post-translational modification of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has b...

Formation of high-order oligomers is required for functional bioactivity of an African bat henipavirus surface glycoprotein.

PubMed

Behner, Laura; Zimmermann, Louisa; Ringel, Marc; Weis, Michael; Maisner, Andrea

2018-05-01

Hendra virus (HeV) and Nipah virus (NiV) are highly pathogenic henipaviruses originating from fruit bats in Australia and Asia that can cause severe infections in livestock and humans. In recent years, also African bat henipaviruses were identified at the nucleic acid level. To assess their potential to replicate in non-bat species, several studies were performed to characterize the two surface glycoproteins required for virus entry and spread by cell-cell fusion. It has been shown that surface expression and fusion-helper function of the receptor-binding G protein of Kumasi virus (KV), the prototypic Ghanaian bat henipavirus, is reduced compared to other non-African henipavirus G proteins. Immunostainings and pulse-chase analysis revealed a delayed export of KV G from the ER. As defects in oligomerization of viral glycoproteins can be responsible for limited surface transport thereby restricting the bioactivity, we analyzed the oligomerization pattern of KV G. In contrast to HeV and NiV whose G proteins are known to be expressed at a dimer-tetramer ratio of 1:1, KV G almost exclusively formed stable tetramers or higher oligomers. KV G also showed less stringent requirements for defined stalk cysteines to form dimers and tetramers. Interestingly, any changes in the oligomeric forms negatively affected the fusion-helper activity although surface expression and receptor binding was unchanged. This clearly indicates that the formation of mostly higher oligomeric KV G forms is not a deficiency responsible for ER retention, but is rather a basic structural feature essential for the bioactivity of this African bat henipavirus glycoprotein. Copyright © 2018 Elsevier B.V. All rights reserved.

The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

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

Zhang, Yanyan; Hu, Yahui; Feng, Yidong

2014-01-15

Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity andmore » multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

PubMed

Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

1998-06-01

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Global analysis of glycoproteins identifies markers of endotoxin tolerant monocytes and GPR84 as a modulator of TNFα expression.

PubMed

Müller, Mario M; Lehmann, Roland; Klassert, Tilman E; Reifenstein, Stella; Conrad, Theresia; Moore, Christoph; Kuhn, Anna; Behnert, Andrea; Guthke, Reinhard; Driesch, Dominik; Slevogt, Hortense

2017-04-12

Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

Glycoprotein-based enzyme-linked immunosorbent assays for serodiagnosis of infectious laryngotracheitis.

PubMed

Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K

2015-05-01

For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate.

PubMed

Lee, Jin; Lim, Kye-Taek

2011-08-01

Di(2-ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP-induced BNL CL. 2 cells. [³H]-thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen-activated protein kinases [extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)], activator protein (AP)-1 (c-Jun and c-Fos), proliferating cell nuclear antigen (PCNA) and cell cycle-related factors (cyclin D1/cyclin-dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]-thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP-induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP-1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate-induced BNL CL. 2 cells. Copyright © 2011 John Wiley & Sons, Ltd.

Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

USDA-ARS?s Scientific Manuscript database

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus.

PubMed

Madoff, D H; Lenard, J

1982-04-01

The intracellular transport and certain posttranslational modifications of the large glycoprotein (G1) of LaCrosse virus (LAC) in BHK cells have been studied. G1 from released LAC virus was characterized by complex oligosaccharides (endo H-resistant) and covalently attached fatty acid. Only a small fraction of total cellular G1 was present on the baby hamster kidney cell surface. Cell-surface G1 contained complex oligosaccharides, while total G1 in infected cells contained largely unprocessed (endo H-sensitive) oligosaccharides. In addition, cell G1 contained significantly less fatty acid than virion-associated G1. Pulse-chase experiments showed that the oligosaccharides of G1 were processed to the complex from much more slowly than the oligosaccharides of the vesicular stomatitis virus (VSV) glycoprotein (G). In addition, transit of LAC G1 to the cell surface and into extracellular virions was two to three fold slower than the transit of VSV G. Thus LAC G1 accumulates intracellularly and is only slowly processed by intracellular processing enzymes. Treatment with monensin caused accumulation in the cell of a form of G1 with partial sensitivity toward endo H, suggesting that monensin may act to inhibit the glycosylation process directly.

Leukoreduced red blood cell transfusions do not induce platelet glycoprotein antibodies in patients with sickle cell disease.

PubMed

Nickel, Robert Sheppard; Winkler, Anne M; Horan, John T; Hendrickson, Jeanne E

2016-09-01

Alloimmunization to red blood cell (RBC) antigens after transfusion is well described in patients with sickle cell disease (SCD). We recently demonstrated that leukocyte-reduced RBC transfusions appeared to induce human leukocyte antigen (HLA) antibodies in some children with SCD; now, we hypothesize that residual platelets contained in transfused RBC products may lead to platelet glycoprotein antibody formation. A cross-sectional study was conducted among never pregnant pediatric patients with SCD who either had received many RBC transfusions or had never received any transfusions. Serum was tested for antibodies to platelet-specific glycoproteins using a commercial enzyme immunoassay. Platelet-specific glycoprotein antibodies were found in 12 of 90 patients (13%) in the transfused group versus 5 of 24 patients (21%) in the never transfused group (p = 0.35). The prevalence of antibodies as well as the median standardized optical density for these two groups was not significantly different for any of the studied platelet glycoprotein antigens. There was no association with the presence of platelet-specific glycoprotein antibodies with either RBC or HLA antibodies. Leukocyte-reduced RBC transfusions do not appear to induce platelet-specific glycoprotein antibodies. The positive platelet-specific glycoprotein antibody results from this study may represent platelet autoantibodies, platelet alloantibodies, or false-positive reactions. A better understanding of the immunobiology of patients with SCD at baseline and after blood product exposure may help improve future transfusion and transplantation. © 2016 AABB.

Structural modification of P-glycoprotein induced by OH radicals: Insights from atomistic simulations

NASA Astrophysics Data System (ADS)

Khosravian, N.; Kamaraj, B.; Neyts, E. C.; Bogaerts, A.

2016-02-01

This study reports on the possible effects of OH radical impact on the transmembrane domain 6 of P-glycoprotein, TM6, which plays a crucial role in drug binding in human cells. For the first time, we employ molecular dynamics (MD) simulations based on the self-consistent charge density functional tight binding (SCC-DFTB) method to elucidate the potential sites of fragmentation and mutation in this domain upon impact of OH radicals, and to obtain fundamental information about the underlying reaction mechanisms. Furthermore, we apply non-reactive MD simulations to investigate the long-term effect of this mutation, with possible implications for drug binding. Our simulations indicate that the interaction of OH radicals with TM6 might lead to the breaking of C-C and C-N peptide bonds, which eventually cause fragmentation of TM6. Moreover, according to our simulations, the OH radicals can yield mutation in the aromatic ring of phenylalanine in TM6, which in turn affects its structure. As TM6 plays an important role in the binding of a range of cytotoxic drugs with P-glycoprotein, any changes in its structure are likely to affect the response of the tumor cell in chemotherapy. This is crucial for cancer therapies based on reactive oxygen species, such as plasma treatment.

Modulatory effect of phytoglycoprotein (38 kDa) on cyclin D1/CDK4 in BNL CL.2 cells induced by N-methyl-N'-nitro-N-nitrosoguanidine.

PubMed

Lee, Jin; Lim, Kye-Taek

2012-02-01

In the developmental stages of cancer, cell transformation occurs after the promotion stage and is a marker of cancer progression. This cell transformation is related to abnormal proliferation during the cancer initiation stage. The purpose of this study was to evaluate the effect of Styrax japonica Siebold et al. Zuccarin (SJSZ) glycoprotein on cell transformation in murine embryonic liver cells (BNL CL.2) following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. To determine abnormal proliferation during the initiation stage, intracellular reactive oxygen species (ROS), phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), activities of cell cycle-related factors [cyclin D1/cyclin dependent kinase (CDK) 4], cell cycle inhibitors (p53, p21, and p27), nuclear factor (NF)-κB, and proliferating cell nuclear antigen (PCNA) were evaluated using Western blot analysis and real-time PCR. Our study demonstrated that SJSZ glycoprotein (50 μg/ml) reduces foci formation with combined treatment [MNNG and 12-O-tetradecanoyl phorbol-13-acetate] of BNL CL.2 cells. With regard to proliferation-related signals, our finding indicated that SJSZ glycoprotein (50 μg/ml) diminished the production of intracellular ROS, activity of phosphorylated ERK, p38 MAPK, NF-κB (p50 and p65), PCNA, and cyclin D1/CDK4 in MNNG-induced BNL CL.2 cells. Taken together, these results lead us to speculate that SJSZ glycoprotein can inhibit abnormal cell proliferation at the initiation stage of hepatocarcinogenesis.

Paramyxovirus glycoprotein incorporation, assembly and budding: a three way dance for infectious particle production.

PubMed

El Najjar, Farah; Schmitt, Anthony P; Dutch, Rebecca Ellis

2014-08-07

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout.

Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production

PubMed Central

El Najjar, Farah; Schmitt, Anthony P.; Dutch, Rebecca Ellis

2014-01-01

Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. PMID:25105277

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

PubMed Central

Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

2016-01-01

Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

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

Backovic, Marija; Longnecker, Richard; Jardetzky, Theodore S

Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which formsmore » 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.« less

Effect of P-glycoprotein on flavopiridol sensitivity

PubMed Central

Boerner, S A; Tourne, M E; Kaufmann, S H; Bible, K C

2001-01-01

Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC 50 s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CHRC5 (higher Pgp) cells were 90.2 ± 6.6 nM and 117 ± 2.3 nM, respectively (P< 0.01), suggesting that Pgp might have a modest effect on flavopiridol action. Consistent with this hypothesis, pretreatment with either quinidine or verapamil (inhibitors of Pgp-mediated transport) sensitized CHRC5 cells to the antiproliferative effects of flavopiridol. Because of concern that colony forming assays might not accurately reflect cytotoxicity, we also examined flavopiridol-treated cells by trypan blue staining and flow cytometry. These assays confirmed that flavopiridol was less toxic to cells expressing higher levels of Pgp. Further experiments revealed that flavopiridol inhibited the binding of [3H]-azidopine to Pgp in isolated membrane vesicles, but only at high concentrations. Collectively, these results identify flavopiridol as a weak substrate for Pgp. © 2001 Cancer Research Campaign www.bjcancer.com PMID:11355953

Developmental localization and the role of hydroxyproline rich glycoproteins during somatic embryogenesis of banana (Musa spp. AAA)

PubMed Central

2011-01-01

Background Hydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody. Results Immunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed

Structural basis for Epstein–Barr virus host cell tropism mediated by gp42 and gHgL entry glycoproteins

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

Sathiyamoorthy, Karthik; Hu, Yao Xiong; Möhl, Britta S.

Herpesvirus entry into host cells is mediated by multiple virally encoded receptor binding and membrane fusion glycoproteins. Despite their importance in host cell tropism and associated disease pathology, the underlying and essential interactions between these viral glycoproteins remain poorly understood. For Epstein–Barr virus (EBV), gHgL/gp42 complexes bind HLA class II to activate membrane fusion with B cells, but gp42 inhibits fusion and entry into epithelial cells. To clarify the mechanism by which gp42 controls the cell specificity of EBV infection, in this paper we determined the structure of gHgL/gp42 complex bound to an anti-gHgL antibody (E1D1). The critical regulator ofmore » EBV tropism is the gp42 N-terminal domain, which tethers the HLA-binding domain to gHgL by wrapping around the exterior of three gH domains. Both the gp42 N-terminal domain and E1D1 selectively inhibit epithelial-cell fusion; however, they engage distinct surfaces of gHgL. Finally, these observations clarify key determinants of EBV host cell tropism.« less

Structural basis for Epstein–Barr virus host cell tropism mediated by gp42 and gHgL entry glycoproteins

DOE PAGES

Sathiyamoorthy, Karthik; Hu, Yao Xiong; Möhl, Britta S.; ...

2016-12-08

Herpesvirus entry into host cells is mediated by multiple virally encoded receptor binding and membrane fusion glycoproteins. Despite their importance in host cell tropism and associated disease pathology, the underlying and essential interactions between these viral glycoproteins remain poorly understood. For Epstein–Barr virus (EBV), gHgL/gp42 complexes bind HLA class II to activate membrane fusion with B cells, but gp42 inhibits fusion and entry into epithelial cells. To clarify the mechanism by which gp42 controls the cell specificity of EBV infection, in this paper we determined the structure of gHgL/gp42 complex bound to an anti-gHgL antibody (E1D1). The critical regulator ofmore » EBV tropism is the gp42 N-terminal domain, which tethers the HLA-binding domain to gHgL by wrapping around the exterior of three gH domains. Both the gp42 N-terminal domain and E1D1 selectively inhibit epithelial-cell fusion; however, they engage distinct surfaces of gHgL. Finally, these observations clarify key determinants of EBV host cell tropism.« less

Research resource: Update and extension of a glycoprotein hormone receptors web application.

PubMed

Kreuchwig, Annika; Kleinau, Gunnar; Kreuchwig, Franziska; Worth, Catherine L; Krause, Gerd

2011-04-01

The SSFA-GPHR (Sequence-Structure-Function-Analysis of Glycoprotein Hormone Receptors) database provides a comprehensive set of mutation data for the glycoprotein hormone receptors (covering the lutropin, the FSH, and the TSH receptors). Moreover, it provides a platform for comparison and investigation of these homologous receptors and helps in understanding protein malfunctions associated with several diseases. Besides extending the data set (> 1100 mutations), the database has been completely redesigned and several novel features and analysis tools have been added to the web site. These tools allow the focused extraction of semiquantitative mutant data from the GPHR subtypes and different experimental approaches. Functional and structural data of the GPHRs are now linked interactively at the web interface, and new tools for data visualization (on three-dimensional protein structures) are provided. The interpretation of functional findings is supported by receptor morphings simulating intramolecular changes during the activation process, which thus help to trace the potential function of each amino acid and provide clues to the local structural environment, including potentially relocated spatial counterpart residues. Furthermore, double and triple mutations are newly included to allow the analysis of their functional effects related to their spatial interrelationship in structures or homology models. A new important feature is the search option and data visualization by interactive and user-defined snake-plots. These new tools allow fast and easy searches for specific functional data and thereby give deeper insights in the mechanisms of hormone binding, signal transduction, and signaling regulation. The web application "Sequence-Structure-Function-Analysis of GPHRs" is accessible on the internet at http://www.ssfa-gphr.de/.

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

PubMed

Lehmann, Marcus; Schoeman, Rogier M; Krohl, Patrick J; Wallbank, Alison M; Samaniuk, Joseph R; Jandrot-Perrus, Martine; Neeves, Keith B

2018-05-01

The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow. © 2018 American Heart Association, Inc.

Changes in intestinal absorption of nutrients and brush border glycoproteins after total parenteral nutrition in rats.

PubMed Central

Miura, S; Tanaka, S; Yoshioka, M; Serizawa, H; Tashiro, H; Shiozaki, H; Imaeda, H; Tsuchiya, M

1992-01-01

The effect of total parenteral nutrition on nutrients absorption and glycoprotein changes of brush border membrane was examined in rat small intestine. In total parenteral nutrition rats, a marked decrease in activity of brush border enzymes was observed mainly in the proximal and middle segments of the intestine. Galactose perfusion of jejunal segment showed that hexose absorption was significantly inhibited, while intestinal absorption of glycine or dipeptide, glycylglycine was not significantly affected by total parenteral nutrition treatment. When brush border membrane glycoprotein profile was examined by [3H]-glucosamine or [3H]-fucose incorporation into jejunal loops, significant changes were observed in the glycoprotein pattern of brush border membrane especially in the high molecular weight range over 120 kDa after total parenteral nutrition treatment, suggesting strong dependency of glycoprotein synthesis on luminal substances. Molecular weight of sucrase isomaltase in brush border membrane detected by specific antibody showed no significant difference, however, in total parenteral nutrition and control rats. Also, molecular weight of specific sodium glucose cotransporter of intestinal brush border membrane detected by selective photoaffinity labelling was not altered in total parenteral nutrition rats. It may be that prolonged absence of oral food intake may produce significant biochemical changes in brush border membrane glycoprotein and absorptive capacity of small intestine, but these changes were not observed in all brush border membrane glycoproteins. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1582592

Carcinoma-specific Ulex europaeus agglutinin-I binding glycoproteins of human colorectal carcinoma and its relation to carcinoembryonic antigen.

PubMed

Matsushita, Y; Yonezawa, S; Nakamura, T; Shimizu, S; Ozawa, M; Muramatsu, T; Sato, E

1985-08-01

Glycoproteins binding to Ulex europaeus agglutinin-I (UEA-I) lectin, which recognizes the terminal alpha-L-fucose residue, were analyzed in 18 cases of human colorectal carcinoma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by the Western blotting method. In the distal large bowel (descending and sigmoid colon and rectum), high-molecular-weight glycoproteins binding to UEA-I existed in carcinoma tissue but not in normal mucosa. In the proximal large bowel (ascending and transverse colon), high-molecular-weight glycoproteins binding to UEA-I were found both in normal mucosa and in carcinoma tissue, whereas those from the carcinoma tissue had an apparently lower molecular weight as compared to the weight of those from the normal mucosa. Thus there is a biochemical difference in UEA-I binding glycoproteins between the normal mucosa and the carcinoma tissue, although in our previous histochemical study no difference was observed in UEA-I binding glycoproteins of the proximal large bowel between the carcinoma tissue and the normal mucosa. Furthermore, carcinoembryonic antigen from the carcinoma tissue was found to have the same electrophoretical mobility as the UEA-I binding glycoproteins.

Contribution of tumor endothelial cells to drug resistance: anti-angiogenic tyrosine kinase inhibitors act as p-glycoprotein antagonists.

PubMed

Bani, MariaRosa; Decio, Alessandra; Giavazzi, Raffaella; Ghilardi, Carmen

2017-05-01

Tumor endothelial cells (TEC) differ from the normal counterpart, in both gene expression and functionality. TEC may acquire drug resistance, a characteristic that is maintained in vitro. There is evidence that TEC are more resistant to chemotherapeutic drugs, substrates of ATP-binding cassette (ABC) transporters. TEC express p-glycoprotein (encoded by ABCB1), while no difference in other ABC transporters was revealed compared to normal endothelia. A class of tyrosine kinase inhibitors (TKI), used as angiostatic compounds, interferes with the ATPase activity of p-glycoprotein, thus impairing its functionality. The exposure of ovarian adenocarcinoma TEC to the TKIs sunitinib or sorafenib was found to abrogate resistance (proliferation and motility) to doxorubicin and paclitaxel in vitro, increasing intracellular drug accumulation. A similar effect has been reported by the p-glycoprotein inhibitor verapamil. No beneficial effect was observed in combination with cytotoxic drugs that are not p-glycoprotein substrates. The current paper reviews the mechanisms of TEC chemoresistance and shows the role of p-glycoprotein in mediating such resistance. Inhibition of p-glycoprotein by anti-angiogenic TKI might contribute to the beneficial effect of these small molecules, when combined with chemotherapy, in counteracting acquired drug resistance.

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

PubMed

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential

Rabies virus glycoprotein as a carrier for anthrax protective antigen

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

Smith, Mary Ellen; Koser, Martin; Xiao Sa

2006-09-30

Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen wasmore » also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.« less

Preferential recognition of avian-like receptors in human influenza A H7N9 viruses.

PubMed

Xu, Rui; de Vries, Robert P; Zhu, Xueyong; Nycholat, Corwin M; McBride, Ryan; Yu, Wenli; Paulson, James C; Wilson, Ian A

2013-12-06

The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu(226), a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to humanlike α2-6-linked receptors and strong preference for a subset of avian-like α2-3-linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.

Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis.

PubMed

Moore, S E; Spiro, R G

1994-04-29

The intracellular site for the degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis has been studied by permeabilizing the plasma membrane of metabolically radiolabeled HepG2 cells with streptolysin O. This pore-forming agent permitted us to examine the breakdown in both the cytosolic and vesicular compartments of the previously recognized (Anumula, K. R., and Spiro, R. G. (1983) J. Biol. Chem. 258, 15274-15282) polymannose components terminating in a di-N-acetylchitobiose sequence (OS-Glc-NAc2) or a single N-acetylglucosamine residue (OS-Glc-NAc1) residue. Pulse-chase studies indicated that although the OS-GlcNAc2 saccharides were about equally distributed between vesicles and cytosol and rapidly disappeared after reaching the Man8 stage, the OS-GlcNAc1 species were found predominantly in the extravesicular compartment and there underwent a distinctive demannosylation sequence resulting in the formation of a Man5GlcNAc isomer (Man alpha 1-->2Man alpha 1-->2Man alpha 1-->3(Man alpha 1-->6)Man beta 1-->4GlcNAc) which was different from the product of Golgi processing enzymes. Further trimming of this cytosolic limit product required its translocation into a vesicular compartment, believed to be lysosomes, in which Man2-4GlcNAc components appeared as the metabolic chase progressed. The accumulation of Glc1Man5GlcNAc in the cytosol during the chase suggested that glucose interferes with the cytosolic-vesicular transfer and this became even more evident by the pronounced pile-up of extravesicular Glc3Man5GlcNAc when the cells were incubated in the presence of castanospermine. Although the biological significance and mechanism of free polymannose oligosaccharide entry into the cytosol is not yet known, the possibility that it may reflect an endoplasmic reticulum-situated degradative process of glycoproteins merits consideration.

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.

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

PubMed Central

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

2008-01-01

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

Involvement of the Golgi apparatus in the synthesis and secretion of hydroxyproline-rich cell wall glycoproteins. [carrot

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

Gardiner, M.; Chrispeels, M.J.

1975-01-01

Pulse labeling of carrot root phloem parenchyma (Daucus carota L. ev. Nantes) tissue with /sup 14/C-proline followed by fractionation of the cytoplasmic organelles on sucrose gradients was used to determine the identiy of the membranous organelles involved in the secretion of the hydroxyproline-rich glycoproteins of the cell wall. Identification of the organelles was done through electron-microscopical observations and through the localization of marker enzymes on the sucrose gradients. Enrichment of the organelles involved in secretion was determined by measuring the percentage of the incorporated radioactivity present as /sup 14/C-hydroxyproline. The Golgi apparatus (dictyosome) was found to be a major sitemore » of glycoprotein transport. This identification was based on the observed enrichment of dictyosomes paralleling the purification of newly synthesized cell-wall glycoproteins. A marker enzyme for the Golgi apparatus, inosinediphosphatase, banded with the newly synthesized cell wall glycoproteins on sequential isopycnic and rate zonal sucrose gradients. Marker enzymes for the endoplasmic reticulum and the plasma memebrane were clearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in the glycosylation of the peptide moiety of this glycoprotein, also banded with the dictyosomes on both kinds of gradients. The results suggest an important role of the Golgi apparatus in the biosynthesis and the secretion of the cell wall glycoproteins of higher plants. (auth)« less

Platelet Glycoprotein lb-1X and Malignancy

DTIC Science & Technology

2010-09-01

supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation factors. This paradigm has been...a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib-IX have been identified, including...08-1-0576 TITLE: Platelet Glycoprotein lb-1X and Malignancy PRINCIPAL INVESTIGATOR: Dr. Jerry Ware

Thiolated polymers: evidence for the formation of disulphide bonds with mucus glycoproteins.

PubMed

Leitner, Verena M; Walker, Greg F; Bernkop-Schnürch, Andreas

2003-09-01

Disulphide bonds between thiolated polymers (thiomers) and cysteine-rich subdomains of mucus glycoproteins are supposed to be responsible for the enhanced mucoadhesive properties of thiomers. This study set out to provide evidence for these covalent interactions using poly(acrylic acid)-cysteine conjugates of 2 and 450 kDa (PAA2-Cys, PAA450-Cys) displaying 402.5-776.0 micromol thiol groups per gram polymer. The effect of the disulphide bond breaker cysteine on thiomer-mucin disulphide bonds was monitored by (1) mucoadhesion studies and (2) rheological studies. Furthermore, (3) diffusion studies and (4) gel filtration studies were performed with thiomer-mucus mixtures. The addition of cysteine significantly (P<0.01) reduced the adhesion of thiomer tablets to porcine mucosa and G'/G" values of thiomer-mucin mixtures, whereas unthiolated controls were not influenced. These results indicate the cleavage of disulphide bonds between thiomer and mucus glycoproteins. Diffusion studies demonstrated that a 12.8-fold higher concentration of the thiomer (PAA2-Cys) remains in the mucin gel than the corresponding unmodified polymer. Gel filtration studies showed that PAA2-Cys was able to form disulphide bonds with mucin glycoproteins resulting in an altered elution profile of the mucin/PAA2-Cys mixture in comparison to mucin alone or mucin/PAA2 mixture. According to these results, the study provides evidence for the formation of covalent bonds between thiomer and mucus glycoproteins.

Methylation of Notch3 modulates chemoresistance via P-glycoprotein.

PubMed

Gu, Xiaoting; Lu, Yangfan; He, Dongxu; Lu, Chunxiao; Jin, Jian; Lu, Xiaojie; Ma, Xin

2016-12-05

The global gene expression and DNA methylation of genes in adriamycin-resistant human breast cancer cells (MCF-7/ADM cells) are similar to those in paclitaxel-resistant MCF-7 cells (MCF-7/PTX) and are significantly different from those in wild-type MCF-7 cells. DNA methylation is associated with chemoresistance in breast cancer and changes the characteristics of chemoresistant and chemosensitive cells. Here, we showed that the tumor-suppressor gene Notch3 was inactivated due to epigenetic silencing DNA hypermethylation in MCF-7/ADM cells. In addition, the drug efflux pump P-glycoprotein was negatively regulated by Notch3 and highly expressed in MCF-7/ADM cells. Taken together, our findings demonstrated that hypermethylation of Notch3 causes activation of P-glycoprotein in adriamycin-resistant cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

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

McLellan, Jason S.; Yang, Yongping; Graham, Barney S.

2011-09-16

Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in itsmore » postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.« less

Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012*

PubMed Central

Leymarie, Nancy; Griffin, Paula J.; Jonscher, Karen; Kolarich, Daniel; Orlando, Ron; McComb, Mark; Zaia, Joseph; Aguilan, Jennifer; Alley, William R.; Altmann, Friederich; Ball, Lauren E.; Basumallick, Lipika; Bazemore-Walker, Carthene R.; Behnken, Henning; Blank, Michael A.; Brown, Kristy J.; Bunz, Svenja-Catharina; Cairo, Christopher W.; Cipollo, John F.; Daneshfar, Rambod; Desaire, Heather; Drake, Richard R.; Go, Eden P.; Goldman, Radoslav; Gruber, Clemens; Halim, Adnan; Hathout, Yetrib; Hensbergen, Paul J.; Horn, David M.; Hurum, Deanna; Jabs, Wolfgang; Larson, Göran; Ly, Mellisa; Mann, Benjamin F.; Marx, Kristina; Mechref, Yehia; Meyer, Bernd; Möginger, Uwe; Neusüβ, Christian; Nilsson, Jonas; Novotny, Milos V.; Nyalwidhe, Julius O.; Packer, Nicolle H.; Pompach, Petr; Reiz, Bela; Resemann, Anja; Rohrer, Jeffrey S.; Ruthenbeck, Alexandra; Sanda, Miloslav; Schulz, Jan Mirco; Schweiger-Hufnagel, Ulrike; Sihlbom, Carina; Song, Ehwang; Staples, Gregory O.; Suckau, Detlev; Tang, Haixu; Thaysen-Andersen, Morten; Viner, Rosa I.; An, Yanming; Valmu, Leena; Wada, Yoshinao; Watson, Megan; Windwarder, Markus; Whittal, Randy; Wuhrer, Manfred; Zhu, Yiying; Zou, Chunxia

2013-01-01

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods. PMID

Relationship between structure and P-glycoprotein inhibitory activity of dimeric peptides related to the Dmt-Tic pharmacophore.

PubMed

Ambo, Akihiro; Ohkatsu, Hiromichi; Minamizawa, Motoko; Watanabe, Hideko; Sugawara, Shigeki; Nitta, Kazuo; Tsuda, Yuko; Okada, Yoshio; Sasaki, Yusuke

2012-03-15

To develop novel inhibitors of P-glycoprotein (P-gp), dimeric peptides related to an opioid peptide containing the Dmt-Tic pharmacophore were synthesized and their P-gp inhibitory activities were analyzed. Of the 30 analogs synthesized, N(α),N(ε)-[(CH(3))(2)Mle-Tic](2)Lys-NH(2) and its D-Lys analog were found to exhibit potent P-gp inhibitory activity, twice that of verapamil, in doxorubicin-resistant K562 cells. Structure-activity studies indicated that the correct hydrophobicity and spacer length between two aromatic rings are important structural elements in this series of analogs for inhibition of P-gp. Copyright © 2012 Elsevier Ltd. All rights reserved.

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

PubMed Central

Mbua, Ngalle Eric; Flanagan-Steet, Heather; Johnson, Steven; Wolfert, Margreet A.; Boons, Geert-Jan; Steet, Richard

2013-01-01

Niemann–Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins. PMID:23733943

Chimeric bovine respiratory syncytial virus with attachment and fusion glycoproteins replaced by bovine parainfluenza virus type 3 hemagglutinin-neuraminidase and fusion proteins.

PubMed

Stope, M B; Karger, A; Schmidt, U; Buchholz, U J

2001-10-01

Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background.

21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

PubMed Central

Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

2008-01-01

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galα(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors. PMID:18456721

Immunoinformatic Analysis of Crimean Congo Hemorrhagic Fever Virus Glycoproteins and Epitope Prediction for Synthetic Peptide Vaccine.

PubMed

Tipu, Hamid Nawaz

2016-02-01

To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Cross-sectional study. Combined Military Hospital, Khuzdar Cantt, in May 2015. Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLAclass I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. HLAA*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. Atotal of 35 nanomers from GP1, and 3 from GP2 were identified. HLAB*0702 bound maximum number of peptides (6), while HLAB*4001 showed strongest binding affinity. HLAspecific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates.

Application of Physiologically Based Pharmacokinetic Modeling in Understanding Bosutinib Drug-Drug Interactions: Importance of Intestinal P-Glycoprotein.

PubMed

Yamazaki, Shinji; Loi, Cho-Ming; Kimoto, Emi; Costales, Chester; Varma, Manthena V

2018-05-08

Bosutinib is an orally available Src/Abl tyrosine kinase inhibitor indicated for the treatment of patients with Ph+ chronic myelogenous leukemia at a clinically recommended dose of 500 mg once daily. Clinical results indicated that increases in bosutinib oral exposures were supra-proportional at the lower doses (50 to 200 mg) and approximately dose-proportional at the higher doses (200 to 600 mg). Bosutinib is a substrate of CYP3A4 and P-glycoprotein and exhibits pH-dependent solubility with moderate intestinal permeability. These findings led us to investigate the factors influencing the underlying pharmacokinetic mechanisms of bosutinib with physiologically-based pharmacokinetic (PBPK) models. Our primary objectives were to: 1) refine the previously developed bosutinib PBPK model based on the latest oral bioavailability data and 2) verify the refined PBPK model with P-glycoprotein kinetics based on the bosutinib drug-drug interaction (DDI) results with ketoconazole and rifampin. Additionally, the verified PBPK model was applied to predict bosutinib DDIs with dual CYP3A/P-glycoprotein inhibitors. The results indicated that 1) the refined PBPK model adequately described the observed plasma concentration-time profiles of bosutinib and 2) the verified PBPK model reasonably predicted the effects of ketoconazole and rifampin on bosutinib exposures by accounting for intestinal P-gp inhibition/induction. These results suggested that bosutinib DDI mechanism could involve not only CYP3A4-mediated metabolism but also P-glycoprotein-mediated efflux on absorption. In summary, P-glycoprotein kinetics could constitute a critical element in the PBPK models to understand the pharmacokinetic mechanism of dual CYP3A/P-glycoprotein substrates such as bosutinib exhibiting nonlinear pharmacokinetics due largely to a saturation of intestinal P-glycoprotein-mediated efflux. The American Society for Pharmacology and Experimental Therapeutics.

A hybrid monolithic column based on boronate-functionalized graphene oxide nanosheets for online specific enrichment of glycoproteins.

PubMed

Zhou, Chanyuan; Chen, Xiaoman; Du, Zhuo; Li, Gongke; Xiao, Xiaohua; Cai, Zongwei

2017-05-19

A hybrid monolithic column based on aminophenylboronic acid (APBA)-functionalized graphene oxide (GO) has been developed and used for selective enrichment of glycoproteins. The APBA/GO composites were homogeneously incorporated into a polymer monolithic column with the help of oligomer matrix and followed by in situ polymerization. The effect of dispersion of APBA/GO composites in the polymerization mixture on the performance of the monolithic column was explored in detail. The presence of graphene oxide not only enlarged the BET surface area from 6.3m 2 /g to 169.4m 2 /g, but also provided abundant boronic acid moieties for glycoprotein extraction, which improved the enrichment selectivity and efficiency for glycoproteins. The APBA/GO hybrid monolithic column was incorporated into a sequential injection system, which facilitated online extraction of proteins. Combining the superior properties of extraordinary surface area of GO and the affinity interaction of APBA to glycoproteins, the APBA/GO hybrid monolithic column showed higher enrichment factors for glycoproteins than other proteins without cis-diol-containing groups. Also, under comparable or even shorter processing time and without the addition of any organic solvent, it showed higher binding capacity toward glycoproteins compared with the conventional boronate affinity monolithic column. The practical applicability of this system was demonstrated by processing of egg white samples for extraction of ovalbumin and ovotransferrin, and satisfactory results were obtained by assay with SDS-PAGE. Copyright © 2017 Elsevier B.V. All rights reserved.

[Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry].

PubMed

Li, Feng; Kang, Jingwu

2014-04-01

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP @ ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. /3-2-Glycoprotein 1 present in serum at low mass concentration around 0.000 01 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP @ ConA has been proven to be an efficient alternative to currently available immobilization supports.

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

PubMed

Xu, Kai; Chan, Yee-Peng; Bradel-Tretheway, Birgit; Akyol-Ataman, Zeynep; Zhu, Yongqun; Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z Hong; Broder, Christopher C; Aguilar, Hector C; Nikolov, Dimitar B

2015-12-01

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.

Unusual neutral oligosaccharides in mature Sindbis virus glycoproteins are synthesized from truncated precursor oligosaccharides in Chinese hamster ovary cells.

PubMed

Davidson, S K; Hunt, L A

1983-03-01

We have previously demonstrated the presence of unusual small asparaginyl-oligosaccharides [(Man)3GlcNAc2-ASN] in the mature glycoproteins of Sindbis virus released from both wild-type and lectin-resistant Chinese hamster ovary cells, but the mechanism of synthesis of these structures was not determined. Gel filtration and endo-beta-N-acetylglucosaminidase analyses of Pronase-digested glycopeptides from [3H]mannose-labelled Sindbis virus released at different times after infection of a phytohaemagglutinin-resistant line of Chinese hamster ovary cells demonstrated that these small asparaginyl-oligosaccharides were present in similar relative amounts in virus released throughout the virus infection, rather than arising primarily at late times when cytopathic effects were maximal. Similar analyses of pulse-labelled, cell-associated viral glycopeptides suggested that these small oligosaccharides on mature virus glycoprotein resulted from the normal alpha 1,2-mannosidase processing of truncated precursor oligosaccharides (containing five rather than nine mannoses), rather than from aberrant processing or degradation of the full-size precursor oligosaccharides or normal intermediates.

Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems.

PubMed

Westhoff, C M; Wylie, D E

2006-01-01

The development and use of heterologous expression systems is critical for deciphering the function of mammalian Rh and Rh-glycoproteins. The studies here use Xenopus oocytes, well known for their ability to readily traffic and express difficult membrane proteins, and S. cerevisiae wild-type strains and mutants that are defective in ammonium transport. Data obtained in both of these expression systems revealed that mammalian Rh-glycoprotein-mediated transport (RhAG, RhBG, and RhCG) is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient, effectively exchanging NH4+ for H+ in a process that results in transport of net NH3. Homology modeling and functional studies suggest that the more recently evolved erythrocyte blood group proteins, RhCE and RhD, may not function directly in ammonia transport and may be evolving a new function in the RBC membrane. The relationship of Rh and Rh-glycoproteins to the Amt/Mep ammonium transporters is substantiated with functional transport data and structural modeling.

Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

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

Mahmoud, Nora F.; Faculty of Pharmacy, Suez Canal University, Ismailia; Jasirwan, Chyntia

2016-03-15

Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highlymore » conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.« less

Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin.

PubMed

Polonoff, E; Machida, C A; Kabat, D

1982-12-10

Addition of asparagine-linked oligosaccharides to nascent murine leukemia virus (MuLV)-encoded membrane glycoproteins was inhibited either completely by tunicamycin or specifically at Asn-X-Thr glycosylation sites by incorporation of the threonine analogue beta-hydroxynorvaline. In conditions of partial analogue substitution, a series of subglycosylated components is formed which are related by a constant apparent Mr difference when assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The total number of asparagine-linked oligosaccharides is then estimated by dividing the measured apparent Mr of one oligosaccharide into the total apparent Mr difference between the complete glycoprotein and the polypeptide chain that is synthesized in cells incubated with tunicamycin. Correct results were obtained using glycoproteins with known numbers of oligosaccharides. Our analyses indicate that the gp70 membrane envelope glycoproteins of certain ecotropic MuLVs contain seven oligosaccharides, whereas the GIX+ antigen-containing variant gp70 contains one fewer Asn-X-Thr-linked oligosaccharide. The membrane glycoprotein encoded by the gag gene of Friend MuLV contains only one asparagine-linked oligosaccharide. Similarly, the gp55 membrane glycoprotein encoded by Friend erythroleukemia virus contains four asparagine-linked oligosaccharides. Pulse-chase and cell surface iodination analyses indicate that MuLV membrane envelope glycoprotein processing by partial proteolysis and transport to the cell surface can be efficiently blocked by structural perturbations caused by incorporation of different amino acid analogues or by loss of oligosaccharides. Our data also suggest that loss of oligosaccharides may expose new antigenic sites in viral membrane glycoproteins and increase their susceptibility to intracellular proteolysis.

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein.

PubMed

Favier, Anne-Laure; Gout, Evelyne; Reynard, Olivier; Ferraris, Olivier; Kleman, Jean-Philippe; Volchkov, Viktor; Peyrefitte, Christophe; Thielens, Nicole M

2016-06-01

Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the

Characterisation of P-glycoprotein-9.1 in Haemonchus contortus.

PubMed

Godoy, Pablo; Che, Hua; Beech, Robin N; Prichard, Roger K

2016-01-28

The existence nematodes of veterinary importance such as Haemonchus contortus resistant to anthelmintic drugs, including the macrocyclic lactones, has become a major concern in animal health. Macrocyclic lactone resistance in H. contortus seems to be multigenic including the active efflux of these drugs by P-glycoproteins, members of the ABC transporter family, present in this parasite. The goals of the present work were to determine the activity of H. contortus P-glycoprotein 9.1 (Hco-PGP-9.1) and its interaction with the avermectins, ivermectin, abamectin, and also the milbemycin, moxidectin. Additionally, the localisation of Hco-PGP-9.1 was sought in adult worms. Hco-Pgp-9.1 was cloned and expressed in mammalian cells and its expression profile was determined at the transcriptional and protein level by qRT-PCR and Western-blot, respectively. The nematode transport activity was assessed using the tracer dye Rhodamine 123. A ligand competition assay between different macrocyclic lactones and Rhodamine 123 was used to establish whether or not there was interaction between Hco-PGP-9.1 and the avermectins (abamectin and ivermectin) or moxidectin. In addition, immunostaining was carried out to localise Hco-PGP-9.1 expression in the transgenic cells and in adult female parasites. Hco-PGP-9.1 was expressed in the cell membrane of the transfected host cells and was able to extrude Rhodamine 123. Ivermectin and abamectin, but not moxidectin, had a pronounced inhibitory effect on the ability of Hco-PGP-9.1 to transport Rhodamine 123. Antibodies raised against Hco-PGP-9.1 epitopes localised to the uterus of adult female H. contortus. These results suggest a strong interaction of the avermectins with Hco-PGP-9.1. However, possibly due to its physico-chemical properties, moxidectin had markedly less effect on Hco-PGP-9.1. Because of the greater interaction of the avermectins than moxidectin with this transporter, it is more likely to contribute to avermectin resistance than

Association between MDR1 gene of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs.

PubMed

Su, Jian-Li; Wang, Cheng-Hong; Kang, Hong-Gang; Zhang, Jing; Wang, Bao-Zhong; Liu, Mei-Rong; Zhao, Jun; Liu, Lin

2017-09-01

The aim of the present study was to examine and discuss the association between multidrug resistance 1 gene ( MDR1 ) of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs. In this study, 126 cases of patients with gastrointestinal tumors admitted to hospital from February 2013 to February 2015 were selected. The expression levels of MDR1 gene were obsreved in the control population and patients before and after treatment by fluoresecent quantitative PCR. The protein expression level of P-glycoprotein was determined using western blotting and enzyme-linked immunosorbent assay. In addition, drug resistance was assessed by ATP-TCA chemosensitivity experiments. The results showed that before treatment, the expression of mRNA in MDR1 of tissues of gastrointestinal tract of the 126 cases was 108-fold larger than that of the gastrointestinal tract of the controls (p<0.05), P-glycoprotein was 87-fold larger than the expression level of the controls (p<0.05). The sensitivity of 126 tumor tissues to different chemotherapeutic drugs was determined, and the results showed that most of the tumor tissues were sensitive to chemotherapeutic drugs, and the sensitivity rate reached 96.4%. Following chemotherapy, the expression of mRNA in MDR1 of tumor tissues and the expression of P-glycoprotein decreased (p<0.05). In conclusion, the MDR1 gene and P-glycoprotein have a positive correlation with the occurrence of gastrointestinal tumors, and a negative correlation between the MDR1 gene and P-glycoprotein with resistance of chemotherapeutic drugs. Therefore, the MDR1 gene and P-glycoprotein can be used as references in the identification and diagnosis of gastrointestinal tumors.

The Lyssavirus glycoprotein: A key to cross-immunity.

PubMed

Buthelezi, Sindisiwe G; Dirr, Heini W; Chakauya, Ereck; Chikwamba, Rachel; Martens, Lennart; Tsekoa, Tsepo L; Stoychev, Stoyan H; Vandermarliere, Elien

2016-11-01

Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. Copyright © 2016 Elsevier Inc. All rights reserved.

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain...consistent with viral TM fusion proteins [9,10]. GPC con- tains a 58 residue hydrophobic N-terminal signal peptide (SP), which directs the precursor to the...including GPC, GP1, and GP2. Various signal peptides , purification tags, and modifications to internal domains were employed for the generation and

Identifying the Viral Genes Encoding Envelope Glycoproteins for Differentiation of Cyprinid herpesvirus 3 Isolates

PubMed Central

Han, Jee Eun; Kim, Ji Hyung; Renault, Tristan; Choresca, Casiano; Shin, Sang Phil; Jun, Jin Woo; Park, Se Chang

2013-01-01

Cyprinid herpes virus 3 (CyHV-3) diseases have been reported around the world and are associated with high mortalities of koi (Cyprinus carpio). Although little work has been conducted on the molecular analysis of this virus, glycoprotein genes identified in the present study seem to be valuable targets for genetic comparison of this virus. Three envelope glycoprotein genes (ORF25, 65 and 116) of the CyHV-3 isolates from the USA, Israel, Japan and Korea were compared, and interestingly, sequence insertions or deletions were observed in these target regions. In addition, polymorphisms were presented in microsatellite zones from two glycoprotein genes (ORF65 and 116). In phylogenetic tree analysis, the Korean isolate was remarkably distinguished from USA, Israel, Japan isolates. These findings may be suitable for many applications including isolates differentiation and phylogeny studies. PMID:23435236

Identifying the viral genes encoding envelope glycoproteins for differentiation of Cyprinid herpesvirus 3 isolates.

PubMed

Han, Jee Eun; Kim, Ji Hyung; Renault, Tristan; Choresca, Casiano; Shin, Sang Phil; Jun, Jin Woo; Park, Se Chang

2013-01-31

Cyprinid herpes virus 3 (CyHV-3) diseases have been reported around the world and are associated with high mortalities of koi (Cyprinus carpio). Although little work has been conducted on the molecular analysis of this virus, glycoprotein genes identified in the present study seem to be valuable targets for genetic comparison of this virus. Three envelope glycoprotein genes (ORF25, 65 and 116) of the CyHV-3 isolates from the USA, Israel, Japan and Korea were compared, and interestingly, sequence insertions or deletions were observed in these target regions. In addition, polymorphisms were presented in microsatellite zones from two glycoprotein genes (ORF65 and 116). In phylogenetic tree analysis, the Korean isolate was remarkably distinguished from USA, Israel, Japan isolates. These findings may be suitable for many applications including isolates differentiation and phylogeny studies.

Structures of the Oligosaccharides of the Glycoprotein Coded by Early Region E3 of Adenovirus 2

PubMed Central

Kornfeld, Rosalind; Wold, William S. M.

1981-01-01

Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-3H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-β-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man9GlcNAc and Man8GlcNAc and small amounts of Man7GlcNAc and Man6GlcNAc. The pulse-chase sample had predominantly Man8GlcNAc and much less Man9GlcNAc, indicating that processing of the Man9GlcNAc to Man8GlcNAc had occurred during the chase period. Thus, Man8GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with α-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man9GlcNAc were identical to those of the lipid-linked Glc3Man9GlcNAc2 donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell-coded glycoprotein. Images PMID:7321093

Structures of the oligosaccharides of the glycoprotein coded by early region E3 of adenovirus 2.

PubMed

Kornfeld, R; Wold, W S

1981-11-01

Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-(3)H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-beta-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man(9)GlcNAc and Man(8)GlcNAc and small amounts of Man(7)GlcNAc and Man(6)GlcNAc. The pulse-chase sample had predominantly Man(8)GlcNAc and much less Man(9)GlcNAc, indicating that processing of the Man(9)GlcNAc to Man(8)GlcNAc had occurred during the chase period. Thus, Man(8)GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with alpha-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man(9)GlcNAc were identical to those of the lipid-linked Glc(3)Man(9)GlcNAc(2) donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell

Releasing N-glycan from peptide N-terminus by N-terminal succinylation assisted enzymatic deglycosylation.

PubMed

Weng, Yejing; Sui, Zhigang; Jiang, Hao; Shan, Yichu; Chen, Lingfan; Zhang, Shen; Zhang, Lihua; Zhang, Yukui

2015-04-22

Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests. Therefore, in this study, we developed a novel strategy to identify PGANs by releasing N-glycans through the N-terminal site-selective succinylation assisted enzymatic deglycosylation. The obtained PGANs information is beneficial to not only achieve the deep coverage analysis of glycoproteomes, but also discover the new biological functions of such modification.

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.

Calreticulin discriminates the proximal region at the N-glycosylation site of Glc1Man9GlcNAc2 ligand

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

Hirano, Makoto; Adachi, Yuka; Ito, Yukishige

Calreticulin (CRT) is well known as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However, whether CRT can directly interact with the aglycone moiety (protein portion) of the glycoprotein remains controversial. To improve our understanding of CRT interactions, structure-defined G1M9-derivatives with different aglycones (–OH, –Gly–NH{sub 2}, and –Gly–Glu–{sup t}Bu) were used as CRT ligands, and their interactions with recombinant CRT were analyzed using thermal shift analysis. The results showed that CRT binds strongly to a G1M9-ligand in the order –Gly–Glu–{sup t}Bu > –Gly–NH{sub 2} > –OH, which is the same as that of the reglucosylation of Man9GlcNAc2 (M9)-derivatives by themore » folding sensor enzyme UGGT (UDP-glucose: glycoprotein glucosyltransferase). Our results indicate that, similar to UGGT, CRT discriminates the proximal region at the N-glycosylation site, suggesting a similar mechanism mediating the recognition of aglycone moieties in the ER glycoprotein quality control system. - Highlights: • Glc1Man9GlcNAc2 (G1M9) ligands with different aglycones were chemically prepared. • Calreticulin (CRT) discriminates the aglycone of Glc1Man9GlcNAc2 (G1M9) ligand. • CRT binds with G1M9 ligands in a similar manner to folding sensor enzyme.« less

LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

PubMed Central

2015-01-01

Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC–MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC–ESI–MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts. PMID:25134008

Efficacy of soluble glycoprotein fraction from Allium sativum purified by size exclusion chromatography on murine Schistosomiasis mansoni.

PubMed

Aly, Ibrahim; Taher, Eman E; El-Sayed, Hoda; Mohammed, Faten A; ELnain, Gehan; Hamad, Rabab S; Bayoumy, Elsayed M

2017-06-01

In this work, the efficiency of crude MeOH extracts and soluble glycoprotein fraction of Allium sativum purified by size-exclusion chromatography (SEC) on parasitological, histopathological and some biochemical parameters in Schistosoma mansoni infected mice were investigated. Animals were infected by tail immersion with 100 cercariae/each mouse and divided into five groups in addition to the normal control. The results revealed a significant decrease in mean worm burden in all treated mice especially in the group treated with soluble glycoprotein fraction of A. sativum as compared to infected non-treated control with the disappearance of female worms. Administration of the studied extracts revealed remarkable amelioration in the levels of all the measured parameters in S. mansoni infected mice. In addition, treatment of mice with crude A. sativum MeOH extract and soluble glycoprotein fraction of A. sativum decreased significantly the activities of studied enzymes as compared to the infected untreated group. The highest degrees of enhancement in pathological changes was observed in the treated one with soluble glycoprotein fraction of A. sativum compared to the infected group represented by small sized, late fibro-cellular granuloma, the decrease in cellular constituents and degenerative changes in eggs. In conclusion, A. sativum treatment had effective schistosomicidal activities, through reduction of worm burden and tissue eggs, especially when it was given in purified glycoprotein fraction. Moreover, the soluble glycoprotein fraction of A. sativum largely modulates both the size and the number of granulomas. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Novel Endo-β-N-Acetylglucosaminidase Releases Specific N-Glycans Depending on Different Reaction Conditions

PubMed Central

De Moura Bell, Juliana Maria Leite Nobrega; Frese, Steven A.; Liu, Yan; Mills, David A.; Block, David E.; Barile, Daniela

2015-01-01

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant micro-biome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N-glycans in protein functionality. Endo-β-N-acetylglucosaminidase (EndoBI-1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat-stable enzyme that cleaves the N-N′-diacetyl chitobiose moiety found in the N-glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI-1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI-1 is able to cleave a high diversity of N-glycan structures. Nano-LC-Chip–Q-TOF MS data also revealed that different reaction conditions resulted in different N-glycan compositions released, thus modifying the relative abundance of N-glycan types. In general, more sialylated N-glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI-1 is able to release a wide variety of N-glycans, whose compositions can be selectively manipulated using different processing conditions. PMID:26101185

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

PubMed

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

2015-01-01

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

Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins.

PubMed Central

García-Barreno, B; Palomo, C; Peñas, C; Delgado, T; Perez-Breña, P; Melero, J A

1989-01-01

Monoclonal antibodies directed against the glycoproteins of human respiratory syncytial virus were used in competitive enzyme-linked immunosorbent assays for topological mapping of epitopes. Whereas epitopes of the F glycoprotein could be ascribed to five nonoverlapping antigenic sites, anti-G antibodies recognized unique epitopes, many of whose competition profiles overlapped extensively. Variant viruses selected with a neutralizing (47F) anti-F antibody lost the binding for only 47F and 49F antibodies, which mapped in the same antigenic area. In contrast, viruses selected with an anti-G antibody lost the capacity to bind most of the anti-G antibodies, and their G protein was not recognized by an anti-virus antiserum, indicating major changes in the antigenic structure of the G molecule. Finally, we found great antigenic variation of the G protein among viral isolates. This occurred even within viruses of the same subtype with only limited divergence of amino acid sequence between strains. All of these data indicate marked differences in the antigenic organization of the G and F glycoproteins of respiratory syncytial virus; we discuss these differences in terms of the chemical structure of the glycoproteins. Images PMID:2463385

Studies on the site of biosynthesis of acidic glycoproteins of guinea-pig serum

PubMed Central

Simkin, J. L.; Jamieson, J. C.

1967-01-01

1. Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an α-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. l-[U-14C]Leucine or -valine and d-[1-14C]glucosamine were used as precursors. 2. A lag of about 10min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. 3. Site of synthesis was investigated by studies in vivo up to 17min. after injection of precursor. Particulate subcellular fractions isolated from liver, spleen and kidney or homogenates of the latter two tissues were extracted with Lubrol. Extracts were allowed to react by double diffusion with antisera to fraction I or to subfractions isolated from it, and gels were subsequently subjected to radioautography. With either amino acid or glucosamine as precursor, only extracts of the microsome fraction of liver formed precipitin lines that were appreciably radioactive. 4. The role of the microsome fraction of liver in the synthesis of these glycoproteins was confirmed by immunological studies after incubation of liver slices with leucine or glucosamine. Incorporation of leucine was also investigated in a cell-free microsome system. 5. Material was also precipitated from certain Lubrol extracts of liver microsomes by direct addition of antiserum and its radioactivity measured. Degradation of material thus precipitated and use of heterologous immune systems showed that labelling of precipitin lines represented biosynthesis. 6. A study of extraction procedures suggested that the substances present in the microsome fraction of liver that react with specific antisera are associated with membranous structures. 7. Most or all precipitin lines formed by Lubrol extracts of liver microsomes interacted with precipitin lines given by guinea

Decreased levels of alpha-1-acid glycoprotein are related to the mortality of septic patients in the emergency department.

PubMed

Barroso-Sousa, Romualdo; Lobo, Romulo R; Mendonça, Patricia R; Memória, Renan R; Spiller, Fernando; Cunha, Fernando Q; Pazin-Filho, Antonio

2013-01-01

To determine the validity of alpha-1-acid glycoprotein as a novel biomarker for mortality in patients with severe sepsis. We prospectively included patients with severe sepsis or septic shock at the emergency department at a single tertiary referral teaching hospital. All of the patients were enrolled within the first 24 hours of emergency department admission, and clinical data and blood samples were obtained. As the primary outcome, we investigated the association of serum levels of alpha-1-acid glycoprotein and 96-hour mortality with logistic regression analysis and generalized estimating equations adjusted for age, sex, shock status and Acute Physiology and Chronic Health Evaluation II score. Patients with septic shock had lower alpha-1-acid glycoprotein levels at the time of emergency department admission compared to patients without shock (respectively, 149.1 ±42.7 vs. 189.8 ±68.6; p = 0.005). Similarly, non-survivors in the first 96 hours were also characterized by lower levels of alpha-1-acid glycoprotein at the time of emergency department admission compared to survivors (respectively, 132.18 ±50.2 vs. 179.8 ±61.4; p = 0.01). In an adjusted analysis, alpha-1-acid glycoprotein levels ≤120 mg/dL were significantly associated with 96-hour mortality (odds ratio = 14.37; 95% confidence interval = 1.58 to 130.21). Septic shock patients exhibited lower circulating alpha-1-acid glycoprotein levels than patients without shock. Alpha-1-acid glycoprotein levels were independently associated with 96-hour mortality in individuals with severe sepsis.

Determinant for Endoplasmic Reticulum Retention in the Luminal Domain of the Human Cytomegalovirus US3 Glycoprotein

PubMed Central

Lee, Sungwook; Park, Boyoun; Ahn, Kwangseog

2003-01-01

US3 of human cytomegalovirus is an endoplasmic reticulum resident transmembrane glycoprotein that binds to major histocompatibility complex class I molecules and prevents their departure. The endoplasmic reticulum retention signal of the US3 protein is contained in the luminal domain of the protein. To define the endoplasmic reticulum retention sequence in more detail, we have generated a series of deletion and point mutants of the US3 protein. By analyzing the rate of intracellular transport and immunolocalization of the mutants, we have identified Ser58, Glu63, and Lys64 as crucial for retention, suggesting that the retention signal of the US3 protein has a complex spatial arrangement and does not comprise a contiguous sequence of amino acids. We also show that a modified US3 protein with a mutation in any of these amino acids maintains its ability to bind class I molecules; however, such mutated proteins are no longer retained in the endoplasmic reticulum and are not able to block the cell surface expression of class I molecules. These findings indicate that the properties that allow the US3 glycoprotein to be localized in the endoplasmic reticulum and bind major histocompatibility complex class I molecules are located in different parts of the molecule and that the ability of US3 to block antigen presentation is due solely to its ability to retain class I molecules in the endoplasmic reticulum. PMID:12525649

Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

PubMed

Ji, Xin; Olinger, Gene G; Aris, Sheena; Chen, Ying; Gewurz, Henry; Spear, Gregory T

2005-09-01

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

PubMed

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Rajendran, Madhusudan; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R; García-Sastre, Adolfo; Basler, Christopher F; Munoz-Fontela, Cesar; Krammer, Florian

2017-08-15

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 -/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro , suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

PubMed Central

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R.; García-Sastre, Adolfo; Basler, Christopher F.

2017-01-01

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2−/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies

Effect of an aqueous extract of Scoparia dulcis on plasma and tissue glycoproteins in streptozotocin induced diabetic rats.

PubMed

Latha, M; Pari, L

2005-02-01

The influence of Scoparia dulcis, a traditionally used plant for the treatment of diabetes mellitus, was examined in streptozotocin diabetic rats on dearrangement in glycoprotein levels. Diabetes was induced in male Wistar rats by a single intraperitoneal injection of streptozotocin. An aqueous extract of Scoparia dulcis plant was administered orally for 6 weeks. The effect of the Scoparia dulcis extract on blood glucose, plasma insulin, plasma and tissue glycoproteins studied was in comparison to glibenclamide. The levels of blood glucose and plasma glycoproteins were increased significantly whereas the level of plasma insulin was significantly decreased in diabetic rats. There was a significant decrease in the level of sialic acid and elevated levels of hexose, hexosamine and fucose in the liver and kidney of streptozotocin diabetic rats. Oral administration of Scoparia dulcis plant extract (SPEt) to diabetic rats led to decreased levels of blood glucose and plasma glycoproteins. The levels of plasma insulin and tissue sialic acid were increased whereas the levels of tissue hexose, hexosamine and fucose were near normal. The present study indicates that Scoparia dulcis possesses a significant beneficial effect on glycoproteins in addition to its antidiabetic effect.

The G glycoprotein of respiratory syncytial virus depresses respiratory rates through the CX3C motif and substance P.

PubMed

Tripp, Ralph A; Dakhama, Azzeddine; Jones, Les P; Barskey, Albert; Gelfand, Erwin W; Anderson, Larry J

2003-06-01

Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease.

The G Glycoprotein of Respiratory Syncytial Virus Depresses Respiratory Rates through the CX3C Motif and Substance P

PubMed Central

Tripp, Ralph A.; Dakhama, Azzeddine; Jones, Les P.; Barskey, Albert; Gelfand, Erwin W.; Anderson, Larry J.

2003-01-01

Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease. PMID:12743318

Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections.

PubMed

Xiong, Xiaoli; Tortorici, M Alejandra; Snijder, Joost; Yoshioka, Craig; Walls, Alexandra C; Li, Wentao; McGuire, Andrew T; Rey, Félix A; Bosch, Berend-Jan; Veesler, David

2017-11-01

decorated with 78 N-linked glycans obstructing the protein surface to limit accessibility to neutralizing antibodies in a way reminiscent of what has recently been described for a human respiratory coronavirus. PDCoV S is largely protease-resistant which distinguishes it from most other characterized coronavirus S glycoproteins and suggests that enteric coronaviruses have evolved to fine-tune fusion activation in the protease-rich environment of the small intestine of infected hosts. Copyright © 2017 American Society for Microbiology.

Sindbis virus glycoproteins are abnormally glycosylated in Chinese hamster ovary cells deprived of glucose.

PubMed

Davidson, S K; Hunt, L A

1985-07-01

We have previously demonstrated that Sindbis virus infection of Chinese hamster ovary (CHO) cells altered the protein glycosylation machinery of the cell, so that both normal, full-size (nine mannose-containing) oligosaccharides and abnormal, "truncated' (five mannose-containing) oligosaccharides are transferred from lipid-linked precursors to newly synthesized viral membrane glycoproteins. In the present studies, we have examined the precursor oligosaccharides on viral glycoproteins that were pulse-labelled with [3H]mannose in the presence or absence of glucose, since glucose starvation of uninfected CHO cells has been reported to induce synthesis of truncated precursor oligosaccharides. Pulse-labelling in the absence of glucose led to a greater than 10-fold increase in the relative amount of the truncated precursor oligosaccharides being transferred to the newly synthesized viral glycoproteins and to an apparent underglycosylation of some precursor viral polypeptides, with some asparaginyl sites not acquiring covalently linked oligosaccharides. The mature virion glycoproteins from CHO cells which were pulse-labelled in the absence of glucose and then 'chased' in the presence of glucose contained proportionately more unusual Man3GlcNAc2-size oligosaccharides. These small neutral-type oligosaccharides were apparently not as good a substrate for further processing into complex acidic-type oligosaccharides as the normal Man5GlcNAc2 intermediate that results from the full-size precursor oligosaccharides.

A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

PubMed

Oppliger, Joel; da Palma, Joel Ramos; Burri, Dominique J; Bergeron, Eric; Khatib, Abdel-Majid; Spiropoulou, Christina F; Pasquato, Antonella; Kunz, Stefan

2016-01-15

Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human

An integrative strategy for quantitative analysis of the N-glycoproteome in complex biological samples.

PubMed

Wang, Ji; Zhou, Chuang; Zhang, Wei; Yao, Jun; Lu, Haojie; Dong, Qiongzhu; Zhou, Haijun; Qin, Lunxiu

2014-01-15

The complexity of protein glycosylation makes it difficult to characterize glycosylation patterns on a proteomic scale. In this study, we developed an integrated strategy for comparatively analyzing N-glycosylation/glycoproteins quantitatively from complex biological samples in a high-throughput manner. This strategy entailed separating and enriching glycopeptides/glycoproteins using lectin affinity chromatography, and then tandem labeling them with 18O/16O to generate a mass shift of 6 Da between the paired glycopeptides, and finally analyzing them with liquid chromatography-mass spectrometry (LC-MS) and the automatic quantitative method we developed based on Mascot Distiller. The accuracy and repeatability of this strategy were first verified using standard glycoproteins; linearity was maintained within a range of 1:10-10:1. The peptide concentration ratios obtained by the self-build quantitative method were similar to both the manually calculated and theoretical values, with a standard deviation (SD) of 0.023-0.186 for glycopeptides. The feasibility of the strategy was further confirmed with serum from hepatocellular carcinoma (HCC) patients and healthy individuals; the expression of 44 glycopeptides and 30 glycoproteins were significantly different between HCC patient and control serum. This strategy is accurate, repeatable, and efficient, and may be a useful tool for identification of disease-related N-glycosylation/glycoprotein changes.

Decreased levels of alpha-1-acid glycoprotein are related to the mortality of septic patients in the emergency department

PubMed Central

Barroso-Sousa, Romualdo; Lobo, Romulo R.; Mendonça, Patricia R.; Memória, Renan R.; Spiller, Fernando; Cunha, Fernando Q.; Pazin-Filho, Antonio

2013-01-01

OBJECTIVE: To determine the validity of alpha-1-acid glycoprotein as a novel biomarker for mortality in patients with severe sepsis. METHODS: We prospectively included patients with severe sepsis or septic shock at the emergency department at a single tertiary referral teaching hospital. All of the patients were enrolled within the first 24 hours of emergency department admission, and clinical data and blood samples were obtained. As the primary outcome, we investigated the association of serum levels of alpha-1-acid glycoprotein and 96-hour mortality with logistic regression analysis and generalized estimating equations adjusted for age, sex, shock status and Acute Physiology and Chronic Health Evaluation II score. RESULTS: Patients with septic shock had lower alpha-1-acid glycoprotein levels at the time of emergency department admission compared to patients without shock (respectively, 149.1±42.7 vs. 189.8±68.6; p = 0.005). Similarly, non-survivors in the first 96 hours were also characterized by lower levels of alpha-1-acid glycoprotein at the time of emergency department admission compared to survivors (respectively, 132.18±50.2 vs. 179.8±61.4; p = 0.01). In an adjusted analysis, alpha-1-acid glycoprotein levels ≤120 mg/dL were significantly associated with 96-hour mortality (odds ratio = 14.37; 95% confidence interval = 1.58 to 130.21). CONCLUSION: Septic shock patients exhibited lower circulating alpha-1-acid glycoprotein levels than patients without shock. Alpha-1-acid glycoprotein levels were independently associated with 96-hour mortality in individuals with severe sepsis. PMID:24037010

Cooperative role of calnexin and TigA in Aspergillus oryzae glycoprotein folding.

PubMed

Wang, Ning; Seko, Akira; Takeda, Yoichi; Kikuma, Takashi; Ito, Yukishige

2015-10-01

Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G1M9GN2-proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae, which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G1M9GN2-ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G1M9GN2-RNase was chemoenzymatically synthesized from G1M9GN-oxazoline and GN-RNase. Denatured G1M9GN2-RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae, TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Intrinsic protein fluorescence interferes with detection of tear glycoproteins in SDS-polyacrylamide gels using extrinsic fluorescent dyes.

PubMed

Zhao, Zhenjun; Aliwarga, Yulina; Willcox, Mark D P

2007-12-01

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

PubMed Central

Zhao, Zhenjun; Aliwarga, Yulina; Willcox, Mark DP

2007-01-01

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1–10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce. PMID:18166676

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

PubMed Central

McGrath, M; Witte, O; Pincus, T; Weissman, I L

1978-01-01

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques. Images PMID:205680

Quinovosamycins: New tunicamycin-type antibiotics in which the alpha, beta-1", 11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine.

USDA-ARS?s Scientific Manuscript database

Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall...

All-Atom Internal Coordinate Mechanics (ICM) Force Field for Hexopyranoses and Glycoproteins.

PubMed

Arnautova, Yelena A; Abagyan, Ruben; Totrov, Maxim

2015-05-12

We present an extension of the all-atom internal-coordinate force field, ICMFF, that allows for simulation of heterogeneous systems including hexopyranose saccharides and glycan chains in addition to proteins. A library of standard glycan geometries containing α- and β-anomers of the most common hexapyranoses, i.e., d-galactose, d-glucose, d-mannose, d-xylose, l-fucose, N -acetylglucosamine, N -acetylgalactosamine, sialic, and glucuronic acids, is created based on the analysis of the saccharide structures reported in the Cambridge Structural Database. The new force field parameters include molecular electrostatic potential-derived partial atomic charges and the torsional parameters derived from quantum mechanical data for a collection of minimal molecular fragments and related molecules. The ϕ/ψ torsional parameters for different types of glycosidic linkages are developed using model compounds containing the key atoms in the full carbohydrates, i.e., glycosidic-linked tetrahydropyran-cyclohexane dimers. Target data for parameter optimization include two-dimensional energy surfaces corresponding to the ϕ/ψ glycosidic dihedral angles in the disaccharide analogues, as determined by quantum mechanical MP2/6-31G** single-point energies on HF/6-31G** optimized structures. To achieve better agreement with the observed geometries of glycosidic linkages, the bond angles at the O-linkage atoms are added to the internal variable set and the corresponding bond bending energy term is parametrized using quantum mechanical data. The resulting force field is validated on glycan chains of 1-12 residues from a set of high-resolution X-ray glycoprotein structures based on heavy atom root-mean-square deviations of the lowest-energy glycan conformations generated by the biased probability Monte Carlo (BPMC) molecular mechanics simulations from the native structures. The appropriate BPMC distributions for monosaccharide-monosaccharide and protein-glycan linkages are derived from

Evaluation of oxidative stress in placenta of fetal cardiac dysfunction rat model and antioxidant defenses of maternal vitamin C supplementation with the impacts on P-glycoprotein.

PubMed

Li, Yifei; Fang, Jie; Zhou, Kaiyu; Wang, Chuan; Mu, Dezhi; Hua, Yimin

2014-06-01

The oxidative stress of placenta during fetal heart dysfunction (FHD) is lack of evaluation. So, we carried out an experiment to explore whether vitamin C (VitC) can be supplied for placental protection under FHD and its impacts on P-glycoprotein expression. Fetal heart dysfunction was induced by two intra-amniotic injections of isoproterenol, then (VitC) was supplied. Hematoxylin-eosin (HE) staining was used to evaluate placental histology, and oxidative stress was measured by total antioxidant capacity, total superoxide dismutase and level of advanced oxidation protein products (AOPP), as well as apoptosis rate. Real-time polymerase chain reaction was adopted to measure the expressions of superoxide dismutase-1 (Sod-1), glutathione peroxidase-1 (Gpx-1) and endothelial nitric oxide synthase (eNOS) in placenta. Finally, western blot was performed to detect P-glycoprotein expression. All isoproterenol twice-treated fetuses exhibited significant (P < 0.05) contractile dysfunction by fetal echocardiography compared to others. The HE staining showed severe placental hydrops in the FHD group, and that hydrops could be reduced by VitC treatment. Total antioxidant capacity and total Sod-1 decreased in FHD and elevated after VitC supplementation. Also, level of AOPP increased in FHD and dropped after VitC supplementation. Analysis of apoptosis demonstrated that there was a mild increase in apoptosis rate of FHD. Reductions of Sod-1 and eNOS mRNA expression were confirmed in FHD, but these could recovered after VitC supplementation, with the same tendency of the P-glycoprotein. Severe oxidative injuries were identified in placentas of FHD with P-glycoprotein repression. VitC administration can reduce the oxidative stress and rebuild the protective mechanism of placenta. © 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.

Intercellular transfer of P-glycoprotein from the drug resistant human bladder cancer cell line BIU-87 does not require cell-to-cell contact.

PubMed

Zhou, Hui-liang; Zheng, Yong-jun; Cheng, Xiao-zhi; Lv, Yi-song; Gao, Rui; Mao, Hou-ping; Chen, Qin

2013-09-01

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

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

Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

2013-09-15

Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs.more » Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.« less

Differential interaction of Escherichia coli heat-labile toxin and cholera toxin with pig intestinal brush border glycoproteins depending on their ABH and related blood group antigenic determinants.

PubMed

Balanzino, L E; Barra, J L; Monferran, C G; Cumar, F A

1994-04-01

The ability of glycoproteins from pig intestinal brush border membranes (BBM) to bind cholera toxin (CT) or heat-labile toxins from strains of Escherichia coli isolated from human (LTh) or pig (LTp) intestines was studied. Glycoproteins capable of binding the toxins are also recognized by antibodies or lectins specific for ABO(H) blood group and related antigens. Pigs expressing A, H, or I antigenic determinants were used for comparison. The toxin-binding capacity of a glycoprotein depends on the toxin type and the blood group epitope borne by the glycoprotein. LTh and LTp preferably bound to several blood group A-active glycoproteins rather than H-active glycoproteins. By contrast, CT practically did not recognize either blood group A- or blood group H-active glycoproteins, while glycoproteins from pigs expressing I antigenic determinants were able to interact with LTh, LTp, and CT. LTh, LTp, or CT glycoprotein binding was selectively inhibited by specific lectins or monosaccharides. Affinity purification of the toxin binding brush border glycoproteins on the basis of their blood group reactivity suggests that such glycoproteins are hydrolytic enzymes. BBM from A+ pigs contain about 27 times more LTh binding sites, in addition to those recognized by CT, than an equivalent membrane preparation from H+ pigs. The present findings may help clarify some previous unclear results on LTh binding to intestinal BBM glycoproteins obtained by use of animals not typed by their ABO(H) blood group phenotype.

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.

Molecular Chaperone BiP Interacts with Borna Disease Virus Glycoprotein at the Cell Surface▿ †

PubMed Central

Honda, Tomoyuki; Horie, Masayuki; Daito, Takuji; Ikuta, Kazuyoshi; Tomonaga, Keizo

2009-01-01

Borna disease virus (BDV) is characterized by highly neurotropic infection. BDV enters its target cells using virus surface glycoprotein (G), but the cellular molecules mediating this process remain to be elucidated. We demonstrate here that the N-terminal product of G, GP1, interacts with the 78-kDa chaperone protein BiP. BiP was found at the surface of BDV-permissive cells, and anti-BiP antibody reduced BDV infection as well as GP1 binding to the cell surface. We also reveal that BiP localizes at the synapse of neurons. These results indicate that BiP may participate in the cell surface association of BDV. PMID:19776128

Mutational Analysis of Lassa Virus Glycoprotein Highlights Regions Required for Alpha-Dystroglycan Utilization.

PubMed

Acciani, Marissa; Alston, Jacob T; Zhao, Guohui; Reynolds, Hayley; Ali, Afroze M; Xu, Brian; Brindley, Melinda A

2017-09-15

Lassa virus (LASV) is an enveloped RNA virus endemic to West Africa and responsible for severe cases of hemorrhagic fever. Virus entry is mediated by the glycoprotein complex consisting of a stable-signal peptide, a receptor-binding subunit, GP1, and a viral-host membrane fusion subunit, GP2. Several cellular receptors can interact with the GP1 subunit and mediate viral entry, including alpha-dystroglycan (αDG) and lysosome-associated membrane protein 1 (LAMP1). In order to define the regions within GP1 that interact with the cellular receptors, we implemented insertional mutagenesis, carbohydrate shielding, and alanine scanning mutagenesis. Eighty GP constructs were engineered and evaluated for GP1-GP2 processing, surface expression, and the ability to mediate cell-to-cell fusion after low-pH exposure. To examine virus-to-cell entry, 49 constructs were incorporated onto vesicular stomatitis virus (VSV) pseudoparticles and transduction efficiencies were monitored in HAP1 and HAP1-ΔDAG1 cells that differentially produce the αDG cell surface receptor. Seven constructs retained efficient transduction in HAP1-ΔDAG1 cells yet poorly transduced HAP1 cells, suggesting that they are involved in αDG utilization. Residues H141, N146, F147, and Y150 cluster at the predicted central core of the trimeric interface and are important for GP-αDG interaction. Additionally, H92A-H93A, 150HA, 172HA, and 230HA displayed reduced transduction in both HAP1 and HAP1-ΔDAG1 cells, despite efficient cell-to-cell fusion activity. These mutations may interfere with interactions with the endosomal receptor LAMP1 or interfere at another stage in entry that is common to both cell lines. Insight gained from these data can aid in the development of more-effective entry inhibitors by blocking receptor interactions. IMPORTANCE Countries in which Lassa virus is endemic, such as Nigeria, Sierra Leone, Guinea, and Liberia, usually experience a seasonal outbreak of the virus from December to March

Structural approaches to the study of oligosaccharides in glycoprotein quality control.

PubMed

Ito, Yukishige; Hagihara, Shinya; Matsuo, Ichiro; Totani, Kiichiro

2005-10-01

High-mannose-type oligosaccharides have been shown to play important roles in protein quality control. Several intracellular proteins, such as lectins, chaperones and glycan-processing enzymes, are involved in this process. These include calnexin/calreticulin, UDP-glucose:glycoprotein glucosyltransferase (UGGT), cargo receptors (such as VIP36 and ERGIC-53), mannosidase-like proteins (e.g. EDEM and Htm1p) and ubiquitin ligase (Fbs). They are thought to recognize high-mannose-type glycans with subtly different structures, although the precise specificities are yet to be clarified. In order to gain a clear understanding of these protein-carbohydrate interactions, comprehensive synthesis of high-mannose-type glycans was conducted. In addition, two approaches to the synthesis of artificial glycoproteins with homogeneous oligosaccharides were investigated. Furthermore, a novel substrate of UGGT was discovered.

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

PubMed Central

Dong, Li-Li; Tang, Ru; Zhai, Yu-Jia; Malla, Tejsu; Hu, Kai

2017-01-01

AIM To investigate whether DNA vaccine encoding herpes simplex virus 1 (HSV-1) glycoprotein C (gC) and glycoprotein D (gD) will achieve better protective effect against herpes simplex keratitis (HSK) than DNA vaccine encoding gD alone. METHODS DNA vaccine expressing gD or gC combined gD (gD.gC) were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS Fusion protein gD.gC could be expressed successfully in cultured 293T cells. And, pRSC-gC.gD-IL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and sIgA production, enhanced cytotoxicities of splenocytes and nature killer cells (NK), when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future. PMID:29181304

Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

PubMed

Weber, J M; Sircar, S; Horvath, J; Dion, P

1989-11-01

Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

Relationship between SU Subdomains That Regulate the Receptor-Mediated Transition from the Native (Fusion-Inhibited) to the Fusion-Active Conformation of the Murine Leukemia Virus Glycoprotein

PubMed Central

Lavillette, Dimitri; Ruggieri, Alessia; Boson, Bertrand; Maurice, Marielle; Cosset, François-Loïc

2002-01-01

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All

Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

PubMed Central

Mardones, Gonzalo A.; Bonifacino, Juan S.

2014-01-01

Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons. PMID:24831812

Biologically active peptides of the vesicular stomatitis virus glycoprotein.

PubMed Central

Schlegel, R; Wade, M

1985-01-01

A peptide corresponding to the amino-terminal 25 amino acids of the mature vesicular stomatitis virus glycoprotein has recently been shown to be a pH-dependent hemolysin. In the present study, we analyzed smaller constituent peptides and found that the hemolytic domain resides within the six amino-terminal amino acids. Synthesis of variant peptides indicates that the amino-terminal lysine can be replaced by another positively charged amino acid (arginine) but that substitution with glutamic acid results in the total loss of the hemolytic function. Peptide-induced hemolysis was dependent upon buffer conditions and was inhibited when isotonicity was maintained with mannitol, sucrose, or raffinose. In sucrose, all hemolytic peptides were also observed to mediate hemagglutination. The large 25-amino acid peptide is also a pH-dependent cytotoxin for mammalian cells and appears to effect gross changes in cell permeability. Conservation of the amino terminus of vesicular stomatitis virus and rabies virus suggests that the membrane-destabilizing properties of this domain may be important for glycoprotein function. Images PMID:2981356

A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate-protein interaction profiling and cell surface carbohydrate expression evaluation

NASA Astrophysics Data System (ADS)

Wang, Yangzhong; Chen, Zhuhai; Liu, Yang; Li, Jinghong

2013-07-01

A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and

[Research progress on ebola virus glycoprotein].

PubMed

Ding, Guo-Yong; Wang, Zhi-Yu; Gao, Lu; Jiang, Bao-Fa

2013-03-01

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

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

Grose, C.; Jackson, W.; Traugh, J.A.

1989-09-01

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an inmore » vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.« less

Fibronectin in cell adhesion and migration via N-glycosylation

PubMed Central

Hsiao, Cheng-Te; Cheng, Hung-Wei; Huang, Chi-Ming; Li, Hao-Ru; Ou, Meng-Hsin; Huang, Jie-Rong; Khoo, Kay-Hooi; Yu, Helen Wenshin; Chen, Yin-Quan; Wang, Yang-Kao; Chiou, Arthur; Kuo, Jean-Cheng

2017-01-01

Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate all steps of wound healing. In order to seek safe sources of plasma fibronectin for its practical use in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that both have a similar ability as a suitable substrate for the stimulation of cell adhesion and for directing cell migration. In addition, we also defined the N-glycosylation sites and N-glycans present on homo and porcine plasma fibronectin. These N-glycosylation modifications of the plasma fibronectin synergistically support the integrin-mediated signals to bring about mediating cellular adhesion and directed cell migration. This study not only determines the important function of N-glycans in both homo and porcine plasma fibronectin-mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin if it was applied as a material for clinical wound healing and tissue repair. PMID:29050309

Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation

PubMed Central

Artenjak, Andrej; Leonardi, Adrijana; Križaj, Igor; Ambrožič, Aleš; Sodin-Semrl, Snezna; Božič, Borut; Čučnik, Saša

2014-01-01

Patient biological material for isolation of β2-glycoprotein I (β2GPI) and high avidity IgG anti-β2-glycoprotein I antibodies (HAv anti-β2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked β2GPI and HAv aβ2GPI to gain unmodified proteins in higher yields/purity. Isolation of β2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aβ2GPI affinity chromatographies with immobilized protein G and human β2GPI were used. The unknown protein found during isolation was identified by liquid chromatography electrospray ionization mass spectrometry and the nonredundant National Center for Biotechnology Information database. The average mass of the isolated unnicked purified β2GPI increased from 6.56 mg to 9.94 mg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from β2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-β2GPI from different matrixes was 13.8%, as determined by our in-house anti-β2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked β2GPI and HAv anti-β2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%. PMID:24741579

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Intrahypothalamic Injection of the HIV-1 Envelope Glycoprotein Induces Fever via Interaction with the Chemokine System

PubMed Central

Addou, Saad; Yondorf, Menachem; Geller, Ellen B.; Eisenstein, Toby K.; Adler, Martin W.

2010-01-01

Wasting syndrome is a common complication of HIV infection and is marked by progressive weight loss and weakness, often associated with fever. The mechanisms involved in the pathogenesis of these syndromes are not well defined, and neither are the brain areas involved. The present study tests a new hypothesis: that the preoptic anterior hypothalamus (POAH), the main brain area for thermoregulation and fever, has a role in the pathogenesis of fever induced by glycoprotein 120 (gp120), the surface envelope protein used by the HIV to gain access into immune cells, and that the CXC chemokine receptors (CXCR4) that serve as a coreceptor for HIV entry mediate the effect. A sterilized stainless steel C313G cannula guide was implanted into the POAH, and a biotelemetry system was used to monitor the body temperature (Tb) changes. The administration of gp120 into the POAH induced fever in a dose-dependent manner. To demonstrate possible links between the gp120 and CXCR4 in generating the fever, we pretreated the rats with 1,1′-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] octohydrobromide dihydrate (AMD 3100), an antagonist of stromal cell-derived growth factor (SDF)-1α/CXCL12, acting at its receptor, CXCR4, 30 min before administration of gp120. AMD 3100 significantly reduced the gp120-induced fever. The present studies show that the presence of HIV-1 envelope glycoprotein gp120 in the POAH provokes fever via interaction CXCR4 pathway. PMID:19906780

A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

PubMed

York, Joanne; Nunberg, Jack H

2018-01-01

For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by Apolipoprotein N-Acyltransferase BCG_2070c

PubMed Central

2013-01-01

Background Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. Results We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Conclusions Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria. PMID:24093492

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase BCG_2070c.

PubMed

Brülle, Juliane K; Tschumi, Andreas; Sander, Peter

2013-10-05

Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

An integrative strategy for quantitative analysis of the N-glycoproteome in complex biological samples

PubMed Central

2014-01-01

Background The complexity of protein glycosylation makes it difficult to characterize glycosylation patterns on a proteomic scale. In this study, we developed an integrated strategy for comparatively analyzing N-glycosylation/glycoproteins quantitatively from complex biological samples in a high-throughput manner. This strategy entailed separating and enriching glycopeptides/glycoproteins using lectin affinity chromatography, and then tandem labeling them with 18O/16O to generate a mass shift of 6 Da between the paired glycopeptides, and finally analyzing them with liquid chromatography-mass spectrometry (LC-MS) and the automatic quantitative method we developed based on Mascot Distiller. Results The accuracy and repeatability of this strategy were first verified using standard glycoproteins; linearity was maintained within a range of 1:10–10:1. The peptide concentration ratios obtained by the self-build quantitative method were similar to both the manually calculated and theoretical values, with a standard deviation (SD) of 0.023–0.186 for glycopeptides. The feasibility of the strategy was further confirmed with serum from hepatocellular carcinoma (HCC) patients and healthy individuals; the expression of 44 glycopeptides and 30 glycoproteins were significantly different between HCC patient and control serum. Conclusions This strategy is accurate, repeatable, and efficient, and may be a useful tool for identification of disease-related N-glycosylation/glycoprotein changes. PMID:24428921

Identification of a mouse synaptic glycoprotein gene in cultured neurons.

PubMed

Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

2005-10-01

Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

Strategies to induce broadly protective antibody responses to viral glycoproteins.

PubMed

Krammer, F

2017-05-01

Currently, several universal/broadly protective influenza virus vaccine candidates are under development. Many of these vaccines are based on strategies to induce protective antibody responses against the surface glycoproteins of antigenically and genetically diverse influenza viruses. These strategies might also be applicable to surface glycoproteins of a broad range of other important viral pathogens. Areas covered: Common strategies include sequential vaccination with divergent antigens, multivalent approaches, vaccination with glycan-modified antigens, vaccination with minimal antigens and vaccination with antigens that have centralized/optimized sequences. Here we review these strategies and the underlying concepts. Furthermore, challenges, feasibility and applicability to other viral pathogens are discussed. Expert commentary: Several broadly protective/universal influenza virus vaccine strategies will be tested in humans in the coming years. If successful in terms of safety and immunological readouts, they will move forward into efficacy trials. In the meantime, successful vaccine strategies might also be applied to other antigenically diverse viruses of concern.

"Dermatitis" defined.

PubMed

Smith, Suzanne M; Nedorost, Susan T

2010-01-01

The term "dermatitis" can be defined narrowly or broadly, clinically or histologically. A common and costly condition, dermatitis is underresourced compared to other chronic skin conditions. The lack of a collectively understood definition of dermatitis and its subcategories could be the primary barrier. To investigate how dermatologists define the term "dermatitis" and determine if a consensus on the definition of this term and other related terms exists. A seven-question survey of dermatologists nationwide was conducted. Of respondents (n  =  122), half consider dermatitis to be any inflammation of the skin. Nearly half (47.5%) use the term interchangeably with "eczema." Virtually all (> 96%) endorse the subcategory "atopic" under the terms "dermatitis" and "eczema," but the subcategories "contact," "drug hypersensitivity," and "occupational" are more highly endorsed under the term "dermatitis" than under the term "eczema." Over half (55.7%) personally consider "dermatitis" to have a broad meaning, and even more (62.3%) believe that dermatologists as a whole define the term broadly. There is a lack of consensus among experts in defining dermatitis, eczema, and their related subcategories.

36 kDa glycoprotein isolated from Rhus verniciflua stokes inhibits G/GO-induced mitochondrial apoptotic signal pathways in BNL CL.2 cells.

PubMed

Lee, Sei-Jung; Oh, Phil-Sun; Lim, Kwang; Lim, Kye-Taek

2005-12-01

Rhus verniciflua Stokes is one of the medicinal plants traditionally used to heal and treat hepatic and inflammatory diseases. We found that a glycoprotein isolated from the fruit has a molecular weight of 36 kDa and consists of a carbohydrate component (38.75%) and a protein (61.25%), and that the glycoprotein has a strong scavenging activity against hydroxyl radicals without any pro-oxidant activity in the cell-free system. In glucose/glucose oxidase (G/GO)-induced BNL CL.2 cells, the results showed that Rhus verniciflua Stokes glycoprotein has dose-dependent blocking activities against G/GO-induced cytotoxicity and apoptosis, increasing the glutathione (GSH) peroxidase activity. In the activity of the mitochondrial apoptotic mediators (cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)), the glycoprotein (100 microg/ml) showed an inhibitory effect on cytochrome c release, caspase-9/3 activation, and PARP cleavage. Moreover, Rhus verniciflua Stokes glycoprotein has a stimulating effect on the nitric oxide production. Here, we speculate that this glycoprotein is one of the natural antioxidants and of the modulators of apoptotic signal pathways in BNL CL.2 cells.

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

PubMed Central

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

2013-01-01

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

Increased levels of fucosyltransferase IX and carbohydrate Lewis(x) adhesion determinant in human NT2N neurons.

PubMed

Brito, Catarina; Escrevente, Cristina; Reis, Celso A; Lee, Virginia M-Y; Trojanowski, John Q; Costa, Júlia

2007-05-01

The expression of the fucosylated carbohydrate Lewis(x) (Le(x)) determinant (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc-R) has been found in glycoproteins, proteoglycans, and glycolipids from the nervous system. Evidence suggests its association with cell-cell recognition, neurite outgrowth, and neuronal migration during central nervous system development. In the present work, we detected increased levels of Le(x) in differentiated human NT2N neurons cultured in vitro. To identify which fucosyltransferase (FUT) synthesized the Le(x) in NT2N neurons, RT-PCR, FUT substrate specificity and Western blot analysis were carried out. Strong activity toward acceptors Galbeta4GlcNAc-O-R and Fucalpha2Galbeta4GlcNAc-O-R [R = -(CH(2))(3)NHCO(CH(2))(5)NH-biotin], together with strong FUT9 detection by Western blot and presence of transcripts showed that FUT9 was the enzyme associated with Le(x) biosynthesis in NT2N neurons. Le(x) was detected at the plasma membrane of NT2N neurons, in lysosomes marked with lysosomal-associated membrane protein 1 (LAMP-1), and it was found for the first time to colocalize with the tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) that defines the TI-VAMP exocytic compartment that is involved in neurite outgrowth. Furthermore, incubation with anti-Le(x) monoclonal antibody L5 led to impaired adhesion of NT2N neurons to the surface matrix and inhibited neurite initiation. In conclusion, FUT9 and its product Le(x) are detected specifically in human NT2N neurons and our results indicate that they underlie cell differentiation, cell adhesion, and initiation of neurite outgrowth in those neurons. (c) 2007 Wiley-Liss, Inc.

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

PubMed

Rubak, Peter; Kristensen, Steen D; Hvas, Anne-Mette

2017-06-01

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10 9 /L) than healthy individuals (240 × 10 9 /L, p < 0.01), but a higher MPV (p = 0.03) and IPF (p < 0.01). IPC was similar for the two groups (p = 0.74). PRP had significantly lower MPV (p < 0.01) and significantly higher platelet count and IPC (both p-values <0.03) when compared with whole blood. IPF was similar for PRP and whole blood (p = 0.18). COX-1 expression was 10 times higher and COX-2 expression was 50% higher in PRP than in whole blood (p COX-1 < 0.01, p COX-2 < 0.01). Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p COX-1 < 0.01) and PRP, though this was nonsignificant in PRP (p COX-1 = 0.17). In ITP patients, positive correlations were found between platelet turnover and COX-1 expression (all p-values <0.01, rho = 0.80-0.94), whereas healthy individuals showed significant though weaker

'Leukodystrophy-like' phenotype in children with myelin oligodendrocyte glycoprotein antibody-associated disease.

PubMed

Hacohen, Yael; Rossor, Thomas; Mankad, Kshitij; Chong, Wk 'Kling'; Lux, Andrew; Wassmer, Evangeline; Lim, Ming; Barkhof, Frederik; Ciccarelli, Olga; Hemingway, Cheryl

2018-04-01

To review the demographics and clinical and paraclinical parameters of children with myelin oligodendrocyte glycoprotein (MOG) antibody-associated relapsing disease. In this UK-based, multicentre study, 31 children with MOG antibody-associated relapsing disease were studied retrospectively. Of the 31 children studied, 14 presented with acute disseminated encephalomyelitis (ADEM); they were younger (mean 4.1y) than the remainder (mean 8.5y) who presented with optic neuritis and/or transverse myelitis (p<0.001). Similarly, children who had an abnormal brain magnetic resonance imaging (MRI) at onset (n=20) were younger than patients with normal MRI at onset (p=0.001) or at follow-up (p<0.001). 'Leukodystrophy-like' MRI patterns of confluent largely symmetrical lesions was seen during the course of the disease in 7 out of 14 children with a diagnosis of ADEM, and was only seen in children younger than 7 years of age. Their disability after a 3-year follow-up was mild to moderate, and most patients continued to relapse, despite disease-modifying treatments. MOG antibody should be tested in children presenting with relapsing neurological disorders associated with confluent, bilateral white matter changes, and distinct enhancement pattern. Children with MOG antibody-associated disease present with age-related differences in phenotypes, with a severe leukoencephalopathy phenotype in the very young and normal intracranial MRI in the older children. This finding suggests a susceptibility of the very young and myelinating brain to MOG antibody-mediated mechanisms of damage. Myelin oligodendrocyte glycoprotein (MOG) antibody-associated demyelination manifest with an age-related phenotype. Children with MOG antibody and 'leukodystrophy-like' imaging patterns tend to have poor response to second-line immunotherapy. © 2017 Mac Keith Press.

Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes.

PubMed

Johnson, Kim L; Cassin, Andrew M; Lonsdale, Andrew; Wong, Gane Ka-Shu; Soltis, Douglas E; Miles, Nicholas W; Melkonian, Michael; Melkonian, Barbara; Deyholos, Michael K; Leebens-Mack, James; Rothfels, Carl J; Stevenson, Dennis W; Graham, Sean W; Wang, Xumin; Wu, Shuangxiu; Pires, J Chris; Edger, Patrick P; Carpenter, Eric J; Bacic, Antony; Doblin, Monika S; Schultz, Carolyn J

2017-06-01

The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SP n EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots. © 2017 American Society of Plant Biologists. All Rights Reserved.

Determination of Cytomegalovirus Prevalence and Glycoprotein B Genotypes Among Ulcerative Colitis Patients in Ahvaz, Iran

PubMed Central

Taherkhani, Reza; Farshadpour, Fatemeh; Makvandi, Manoochehr; Hamidifard, Mojtaba; Esmailizadeh, Mahdi; Ahmadi, Bijan; Heidari, Hamid

2015-01-01

Background: The human cytomegalovirus (HCMV) is a common pathogen which usually remains asymptomatic in the healthy adults; however, it can cause a symptomatic disease in the immunocompromised patients. The risk of infection with HCMV increases in ulcerative colitis (UC) patients as a result of receiving immunosuppressive agents. Objectives: This study aimed to determine the prevalence and the glycoprotein B genotypes of HCMV among the patients with HCMV disease superimposed on an UC flare that required hospitalization in Imam Khomeini Hospital in Ahvaz, Iran, during 2010- 2012. Patients and Methods: In this case-control study, formalin-fixed paraffin-embedded intestinal tissue samples were taken from 98 patients with UC disease including 53 males and 45 females (mean age ± standard deviation, 38.95 ± 17.93) and 67 control patients with noninflammatory disease who were referred to Imam Khomeini Hospital during 2010-2012. Detection of HCMV genome in intestinal samples was carried out by seminested polymerase chain reaction. Glycoprotein B genotypes were determined by sequencing. Results: Among 98 patients with UC, only 12 (12.2%) patients were positive for HCMV genome, while the HCMV genome was not detected in any of the controls. (P = 0.002). The distribution of HCMV gB genotypes in 12 CMV-positive UC patients was as follow: gB1, 11 (91.7%) and gB3, 1 (8.3%). The most prevalent genotype in CMV-positive UC patients was gB1. Conclusions: In this study, high prevalence of 91.7% HCMV gB1 genotype was predominant among HCMV-positive UC patients, which suggests that there might be an association between HCMV gB genotype 1 and UC disease. PMID:25793098

Determination of cytomegalovirus prevalence and glycoprotein B genotypes among ulcerative colitis patients in ahvaz, iran.

PubMed

Taherkhani, Reza; Farshadpour, Fatemeh; Makvandi, Manoochehr; Hamidifard, Mojtaba; Esmailizadeh, Mahdi; Ahmadi, Bijan; Heidari, Hamid

2015-02-01

The human cytomegalovirus (HCMV) is a common pathogen which usually remains asymptomatic in the healthy adults; however, it can cause a symptomatic disease in the immunocompromised patients. The risk of infection with HCMV increases in ulcerative colitis (UC) patients as a result of receiving immunosuppressive agents. This study aimed to determine the prevalence and the glycoprotein B genotypes of HCMV among the patients with HCMV disease superimposed on an UC flare that required hospitalization in Imam Khomeini Hospital in Ahvaz, Iran, during 2010- 2012. In this case-control study, formalin-fixed paraffin-embedded intestinal tissue samples were taken from 98 patients with UC disease including 53 males and 45 females (mean age ± standard deviation, 38.95 ± 17.93) and 67 control patients with noninflammatory disease who were referred to Imam Khomeini Hospital during 2010-2012. Detection of HCMV genome in intestinal samples was carried out by seminested polymerase chain reaction. Glycoprotein B genotypes were determined by sequencing. Among 98 patients with UC, only 12 (12.2%) patients were positive for HCMV genome, while the HCMV genome was not detected in any of the controls. (P = 0.002). The distribution of HCMV gB genotypes in 12 CMV-positive UC patients was as follow: gB1, 11 (91.7%) and gB3, 1 (8.3%). The most prevalent genotype in CMV-positive UC patients was gB1. In this study, high prevalence of 91.7% HCMV gB1 genotype was predominant among HCMV-positive UC patients, which suggests that there might be an association between HCMV gB genotype 1 and UC disease.

Effects of nelfinavir and its M8 metabolite on lymphocyte P-glycoprotein activity during antiretroviral therapy.

PubMed

Donahue, John P; Dowdy, David; Ratnam, Krishna K; Hulgan, Todd; Price, James; Unutmaz, Derya; Nicotera, Janet; Raffanti, Steven; Becker, Mark; Haas, David W

2003-01-01

The efflux pump P-glycoprotein decreases drug penetration into cells and tissues. To determine whether nelfinavir or its metabolites inhibit P-glycoprotein in lymphocytes from a healthy volunteer, whole blood cells from human immunodeficiency virus-negative donors were incubated either in human plasma to which nelfinavir or its M8 metabolite were added ex vivo or in plasma from human immunodeficiency virus-positive patients receiving nelfinavir. The 50% P-glycoprotein inhibitory concentrations of purified nelfinavir and M8 were 10.9 micromol/L and 29.5 micromol/L, respectively, for CD4(+) T cells and 19.3 micromol/L and >48 micromol/L, respectively, for CD8(+) T cells. Significant inhibitory activity was present in plasma from 27 of 46 patients (59%) receiving nelfinavir. Plasma nelfinavir concentrations correlated with percent inhibition on CD4(+) (rho = 0.85, P <.0001) and CD8(+) (rho = 0.83, P <.0001) T cells. The M8 concentrations correlated weakly with both inhibition and nelfinavir concentrations. On the basis of our findings in lymphocytes from a healthy volunteer exposed to plasma from human immunodeficiency virus-positive patients, we believe it is likely that CD4(+) and CD8(+) lymphocytes in patients receiving nelfinavir as therapy for human immunodeficiency virus may have P-glycoprotein inhibited by plasma concentrations of nelfinavir.

A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis.

PubMed

Ciocchini, Andrés E; Serantes, Diego A Rey; Melli, Luciano J; Guidolin, Leticia S; Iwashkiw, Jeremy A; Elena, Sebastián; Franco, Cristina; Nicola, Ana M; Feldman, Mario F; Comerci, Diego J; Ugalde, Juan E

2014-08-27

Brucellosis is a highly contagious zoonosis that affects livestock and human beings. Laboratory diagnosis of bovine brucellosis mainly relies on serological diagnosis using serum and/or milk samples. Although there are several serological tests with different diagnostic performance and capacity to differentiate vaccinated from infected animals, there is still no standardized reference antigen for the disease. Here we validate the first recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of bovine brucellosis. This antigen can be produced in homogeneous batches without the need of culturing pathogenic brucellae; all characteristics that make it appropriate for standardization. An indirect immunoassay based on the detection of anti O-polysaccharide IgG antibodies in bovine samples was developed coupling OAg-AcrA to magnetic beads or ELISA plates. As a proof of concept and to validate the antigen, we analyzed serum, whole blood and milk samples obtained from non-infected, experimentally infected and vaccinated animals included in a vaccination/infection trial performed in our laboratory as well as more than 1000 serum and milk samples obtained from naturally infected and S19-vaccinated animals from Argentina. Our results demonstrate that OAg-AcrA-based assays are highly accurate for diagnosis of bovine brucellosis, even in vaccinated herds, using different types of samples and in different platforms. We propose this novel recombinant glycoprotein as an antigen suitable for the development of new standard immunological tests for screening and confirmatory diagnosis of bovine brucellosis in regions or countries with brucellosis-control programs. Copyright © 2014 Elsevier B.V. All rights reserved.

An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

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

Tiwari, Vaibhav; Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612; Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766

2009-12-18

Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surfacemore » HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.« less

Characterization of a family of structurally related glycoproteins expressing beta 1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells.

PubMed

Laferté, S; Loh, L C

1992-04-01

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the

Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus.

PubMed

Bradel-Tretheway, Birgit G; Liu, Qian; Stone, Jacquelyn A; McInally, Samantha; Aguilar, Hector C

2015-07-01

Hendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within the Paramyxoviridae family. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses. Viral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly

Surface glycoproteins of an African henipavirus induce syncytium formation in a cell line derived from an African fruit bat, Hypsignathus monstrosus.

PubMed

Krüger, Nadine; Hoffmann, Markus; Weis, Michael; Drexler, Jan Felix; Müller, Marcel Alexander; Winter, Christine; Corman, Victor Max; Gützkow, Tim; Drosten, Christian; Maisner, Andrea; Herrler, Georg

2013-12-01

Serological screening and detection of genomic RNA indicates that members of the genus Henipavirus are present not only in Southeast Asia but also in African fruit bats. We demonstrate that the surface glycoproteins F and G of an African henipavirus (M74) induce syncytium formation in a kidney cell line derived from an African fruit bat, Hypsignathus monstrosus. Despite a less broad cell tropism, the M74 glycoproteins show functional similarities to glycoproteins of Nipah virus.

Novel aspects of sialoglycan recognition by the Siglec-like domains of streptococcal SRR glycoproteins.

PubMed

Bensing, Barbara A; Khedri, Zahra; Deng, Lingquan; Yu, Hai; Prakobphol, Akraporn; Fisher, Susan J; Chen, Xi; Iverson, Tina M; Varki, Ajit; Sullam, Paul M

2016-11-01

Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity. Here we examine the binding properties of four additional BRs from Streptococcus sanguinis or Streptococcus mitis and characterize the molecular determinants of ligand selectivity and affinity. Each BR has two domains that are essential for sialoglycan binding by GspB. One domain is structurally similar to the glycan-binding module of mammalian Siglecs (sialic acid-binding immunoglobulin-like lectins), including an arginine residue that is critical for glycan recognition, and that resides within a novel, conserved YTRY motif. Despite low sequence similarity to GspB, one of the BRs selectively binds sialyl-T antigen. Although the other three BRs are highly similar to Hsa, each displayed a unique ligand repertoire, including differential recognition of sialyl Lewis antigens and sulfated glycans. These differences in glycan selectivity were closely associated with differential binding to salivary and platelet glycoproteins. Specificity of sialoglycan adherence is likely an evolving trait that may influence the propensity of streptococci expressing Siglec-like adhesins to cause infective endocarditis. Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Hemocytes and Plasma of the Eastern Oyster (Crassostrea virginica) Display a Diverse Repertoire of Sulfated and Blood Group A-modified N-Glycans*

PubMed Central

Kurz, Simone; Jin, Chunsheng; Hykollari, Alba; Gregorich, Daniel; Giomarelli, Barbara; Vasta, Gerardo R.; Wilson, Iain B. H.; Paschinger, Katharina

2013-01-01

The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal β1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1. PMID:23824194

COMPOSITION OF GLYCOPROTEINS SECRETED BY TRACHEAL EXPLANTS FROM VARIOUS ANIMAL SPECIES

EPA Science Inventory

The acidic and neutral glycoproteins secreted by cultured tracheal explants from pigs, sheep, rats, mice, monkeys, guinea pigs, dogs, and chickens were purified and fractionated by column chromatography on DEAE-cellulose and by electrophoresis on cellulose acetate. The ratios of ...

High efficiency labeling of glycoproteins on living cells

PubMed Central

Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

2010-01-01

We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

Simultaneous Release and Labeling of O- and N-Glycans Allowing for Rapid Glycomic Analysis by Online LC-UV-ESI-MS/MS.

PubMed

Wang, Chengjian; Lu, Yu; Han, Jianli; Jin, Wanjun; Li, Lingmei; Zhang, Ying; Song, Xuezheng; Huang, Linjuan; Wang, Zhongfu

2018-05-24

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by β-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.

Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins.

PubMed

Stone, Jacquelyn A; Vemulapati, Bhadra M; Bradel-Tretheway, Birgit; Aguilar, Hector C

2016-12-01

The paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions. Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F

Mechanical circulatory support is associated with loss of platelet receptors glycoprotein Ibα and glycoprotein VI.

PubMed

Lukito, P; Wong, A; Jing, J; Arthur, J F; Marasco, S F; Murphy, D A; Bergin, P J; Shaw, J A; Collecutt, M; Andrews, R K; Gardiner, E E; Davis, A K

2016-11-01

Essentials Relationship of acquired von Willebrand disease (VWD) and platelet dysfunction is explored. Patients with ventricular assist devices and on extracorporeal membrane oxygenation are investigated. Acquired VWD and platelet receptor shedding is demonstrated in the majority of patients. Loss of platelet adhesion receptors glycoprotein (GP) Ibα and GPVI may increase bleeding risk. Background Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. Objectives To investigate whether loss of platelet receptors occurs in vivo, and the relationship with acquired von Willebrand syndrome (AVWS). Methods Platelet counts, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 21 continuous flow VAD (CF-VAD), 20 ECMO, 12 heart failure and seven aortic stenosis patients. Levels of platelet receptors were measured by flow cytometry or ELISA. Results The loss of high molecular weight VWF multimers was observed in 18 of 19 CF-VAD and 14 of 20 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Conclusions These data link AVWS and increased platelet receptor shedding in patients with CF-VADs or ECMO for the first time. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CF-VAD and ECMO patients may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions. © 2016 International Society on Thrombosis and Haemostasis.

Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis.

PubMed

York, Joanne; Nunberg, Jack H

2016-09-15

Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact virion. We find that

Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

PubMed Central

Konrad, Zvia; Eichler, Jerry

2002-01-01

Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane. PMID:12069685

Defining "Sector 3" Poultry Layer Farms in Relation to H5N1-HPAI-An Example from Java, Indonesia.

PubMed

Durr, Peter A; Wibowo, Michael Haryadi; Tarigan, Simson; Artanto, Sidna; Rosyid, Murni Nurhasanah; Ignjatovic, Jagoda

2016-05-01

To help guide surveillance and control of highly pathogenic avian influenza subtype H5N1 (H5N1-HPAI), the Food and Agriculture Organization of the United Nations in 2004 devised a poultry farm classification system based on a combination of production and biosecurity practices. Four "Sectors" were defined, and this scheme has been widely adopted within Indonesia to guide national surveillance and control strategies. Nevertheless, little detailed research into the robustness of this classification system has been conducted, particularly as it relates to independent, small to medium-sized commercial poultry farms (Sector 3). Through an analysis of questionnaire data collected as part of a survey of layer farms in western and central Java, all of which were classified as Sector 3 by local veterinarians, we provide benchmark data on what defines this sector. A multivariate analysis of the dataset, using hierarchical cluster analysis, identified three groupings of the farms, which were defined by a combination of production-and biosecurity-related variables, particularly those related to farm size and (the lack of) washing and disinfection practices. Nevertheless, the relationship between production-related variables and positive biosecurity practices was poor, and larger farms did not have an overall higher total biosecurity score than small or medium-sized ones. Further research is required to define the properties of poultry farms in Indonesia that are most closely related to effective biosecurity and the prevention of H5N1-HPAI.

Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization.

PubMed

Maschkowitz, Gregor; Gärtner, Sabine; Hofmann-Winkler, Heike; Fickenscher, Helmut; Winkler, Michael

2018-05-01

Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans -Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization. IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular

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

PubMed

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

2014-10-01

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

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

PubMed Central

Gardner, Thomas J.

2016-01-01

SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

Novel microinjector for carrying bone substitutes for bone regeneration in periodontal diseases.

PubMed

Tsai, Hsiao-Cheng; Li, Yi-Chen; Young, Tai-Horng; Chen, Min-Huey

2016-01-01

Traditionally, guide bone regeneration (GBR) was a widely used method for repairing bone lost from periodontal disease. There were some disadvantages associated with the GBR method, such as the need for a stable barrier membrane and a new creative cavity during the surgical process. To address these disadvantages, the purpose of this study was to evaluate a novel microinjector developed for dental applications. The microinjector was designed to carry bone graft substitutes to restore bone defects for bone regeneration in periodontal diseases. The device would be used to replace the GBR method. In this study, the injected force and ejected volume of substitutes (including air, water, and ethanol) were defined by Hooke's law (n = 3). The optimal particle size of bone graft substitutes was determined by measuring the recycle ratio of bone graft substitutes from the microinjector (n = 3). Furthermore, a novel agarose gel model was used to evaluate the feasibility of the microinjector. The current study found that the injected force was less than 0.4 N for obtaining the ejected volume of approximately 2 mL, and when the particle size of tricalcium phosphate (TCP) was smaller than 0.5 mm, 80% TCP could be ejected from the microinjector. Furthermore, by using an agarose model to simulate the periodontal soft tissue, it was also found that bone graft substitutes could be easily injected into the gel. The results confirmed the feasibility of this novel microinjector for dental applications to carry bone graft substitutes for the restoration of bone defects of periodontal disease. Copyright © 2015. Published by Elsevier B.V.

A recombinant pseudorabies virus expressing rabies virus glycoprotein: safety and immunogenicity in dogs.

PubMed

Yuan, Ziguo; Zhang, Shoufeng; Liu, Ye; Zhang, Fei; Fooks, Anthony R; Li, Qianxue; Hu, Rongliang

2008-03-04

Several recombinant vaccines expressing the rabies virus glycoprotein have been developed, particularly for the oral vaccination of wildlife. While these vaccines induce protective immunity in some animal species such as foxes, they are less effective in others. Pseudorabies virus (PRV) has been licensed for use as a live vaccine in pigs and possesses an excellent safety and efficacy record. We have used it to construct a recombinant virus, rPRV/eGFP/rgp, expressing the rabies virus glycoprotein. This recombinant virus has been shown to be safe for dogs by oral and intramuscular routes of inoculation and was demonstrated to induce immune responses against both pseudorabies and rabies in dogs after a single oral dose of 2 x 10(7.0) plaque forming units (PFU). Neutralizing antibody titers against rabies reached > 0.5 IU/ml and 1:64-1:128 against pseudorabies by 5 weeks post-vaccination in all dogs, indicating that the pseudorabies virus vector infected dogs and replicated in vivo, and that the rabies virus glycoprotein had been expressed and an effective immune response elicited. Antibody titers were maintained for over 6 months. This suggests that pseudorabies virus could be an effective live vector for recombinant rabies oral vaccination.

The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

USGS Publications Warehouse

Bjorklund, H.V.; Higman, K.H.; Kurath, G.

1996-01-01

The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2–33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

USGS Publications Warehouse

Bjorklund, H.V.; Higman, K.H.; Kurath, G.

1996-01-01

The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2-33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

Liposomes Coloaded with Elacridar and Tariquidar To Modulate the P-Glycoprotein at the Blood-Brain Barrier.

PubMed

Nieto Montesinos, Rita; Béduneau, Arnaud; Lamprecht, Alf; Pellequer, Yann

2015-11-02

This study prepared three liposomal formulations coloaded with elacridar and tariquidar to overcome the P-glycoprotein-mediated efflux at the blood-brain barrier. Their pharmacokinetics, brain distribution, and impact on the model P-glycoprotein substrate, loperamide, were compared to those for the coadministration of free elacridar plus free tariquidar. After intravenous administration in rats, elacridar and tariquidar in conventional liposomes were rapidly cleared from the bloodstream. Their low levels in the brain did not improve the loperamide brain distribution. Although elacridar and tariquidar in PEGylated liposomes exhibited 2.6 and 1.9 longer half-lives than free elacridar and free tariquidar, respectively, neither their Kp for the brain nor the loperamide brain distribution was improved. However, the conjugation of OX26 F(ab')2 fragments to PEGylated liposomes increased the Kps for the brain of elacridar and tariquidar by 1.4- and 2.1-fold, respectively, in comparison to both free P-gp modulators. Consequently, the Kp for the brain of loperamide increased by 2.7-fold. Moreover, the plasma pharmacokinetic parameters and liver distribution of loperamide were not modified by the PEGylated OX26 F(ab')2 immunoliposomes. Thus, this formulation represents a promising tool for modulating the P-glycoprotein-mediated efflux at the blood-brain barrier and could improve the brain uptake of any P-glycoprotein substrate that is intended to treat central nervous system diseases.

Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus.

PubMed

Ghiasi, S M; Salmanian, A H; Chinikar, S; Zakeri, S

2011-12-01

While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition, while the study of CCHF is challenging, our protocol should be further used to study CCHFV infection in the knockout mouse model and virus neutralization assays in biosafety level 4 laboratories.

Interaction and interdependent packaging of tegument protein UL11 and glycoprotein e of herpes simplex virus.

PubMed

Han, Jun; Chadha, Pooja; Meckes, David G; Baird, Nicholas L; Wills, John W

2011-09-01

The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing U(L)11 at the U(L)35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro, indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.

Characterization of a family of structurally related glycoproteins expressing beta 1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells.

PubMed Central

Laferté, S; Loh, L C

1992-01-01

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the

Internalization and Axonal Transport of the HIV Glycoprotein gp120

PubMed Central

Berth, Sarah; Caicedo, Hector Hugo; Sarma, Tulika; Morfini, Gerardo

2015-01-01

The HIV glycoprotein gp120, a neurotoxic HIV glycoprotein that is overproduced and shed by HIV-infected macrophages, is associated with neurological complications of HIV such as distal sensory polyneuropathy, but interactions of gp120 in the peripheral nervous system remain to be characterized. Here, we demonstrate internalization of extracellular gp120 in a manner partially independent of binding to its coreceptor CXCR4 by F11 neuroblastoma cells and cultured dorsal root ganglion neurons. Immunocytochemical and pharmacological experiments indicate that gp120 does not undergo trafficking through the endolysosomal pathway. Instead, gp120 is mainly internalized through lipid rafts in a cholesterol-dependent manner, with a minor fraction being internalized by fluid phase pinocytosis. Experiments using compartmentalized microfluidic chambers further indicate that, after internalization, endocytosed gp120 selectively undergoes retrograde but not anterograde axonal transport from axons to neuronal cell bodies. Collectively, these studies illuminate mechanisms of gp120 internalization and axonal transport in peripheral nervous system neurons, providing a novel framework for mechanisms for gp120 neurotoxicity. PMID:25636314

Antibody induction directed against the tumor-associated MUC4 glycoprotein.

PubMed

Cai, Hui; Palitzsch, Björn; Hartmann, Sebastian; Stergiou, Natascha; Kunz, Horst; Schmitt, Edgar; Westerlind, Ulrika

2015-04-13

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti-tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor-associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor-associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem-repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4-based vaccines induced very strong antigen-specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein

PubMed Central

Francica, Joseph R.; Varela-Rohena, Angel; Medvec, Andrew; Plesa, Gabriela; Riley, James L.; Bates, Paul

2010-01-01

Many viruses alter expression of proteins on the surface of infected cells including molecules important for immune recognition, such as the major histocompatibility complex (MHC) class I and II molecules. Virus-induced downregulation of surface proteins has been observed to occur by a variety of mechanisms including impaired transcription, blocks to synthesis, and increased turnover. Viral infection or transient expression of the Ebola virus (EBOV) glycoprotein (GP) was previously shown to result in loss of staining of various host cell surface proteins including MHC1 and β1 integrin; however, the mechanism responsible for this effect has not been delineated. In the present study we demonstrate that EBOV GP does not decrease surface levels of β1 integrin or MHC1, but rather impedes recognition by steric occlusion of these proteins on the cell surface. Furthermore, steric occlusion also occurs for epitopes on the EBOV glycoprotein itself. The occluded epitopes in host proteins and EBOV GP can be revealed by removal of the surface subunit of GP or by removal of surface N- and O- linked glycans, resulting in increased surface staining by flow cytometry. Importantly, expression of EBOV GP impairs CD8 T-cell recognition of MHC1 on antigen presenting cells. Glycan-mediated steric shielding of host cell surface proteins by EBOV GP represents a novel mechanism for a virus to affect host cell function, thereby escaping immune detection. PMID:20844579

A reassessment of the evolutionary timescale of bat rabies viruses based upon glycoprotein gene sequences.

PubMed

Kuzmina, Natalia A; Kuzmin, Ivan V; Ellison, James A; Taylor, Steven T; Bergman, David L; Dew, Beverly; Rupprecht, Charles E

2013-10-01

Rabies, an acute progressive encephalomyelitis caused by viruses in the genus Lyssavirus, is one of the oldest known infectious diseases. Although dogs and other carnivores represent the greatest threat to public health as rabies reservoirs, it is commonly accepted that bats are the primary evolutionary hosts of lyssaviruses. Despite early historical documentation of rabies, molecular clock analyses indicate a quite young age of lyssaviruses, which is confusing. For example, the results obtained for partial and complete nucleoprotein gene sequences of rabies viruses (RABV), or for a limited number of glycoprotein gene sequences, indicated that the time of the most recent common ancestor (TMRCA) for current bat RABV diversity in the Americas lies in the seventeenth to eighteenth centuries and might be directly or indirectly associated with the European colonization. Conversely, several other reports demonstrated high genetic similarity between lyssavirus isolates, including RABV, obtained within a time interval of 25-50 years. In the present study, we attempted to re-estimate the age of several North American bat RABV lineages based on the largest set of complete and partial glycoprotein gene sequences compiled to date (n = 201) employing a codon substitution model. Although our results overlap with previous estimates in marginal areas of the 95 % high probability density (HPD), they suggest a longer evolutionary history of American bat RABV lineages (TMRCA at least 732 years, with a 95 % HPD 436-1107 years).

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine.

PubMed

Risatti, G R; Borca, M V; Kutish, G F; Lu, Z; Holinka, L G; French, R A; Tulman, E R; Rock, D L

2005-03-01

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

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

Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins.

PubMed

Albeck, S; Addadi, I; Weiner, S

1996-01-01