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Glycoprotein
Kinds of Glycoprotein Terms modified by Glycoprotein Selected AbstractsAssembly and Formation of Biomorphic Tin Dioxide by a Biomimetic Sol,Gel Approach Involving GlycoproteinEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 16 2007Qun Dong Abstract Three diverse layers of eggshell membrane (ESM) were introduced in a biogenic sol,gel technique for the synthesis of hierarchical SnO2 nanomaterials with corresponding configurations. Typically, the biomorphic replication of the interwoven inner eggshell membrane was systematically investigated by controlling synthesis conditions such as pH value, dipping time, and calcination temperature. The as-prepared SnO2 tubes consisting of interconnected 5-nm nanocrystallite units were successfully interwoven into ESM-morphic films. Herein, the biomaterial ESM served both as the physical substrate and the functional macromolecule template to realize the precision replication, by the interactions between ESM macromolecules (containing carboxyl, hydroxy, amino groups, etc.) and Sn colloid ingredients. Moreover, some biomacromolecules also acted as the surfactant to yield small-scaled and well-distributed SnO2 nanocrystallites based on the strong bondage of short-chained amino acids within ESM glycoprotein with SnO2 nuclei. This technique can be attributed to a biomimetic sol,gel process and is widely applicable to the synthesis of other functional material systems. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] ASARM-truncated MEPE and AC-100 enhance osteogenesis by promoting osteoprogenitor adhesionJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 9 2008Andrew P. Sprowson Abstract Matrix extracellular phosphoglycoprotein (MEPE) is a member of the SIBLING (Small Integrin-Binding Ligand, N-linked Glycoprotein) family of secreted glycophosphoproteins. Several previous studies have demonstrated that MEPE and its peptide motif, AC-100, may regulate bone mass and influence osteoblast activity, suggesting its potential for inclusion in novel therapeutic strategies aimed at increasing osteogenesis. Our study uses in vitro approaches to assess how adhesion of nonadherent cells is influenced by MEPE and whether response to MEPE is dependent on the maturity of osteoblastic cells. Truncated MEPE (ASARM removed) or AC-100 enhanced the adhesion, spreading, and focal complex formation of unadhered osteoblastic cells leading to increased differentiation and bone formation after 28 days of culture. Furthermore, addition of truncated MEPE or AC-100 to mature osteoblasts had no significant effect on bone formation. Our data supports an action for truncated MEPE and AC-100 in altering the physiology of immature poorly adherent cells which subsequently influences the way in which these cells interact with a substrate to facilitate their survival and/or commitment to the osteoblast lineage. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1256,1262, 2008 [source] Platelet glycoprotein VI facilitates experimental lung metastasis in syngenic mouse modelsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2009S. JAIN Summary.,Background:,Glycoprotein (GP)VI is a key receptor for collagen on the platelet surface. It is a member of the immunoglobulin superfamily and is uniquely expressed on the surface of platelets, where it is assembled with the immunoreceptor tyrosine activation motif subunit, FcR-,. We have previously reported the generation of a murine model of GPVI deficiency that revealed profound defects in collagen-induced platelet aggregation and in platelet activation following adhesion to collagen. Beyond the hemostasis/thrombosis paradigm, platelet receptors are emerging as significant participants in tumorigenesis and inflammation. Objective:,In the current study, we have evaluated a role for platelet GPVI in primary tumor growth and experimental metastasis. Methods:,Primary tumor induction and experimental metastasis assays were performed using syngenic immunocompetent animals and tumor cells derived from the C57BL/6J mouse strain in wild-type (C57BL/6J) and N10 C57BL/6J congenic GPVI-deficient mice. Results:,Using either a Lewis lung carcinoma (D121) or melanoma (B16F10.1) cell line, we observed an approximately 50% reduction in the number of visible tumor foci in GPVI-deficient mice as compared with control C57BL/6J mice. Additional studies were performed to compare the size of subcutaneously implanted tumor cells, that is, primary tumor growth. Here, we observed no noticeable size difference when comparing the presence or absence of platelet GPVI. Conclusions:,The results demonstrate that the presence of platelet GPVI facilitates experimental tumor metastasis but does not contribute to the growth of primary tumors. [source] Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific FabsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2009S. M. JUNG Summary.,Background: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. Objectives: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc2) but not to GPVI monomer (GPVIex) through a phage display method. Results: Ssix Fabs were found: B,F, only reactive with GPVI-Fc2, and A, mainly reactive with GPVI-Fc2, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc2 binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc2. Adding the anti-GPVI monoclonal antibody 204-11 increased the Bmax of m-Fab-F binding to GPVI-Fc2, suggesting that 204-11 binds to GPVI-Fc2 molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. Conclusions: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface. [source] Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expressionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2008P. OHLMANN Summary.,Objectives:,Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia-,reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro. The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. Methods and results:,Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg,1) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s,1) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. Conclusion:,The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion. [source] Collagen promotes sustained glycoprotein VI signaling in platelets and cell linesJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2007M. G. TOMLINSON Summary. Background:,Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR,-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective:,To determine why GPVI,FcR, signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. Methods and results:,Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI,FcR, signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. Conclusion:,The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI,FcR, has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery. [source] Uptake/Efflux Transport of Tramadol Enantiomers and O -Desmethyl-Tramadol: Focus on P -GlycoproteinBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2009Mouna Kanaan P -glycoprotein (P -gp) might be of importance in the analgesic and tolerability profile variability of TMD. Our study investigated the involvement of P -gp in the transepithelial transport of (+)-TMD, (,)-TMD and M1, using a Caco-2 cell monolayer model. The bidirectional transport of racemic TMD and M1 (1,100 µM) across the monolayers was investigated at two pH conditions (pH 6.8/7.4 and 7.4/7.4) in the presence and absence of P -gp inhibitor cyclosporine A (10 µM) and assessed with the more potent and specific P -gp inhibitor GF120918 (4 µM). Analytical quantification was performed by liquid chromatography coupled to the fluorescence detector. A net secretion of (+)-TMD, (,)-TMD and M1 was observed when a pH gradient was applied (TR: Papp(B , A)/Papp(A , B): 1.8,2.7; P < 0.05). However, the bidirectional transport of all compounds was equal in the non-gradient system. In the presence of P -gp inhibitors, a slight but significant increase of secretory flux was observed (up to 26%; P < 0.05) at both pH conditions. In conclusion, (+)-TMD, (,)-TMD and M1 are not P -gp substrates. However, proton-based efflux pumps may be involved in limiting the gastrointestinal absorption of TMD enantiomers as well as enhancing TMD enantiomers and M1 renal excretion. A possible involvement of uptake carriers in the transepithelial transport of TMD enantiomers and M1 is suggested. [source] Suspension Culture Process of MethA Tumor Cell for the Production of Heat-Shock Protein Glycoprotein 96: Process Optimization in Spinner FlasksBIOTECHNOLOGY PROGRESS, Issue 6 2007Ya-Jie Tang Heat-shock proteins (HSPs) act like "chaperones", making sure that the cellapos;s proteins are in the right shape and in the right place at the right time. Heat-shock protein glycoprotein 96 (gp96) is a member of the HSP90 protein family, which chaperones a number of molecules in protein folding and transportation. Heat-shock protein gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Currently, heat-shock protein gp96 was only isolated from murine and human tissues and cell lines. An animal cell suspension culture process for the production of heat-shock protein gp96 by MethA tumor cell was developed for the first time in spinner flasks. Effects of culture medium and condition were studied to enhance the MethA tumor cell density and the production and productivity of heat-shock protein gp96. Initial glucose concentration had a significant effect on the heat-shock protein gp96 accumulation, and an initial glucose level of 7.0 g/L was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Cultures at an initial glutamine concentration of 3 and 6 mM were nutritionally limited by glutamine. At an initial glutamine concentration of 6 mM, the maximal viable cell density of 19.90 × 105 cells/mL and the maximal heat-shock protein gp96 production of 4.95 mg/L was obtained. The initial concentration of RPMI 1640 and serum greatly affected the MethA tumor cell culture process. Specifically cultures with lower initial concentration of RPMI 1640 resulted in lower viable cell density and lower heat-shock protein gp96 production. At an initial serum concentration of 8%, the maximal viable cell density of 19.18 × 105 cells/mL and the maximal heat-shock protein gp96 production of 5.67 mg/L was obtained. The spin rate significantly affected the cell culture process in spinner flasks, and a spin rate of 150 rpm was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Not only the cell density but also the production and productivity of heat-shock protein gp96 attained in this work are the highest reported in the culture of MethA tumor cell. This work offers an effective approach for producing heat-shock protein glycoprotein 96 from the cell culture process. The fundamental information obtained in this study may be useful for the efficient production of heat-shock protein by animal cell suspension culture on a large scale. [source] Production of a Secreted Glycoprotein from an Inducible Promoter System in a Perfusion BioreactorBIOTECHNOLOGY PROGRESS, Issue 5 2004Matthew L. Lipscomb The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated. [source] A Review of Clinical Trials with Eptifibatide in CardiologyCARDIOVASCULAR THERAPEUTICS, Issue 4 2007Uwe Zeymer ABSTRACT Glycoprotein (GP) IIb/IIIa receptor antagonists inhibit the binding of ligands to activated platelet GP IIb/IIIa receptors and, therefore, prevent the formation of platelet thrombi. Additional antithrombin therapy should be given in connection with GP IIb/IIIa administration. Eptifibatide is a small heptapeptide, which is highly selective and rapidly dissociates from its receptor after cessation of therapy. In clinical trials (IMPACT-II and ESPRIT) concomitant administration of eptifibatide to patients undergoing percutaneous coronary intervention (PCI) reduced thrombotic complications. In the PURSUIT trial, in patients with non-ST-elevation acute coronary syndromes, eptifibatide, compared to placebo, significantly reduced the primary endpoint of death and nonfatal myocardial infarction at 30 days. In patients with STEMI eptifibatide has been studied as an adjunct to fibrinolysis and primary PCI; it improved epicardial flow and tissue reperfusion. Current studies are evaluating eptifibatide as upstream therapy in high-risk patients with NSTE-ACS, in the EARLY-ACS and in comparison with abciximab in patients with primary PCI in the EVA-AMI trial. [source] Structural Investigation of the HIV-1 Envelope Glycoprotein gp160 Cleavage Site 3: Role of Site-Specific Mutations,CHEMBIOCHEM, Issue 12 2004Lucia Falcigno Dr. Abstract Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date. In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure,activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2,, while sharing the same N-terminal sequence, containing helix-breaking D -amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity. [source] Identification of rat urinary glycoproteome captured by three lectins using gel and LC-based proteomicsELECTROPHORESIS, Issue 21 2008Pyong-Gon Moon Abstract Many different types of urine proteome studies have been done, but urine glycoprotein studies are insufficient. Therefore, we studied the glycoproteins from rat urine, which could be used to identify biomarkers in an animal model. First, urinary proteins were prepared by using the dialysis and lyophilizing methods from rat urine. Glycoproteins enriched with lectin affinity purification, concanavalin A, jacalin and wheat germ agglutinin from the urinary proteins were separated by means of reverse-phase fast protein LC (FPLC) or 1-D PAGE. Each FPLC fraction and 1-D PAGE gel band were trypsin-digested and analyzed by means of nanoLC-MS/MS. LC-MS/MS analyses were carried out by using linear ion trap MS. A total of 318 rat urinary glycoproteins were identified from the FPLC fractions and gel bands; approximately 90% of identified proteins were confirmed as glycoproteins in Swiss-Prot. Many glycoproteins, known as biomarkers, including C-reactive protein, uromodulin, amyloid beta A4 protein, alpha-1-inhibitor 3, vitamin D-binding protein, kallikrein 3 and fetuin-A were identified in this study. By studying urinary glycoproteins collected from rat, these results may help to assist in identifying urinary biomarkers regarding various types of disease models. [source] STRUCTURE, COMPOSITION AND BIOGENESIS OF PRASINOPHYTE SCALESJOURNAL OF PHYCOLOGY, Issue 2000B. Becker The cell body and flagellar surfaces of prasinophytes are covered by non-mineralyzed scales. Scales consist mainly of acidic polysaccharides containing large amounts of 2-keto sugar acids. Glycoproteins are minor components and probably mainly involved in mediating scale-subunit and scale-membrane interactions. In thecate prasinophytes the cell body scales coalesce to form a rigid cell wall, generally known as a theca. We have studied the polysaccharides and glycoproteins of the thecate prasinophytes Tetraselmis striata and Scherffelia dubia over the last years. New results regarding the structure of carbohydrates and proteins will be presented. [source] Aspartic Proteinase Members Secreted by the Ruminant Placenta: Specificity of Three Radioimmunoassay Systems for the Measurement of Pregnancy-associated GlycoproteinsREPRODUCTION IN DOMESTIC ANIMALS, Issue 6 2002ZS Perényi Contents Pregnancy-associated glycoproteins (PAGs) isolated from the placenta of various ruminant species are enzymatically inactive members of the aspartic proteinase family. The measurement of these proteins in the maternal blood can be a good indicator of the presence of a live embryo. As certain aspartic proteinases are present in biological fluids in physiological and pathological conditions at various concentrations, it was necessary to determine the specificity of three radioimmunoassay (RIA) systems currently used for the detection of PAG molecules. Commercially available members of the aspartic proteinase family like pepsinogen, pepsin, chymosin, rennet, cathepsin D and renin were tested in a wide concentration range (10 ng/ml , 1 mg/ml). Pepsinogen cross-reacted in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 50 ,g/ml and 500 ,g/ml concentrations, respectively. In the presence of pepsin, cross-reaction was observed in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 500 ,g/ml and 1 mg/ml concentrations, respectively. Chymosin and rennet could cross-react in RIA 2 and RIA 3, while renin and cathepsin D did not decrease the binding of the tracer to antisera more, than that of the minimal detection limit. As the plasma/serum concentrations of the examined aspartic proteinases reported in the literature were outside the concentration range where cross-reaction was observed, it can be concluded that these RIA systems were specific for the detection of PAGs in biological fluids. [source] Structure of N-glycosidic Carbohydrates of Secretory Proteins of Tetrahymena thermophilaTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2003BURKHARD BECKER ABSTRACT. Glycoproteins secreted by Tetrahymena into the culture medium were isolated and the N-glycosidic oligosaccharides analyzed using lectin blots and fluorophore-assisted carbohydrate gel electrophoresis (FACE). Lectin blots showed that the glycoproteins secreted by Tetrahymena contain only N-glycosidic structures of the high mannose type. Further analysis using the FACE technology revealed the presence of four different N-glycosidic structures differing only in the number of mannose residues attached to the core chitobiose unit. [source] Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoproteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Jeffrey E. Lee The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature (London), 454, 177,182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B -value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions. [source] Identification of genes encoding N -glycan processing ,- N -acetylglucosaminidases in Trichoplusia ni and Bombyx mori: Implications for glycoengineering of baculovirus expression systemsBIOTECHNOLOGY PROGRESS, Issue 1 2010Christoph Geisler Abstract Glycoproteins produced by non-engineered insects or insect cell lines characteristically bear truncated, paucimannose N -glycans in place of the complex N -glycans produced by mammalian cells. A key reason for this difference is the presence of a highly specific N -glycan processing ,- N -acetylglucosaminidase in insect, but not in mammalian systems. Thus, reducing or abolishing this enzyme could enhance the ability of glycoengineered insects or insect cell lines to produce complex N -glycans. Of the three insect species routinely used for recombinant glycoprotein production, the processing ,- N -acetylglucosaminidase gene has been isolated only from Spodoptera frugiperda. Thus, the purpose of this study was to isolate and characterize the genes encoding this important processing enzyme from the other two species, Bombyx mori and Trichoplusia ni. Bioinformatic analyses of putative processing ,- N -acetylglucosaminidase genes isolated from these two species indicated that each encoded a product that was, indeed, more similar to processing ,- N -acetylglucosaminidases than degradative or chitinolytic ,- N -acetylglucosaminidases. In addition, over-expression of each of these genes induced an enzyme activity with the substrate specificity characteristic of processing, but not degradative or chitinolytic enzymes. Together, these results demonstrated that the processing ,- N -acetylglucosaminidase genes had been successfully isolated from Trichoplusia ni and Bombyx mori. The identification of these genes has the potential to facilitate further glycoengineering of baculovirus-insect cell expression systems for the production of glycosylated proteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Chemoselective Labeling of Engineered Fucosylated GlycoproteinsCHEMBIOCHEM, Issue 15 2007Christian P. R. Hackenberger Dr. O fucose, where art thou? Metabolic oligosaccharide engineering in combination with chemoselective conjugation of biophysical labels has enabled researchers to selectively target and visualize fucose residues in cellular glycoprotein systems. These recent studies advance the chemical reporter strategy for the functional analysis of this important protein modification in a proteomic context. [source] Glycoproteins of drusen and drusen-like lesionsACTA OPHTHALMOLOGICA, Issue 2007RE BONSHEK Purpose: Drusen are a marker of age-related macular degeneration. Lesions similar to drusen, both in histology and their clinical appearance are also seen in choroidal tumours, chronic inflammatory and degenerative conditions of the eye, and in mesangiocapillary glomerulonephritis type II (MCGN-II). This study aims to compare the saccharide composition of these drusen-like lesions in the various ocular pathological groups and in MCGN-II. Methods: Formalin fixed and paraffin wax embedded tissue from 21 eyes was studied. The histological diagnoses included AMD, retinal detachment, malignant melanoma, long-standing uveitis, glaucoma and MCGN II. Glycosylation was examined using a panel of twenty biotinylated lectins and an avidin-peroxidase-DAB-cobalt revealing system, with and without neuraminidase pre-treatment. Results: High mannose, bi/tri-nonbisected and bisected complex N-glycan, N-acetyl glucosaminyl, galactosyl and sialyl residues were found to be expressed by drusen, while treatment with neuraminidase exposed subterminal N-acetyl galactosamine and galactosyl residues. Similar binding patterns were found in the various pathological groups studied. Conclusions: As there was no significant difference in the lectin-binding pattern in drusen in different pathologies, a common pathogenesis or at least a final common pathway for the elaboration of carbohydrate components of drusen is suggested. [source] Boronic Acid Functionalized Core,Satellite Composite Nanoparticles for Advanced Enrichment of Glycopeptides and GlycoproteinsCHEMISTRY - A EUROPEAN JOURNAL, Issue 39 2009Lijuan Zhang Abstract A core,satellite-structured composite material has been successfully synthesized for capturing glycosylated peptides or proteins. This novel hybrid material is composed of a silica-coated ferrite "core" and numerous "satellites" of gold nanoparticles with lots of "anchors". The anchor, 3-aminophenylboronic acid, designed for capturing target molecules, is highly specific toward glycosylated species. The long organic chains bridging the gold surface and the anchors could reduce the steric hindrance among the bound molecules and suppress nonspecific bindings. Due to the excellent structure of the current material, the trap-and-release enrichment of glycosylated samples is quite simple, specific, and effective. Indeed, the composite nanoparticles could be used for enriching glycosylated peptides and proteins with very low concentrations, and the enriched samples can be easily separated from bulk solution by a magnet. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6,% separately, whereas the adsorption capacity of the composite nanoparticles was proven to be more than 79,mg of glycoproteins per gram of the material. Moreover, the new composite nanoparticles were applied to enrich glycosylated proteins from human colorectal cancer tissues for identification of N-glycosylation sites. In all, 194 unique glycosylation sites mapped to 155 different glycoproteins have been identified, of which 165 sites (85.1,%) were newly identified. [source] Differentiation inducing factors in Dictyostelium discoideum: A novel low molecular factor functions at an early stage(s) of differentiationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2009Akiko A. Oohata There are reports that secreted factor(s) are involved in prespore cell differentiation in Dictyostelium discoideum, but the structures and functions of the various factors have not been elucidated. Previously, we described two prespore cell-inducing factors in conditioned medium; one was a glycoprotein named prespore cell-inducing factor (, factor, or PSI-1), and the other, a heat stable dialyzable factor(s). In the present paper, we purified and characterized the most potent prespore cell-inducing activity in dialysates. The factor began to be secreted after the onset of starvation and stopped being secreted once the cells had aggregated, which was earlier than the onset of the , factor gene expression. In addition, unlike , factor, its secretion did not appear to depend on activation of protein kinase A. Interestingly, the purified factor not only induced prespore cell specific genes such as pspA and cotC but also a prestalk-cell specific gene, ecmB in vitro. The purified factor is tentatively designated polyketide-like factor (PLF), because it seems to be a novel polyketide with 208 Da. Half maximal induction of prespore cell was obtained with 26 nmol/L of PLF. We propose that PLF plays a key role in the acquisition of differentiation commitment, before the , factor induces specifically prespore cell differentiation. [source] Expression of Gpr177, a Wnt trafficking regulator, in mouse embryogenesisDEVELOPMENTAL DYNAMICS, Issue 7 2010Hsiao-Man Ivy Yu Abstract Wls/Evi/Srt encoding a multipass transmembrane protein has been identified as a regulator for proper sorting and secretion of Wnt in flies. We have previously demonstrated that Gpr177 is the mouse ortholog required for axis determination. Gpr177 is a transcriptional target of Wnt that is activated to assist its subcellular distribution in a feedback regulatory loop. We, therefore, proposed that reciprocal regulation of Wnt and Gpr177 is essential for the Wnt-dependent developmental and pathogenic processes. Here, we examine the expression pattern of Gpr177 in mouse development. Gpr177 is expressed in a variety of tissues and cell types during organogenesis. Furthermore, Gpr177 is a glycoprotein primarily accumulating in the Golgi apparatus in signal-producing cells. The glycosylation of Gpr177 is necessary for proper transportation in the secretory pathway. Our findings suggest that the Gpr177-mediated regulation of Wnt is crucial for organogenesis in health and disease. Developmental Dynamics 239:2102,2109, 2010. © 2010 Wiley-Liss, Inc. [source] Polarized expression of integrin ,1 in diencephalic roof plate during chick development, a possible receptor for SCO-spondinDEVELOPMENTAL DYNAMICS, Issue 10 2009Teresa Caprile Abstract The roof plate of the caudal diencephalon is formed by the posterior commissure (PC) and the underlying secretory ependyma, the subcommissural organ (SCO). The SCO is composed by radial glial cells bearing processes that cross the PC and attach to the meningeal basement membrane. Since early development, the SCO synthesizes SCO-spondin, a glycoprotein that shares similarities to axonal guidance proteins. In vitro, SCO-spondin promotes neuritic outgrowth through a mechanism mediated by integrin ,1. However, the secretion of SCO-spondin toward the extracellular matrix that surrounds the PC axons and the expression of integrins throughout PC development have not been addressed. Here we provide immunohistochemical evidence to suggest that during chick development SCO cells secrete SCO-spondin through their basal domain, where it is deposited into the extracellular matrix in close contact with axons of the PC that express integrin ,1. Our results suggest that SCO-spondin has a role in the development of the PC through its interaction with integrin ,1. Developmental Dynamics 238:2494,2504, 2009. © 2009 Wiley-Liss, Inc. [source] Fjx1: A notch-inducible secreted ligand with specific binding sites in developing mouse embryos and adult brainDEVELOPMENTAL DYNAMICS, Issue 3 2005Rebecca Rock Abstract The mouse fjx1 gene was identified as a homologue to the Drosophila gene four-jointed (fj). Fj encodes a transmembrane type II glycoprotein that is partially secreted. The gene was found to be a downstream target of the Notch signaling pathway in leg segmentation and planar cell polarity processes during eye development of Drosophila. Here, we show that fjx1 is not only conserved in vertebrates, but we also identified the murine fjx1 gene as a direct target of Notch signaling. In addition to the previously described expression of fjx1 in mouse brain, we show here that fjx1 is expressed in the peripheral nervous system, epithelial cells of multiple organs, and during limb development. The protein is processed and secreted as a presumptive ligand. Through the use of an fjx1-AP fusion protein, we could visualize fjx1 binding sites at complementary locations, supporting the notion that fjx1 may function as a novel signaling molecule. Developmental Dynamics 234:602,612, 2005. © 2005 Wiley-Liss, Inc. [source] Intrastriatal administration of human immunodeficiency virus-1 glycoprotein 120 reduces glial cell-line derived neurotrophic factor levels and causes apoptosis in the substantia nigraDEVELOPMENTAL NEUROBIOLOGY, Issue 12 2006Rachel L. Nosheny Abstract Uninfected neurons of the substantia nigra (SN) degenerate in human immunodeficiency virus (HIV)-positive patients through an unknown etiology. The HIV envelope glycoprotein 120 (gp120) causes apoptotic neuronal cell death in the rodent striatum, but its primary neurotoxic mechanism is still under investigation. Previous studies have shown that gp120 causes neurotoxicity in the rat striatum by reducing brain-derived neurotrophic factor (BDNF). Because glial cell line-derived neurotrophic factor (GDNF) and BDNF are neurotrophic factors crucial for the survival of dopaminergic neurons of the SN, we investigated whether gp120 reduces GDNF and BDNF levels concomitantly to induce apoptosis. Rats received a microinjection of gp120 or vehicle into the striatum and were sacrificed at various time intervals. GDNF but not BDNF immunoreactivity was decreased in the SN by 4 days in gp120-treated rats. In these animals, a significant increase in the number of caspase-3- positive neurons, both tyrosine hydroxylase (TH)-positive and -negative, was observed. Analysis of TH immunoreactivity revealed fewer TH-positive neurons and fibers in a medial and lateral portion of cell group A9 of the SN, an area that projects to the striatum, suggesting that gp120 induces retrograde degeneration of nigrostriatal neurons. We propose that dysfunction of the nigrostriatal dopaminergic system associated with HIV may be caused by a reduction of neurotrophic factor expression by gp120. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Plasma IL-6 concentration is inversely related to insulin sensitivity, and acute-phase proteins associate with glucose and lipid metabolism in healthy subjectsDIABETES OBESITY & METABOLISM, Issue 6 2005M. K. Heliövaara Aim:, It has been shown that atherosclerosis is an inflammatory disease. Recent data suggest that inflammation precedes type 2 diabetes. Hence, we wanted to study the interrelationship between IL-6, insulin sensitivity, lipids and numerous acute-phase proteins. Methods:, Twenty-one healthy individuals [16 males/5 females, age 27.9 ± 1.8 years, body mass index (BMI) 24.1 ± 0.8 kg/m2] participated in the study. Each patient went through a 4-h hyperinsulinaemic (40 mU/m2/min) euglycaemic clamp and 4-h saline infusion. Blood samples were taken before and at the end of the infusions. Results:, Plasma interleukin (IL)-6 concentration correlated inversely with insulin sensitivity (M -value) (r = ,0.49, p < 0.05). Moreover, the plasma levels of IL-6 associated with c-peptide (r = 0.49, p < 0.05), fat% (r = 0.43, p < 0.05) and diastolic blood pressure (r = 0.46, p < 0.05). ,-1-acid glycoprotein was related to HbA1c (r = 0.47, p < 0.05), insulin (r = 0.55, p < 0.01), diastolic blood pressure (r = 0.58, p < 0.01), systolic blood pressure (r = 0.58, p < 0.01) and triglycerides (r = 0.58, p < 0.01). Haptoglobin was correlated with insulin (r = 0.46, p < 0.05), total cholesterol (r = 0.61, p < 0.01), BMI (r = 0.58, p < 0.01), fat% (r = 0.63, p < 0.01) and lipid oxidation during clamp (r = 0.43, p < 0.05). Diastolic blood pressure decreased during the clamp (from 78.3 ± 1.9 to 72.1 ± 2.0 mmHg, p = 0.001). Insulin infusion did not affect the serum levels of most acute-phase proteins. Conclusions:, Our study suggests that low grade inflammation, as reflected by IL-6, A1GP and haptoglobin contributes to the regulation of insulin sensitivity, lipid metabolism and blood pressure in normal human physiology. [source] Association of erosive esophagitis with Helicobacter pylori eradication: a role of salivary bicarbonate and glycoprotein secretionDISEASES OF THE ESOPHAGUS, Issue 4 2009D. B. Namiot SUMMARY In some populations, Helicobacter pylori eradication is associated with development of erosive esophagitis. The aim of this study was to evaluate the contribution of salivary bicarbonate and glycoprotein secretion to the pathogenesis of erosive esophagitis developing after H. pylori eradication. Gastroscopy and saliva collection were performed at recruitment and 12 months after completion of eradication therapy. Eighty-eight patients with duodenal ulcer were recruited to the study. Erosive esophagitis was found in 13 patients (grade A, 8 patients; grade B, 4 patients; grade C, 1 patient). Among the 74 subjects who completed the study, erosive esophagitis was detected in 21 patients (grade A, 15 patients; grade B, 6 patients); they all were successfully eradicated. Bicarbonate and glycoprotein secretion was not found to differ significantly between the subjects with and without erosive esophagitis both before and 1 year after H. pylori eradication. However, it was lower in H. pylori -infected (baseline) than in H. pylori -noninfected erosive esophagitis subjects (1 year after successful eradication) (bicarbonate 2.34 [1.29,3.40)]vs. 3.64 [2.70,4.58]µmol/min and glycoprotein 0.23 [0.15,0.31]vs. 0.35 [0.28,0.43] mg/min, P= 0.04 and P= 0.04, respectively). We conclude that changes in salivary bicarbonate and glycoprotein secretion related to H. pylori eradication do not promote the development of erosive esophagitis in duodenal ulcer patients. [source] Effect of sialic acid content on glycoprotein pI analyzed by two-dimensional electrophoresisELECTROPHORESIS, Issue 17 2010Sķlvia Barrabés Abstract 2-DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, ,1-antitrypsin and ribonuclease 1), which N -glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule. [source] Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluationELECTROPHORESIS, Issue 16 2010Gian Maria D'Amici Abstract Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate®, Helixate NexGen® and Refacto®), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen® showed the presence of impurities, such as Hsp70,kDa, haptoglobin and proapolipoprotein; Refacto® showed glutathione S -transferase and ,-lactamase, whereas Advate® apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants. [source] Lectin-based electrophoretic analysis of the expression of the 35,kDa inter-,-trypsin inhibitor heavy chain H4 fragment in sera of patients with five different malignanciesELECTROPHORESIS, Issue 12 2008Emida Mohamed Abstract A 35,kDa glycoprotein whose abundance was previously demonstrated to be enhanced in sera of patients with endometrial adenocarcinoma (n,=,12), was isolated from pooled sera of three of the cancer patients using champedak galactose-binding lectin affinity chromatography in the present study. Subjecting it to 2-DE and MS/MS, the glycoprotein was identified as the O -glycosylated fragment of inter-,-trypsin inhibitor heavy chain H4 (ITIH4). When compared to control sera (n,=,17), expression of the 35,kDa ITIH4 cleavage fragment was demonstrated to be significantly enhanced in sera of patients with breast carcinoma (n,=,10), epithelial ovarian carcinoma (n,=,10), and germ cell ovarian carcinoma (n,=,10) but not in patients with nasopharyngeal carcinoma (n,=,13) and osteosarcoma (n,=,7). The lectin-based electrophoretic bioanalytical method adopted in the present study may be used to assess the physiological relevance of ITIH4 fragmentation and its correlation with different malignancies, their stages and progression. [source] | ||||||||||||||||||||||||||||||||||||||||