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Oligosaccharide Chains (oligosaccharide + chain)
Selected AbstractsTyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin bindingFEBS JOURNAL, Issue 3 2002Christoph Böhme The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with ,2,6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLex motif. Proximal ,1,6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in >,90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively ,2,3-linked N -acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin,Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans. [source] New variants of polar glycopeptidolipids detected in Mycobacterium simiae, including ,habana' strains, as evidenced by electrospray ionization-ion trap-mass spectrometryJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2008L. Mederos Abstract Aims:, To determine the composition of polar glycopeptidolipids (pGPLs) of Mycobacterium simiae and, particularly, those of ,habana' strains, in a search for specific markers given the immunogenic potential of ,habana' TMC 5135 in experimental tuberculosis. Methods and Results:, pGPLs were determined in free lipid extracts using electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS), working in both negative- and positive-ion mode. In the case of TMC 5135, the presence of the previously characterized GPL-II (containing 2,4-di-O-CH3 glucuronic acid as distal sugar in the oligosaccharide antigenic moiety) and GPL-III (containing 4-O-CH3 glucuronic acid as distal sugar) was confirmed using MS/MS and MS/MS/MS approaches. Interestingly, some ,habana' strains presented variants of GPL-II, designated GPL-II,-A and GPL-II,-B. A di-O-CH3 -deoxy-hexose (tentatively, 2,3-di-O-CH3 -fucose) was identified as the penultimate sugar in the oligosaccharide moiety of GPL-II,-A, whereas in GPL-II,-B the penultimate sugar was fucose (tentative identification). On the contrary, the distal sugar of the oligosaccharide chain of pGPLs of Myco. simiae ATCC 25275T was identified as tri-O-CH3 -glucuronic acid (designated GPL-simT -I, with two variants: GPL-simT -I-A and GPL-simT -I-B), O-CH3 -glucuronic acid (designated GPL-simT -II) and di-O-CH3 -glucuronic acid (GPL-II,-A and GPL-II,-B). The penultimate sugar of the oligosaccharide chain of GPL-simT -I-A and GPL-simT -II was identified as di-O-CH3 -deoxy-hexose (tentatively, 2,3-di-O-CH3 fucose), and that of GPL-simT -I-B as deoxy-hexose (tentatively, fucose). In all strains studied, each [M-H], and [M+Na]+ ion was revealed as a mixture of homologous compounds varying in the number of ,O-CH3 groups present in the oligosaccharide moiety and in the length of the fatty acyl linked to the peptide. Conclusions:, The present work indicates that, within a similar general pattern of pGPLs, different strains of Myco. simiae present some variations, so that new compounds (GPL-II,-A, GPL-II,-B, GPL-simT -I-A, GPL-simT -I-B and GPL-simT -II) were defined. Noteworthy was the fact that the ,habana' strains clearly differed from the type strain of Myco. simiae. Significance and Impact of the Study:, The data obtained can be used in the delineation of the ,habana' group of Myco. simiae, including the quality control of the immunogenic strain ,habana' TMC 5135. [source] SEQUENCE AND STRUCTURAL ANALYSIS OF ,-CARRAGEENAN-DERIVED OLIGOSACCHARIDES BY TWO-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE,JOURNAL OF PHYCOLOGY, Issue 4 2010Wei Zhang ,-Carrageenan was hydrolyzed with mild hydrochloric acid and separated into a series of oligosaccharides, the sequences and structures of which were investigated by double-quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), heteronuclear multiple-quantum coherence (HMQC), and heteronuclear multiple-bond correlation (HMBC) techniques, respectively. The chemical structures and conformations of the individual sugar residues were identified, as well as the sequential connectivity of the oligosaccharides. The interresidue nuclear Overhauser effects (NOEs)/rotating frame Overhauser effects (ROEs) revealed an ordered helical structure of the carrageenan oligosaccharide chains. Therefore, a general two-dimensional (2-D) NMR methodology for the unambiguous sequence and structure analysis of ,-carrageenan-derived oligosaccharides was established in this study. [source] Coatings of Low-Density Lipoprotein and Synthetic Glycoconjugates as Substrata for HepatocytesARTIFICIAL ORGANS, Issue 6 2009Hirofumi Yura Abstract Asialoglycoprotein (ASGP) receptors expressed on rat hepatocytes interact with glycoproteins containing galactose or N-acetylgalactosamine residues at the nonreducing termini of oligosaccharide chains to mediate endocytosis, and cholesterol transport protein with apolipoprotein B (LDL, low-density lipoprotein) in plasma interacts with LDL receptors and heparinoids in the extracellular matrix. We developed novel techniques to prepare galactose- and LDL-immobilized culture plates, using galactose-tagged polystyrene (galactose-carrying polystyrene [GalCPS]: N-p-vinylbenzyl-O-,-D-galactopyranosyl-[1,4]-D-gluconamide) and poly(2-acrylamide-2-methyl-1-propanesulfonate) (PAPS), respectively. Hepatocytes adhered well to plates coated with either GalCPS or LDL, and therefore the GalCPS- and LDL-coated plates were examined as specific substrata for culturing hepatocytes. These cultures promoted the formation of three-dimensional, multicellular aggregates with regulation of excess proliferation of non-parenchymal cells. Furthermore, the LDL coating resulted in higher albumin synthesis and an identical level of lactate dehydrogenase (LDH) compared with cells cultured on collagen- and GalCPS-coated plates. Thus, the two culture systems described here, and especially the LDL-coated plates, have potential for the development of a hybrid artificial liver. [source] The heterogeneity of the glycosylation of alpha-1-acid glycoprotein between the sera and synovial fluid in rheumatoid arthritisBIOMEDICAL CHROMATOGRAPHY, Issue 4 2002Kevin D. Smith Alpha-1-acid glycoprotein (AGP) is a plasma glycoprotein produced by the liver that undergoes increased production and altered glycosylation in several physiological and pathological conditions including rheumatoid arthritis. To date, although present in the synovial fluid of rheumatoid arthritis patients, there has been no evidence for the separate extra-hepatic production of AGP. This study indicates that there could be a localized production of AGP in rheumatoid synovial fluid by demonstrating that the glycosylation patterns of AGP differed between the serum and synovial fluid in the same rheumatoid patient. Serum AGP was largely composed of fucosylated tri- and tetra-antennary oligosaccharide chains while the synovial fluid contained mainly bi-antennary chains that were fucosylated to a lesser extent. This structural heterogeneity of glycosylation resulted in functional diversity; serum but not synovial AGP is able to inhibit binding to the cell adhesion molecule E-selectin through expression of antigen sialyl Lewis X. Copyright © 2002 John Wiley & Sons, Ltd. [source] Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture systemBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Christopher K. Crowell Abstract The manufacture of secreted proteins is complicated by the need for both high levels of expression and appropriate processing of the nascent polypeptide. For glycoproteins, such as erythropoietin (EPO), posttranslational processing involves the addition of oligosaccharide chains. We initially noted that a subset of the amino acids present in the cell culture media had become depleted by cellular metabolism during the last harvest cycle in our batch fed system and hypothesized that by supplementing these nutrients we would improve EPO yields. By increasing the concentration of these amino acids we increased recombinant human erythropoietin (rHuEPO) biosynthesis in the last harvest cycle as expected but, surprisingly, we also observed a large increase in the amount of rHuEPO with a relatively low sialic acid content. To understand the nature of this process we isolated and characterized the lower sialylated rHuEPO pool. Decreased sialylation correlated with an increase in N-linked carbohydrates missing terminal galactose moieties, suggesting that ,-1,4-galactosyltransferase may be rate limiting in our system. To test this hypothesis we supplemented our cultures with varying concentrations of manganese (Mn2+), a cofactor for ,-1,4-galactosyltransferase. Consistent with our hypothesis we found that Mn2+ addition improved galactosylation and greatly reduced the amount of rHuEPO in the lower sialylated fraction. Additionally, we found that Mn2+ addition increased carbohydrate site occupancy and narrowed carbohydrate branching to bi-antennary structures in these lower sialylated pools. Surprisingly Mn2+ only had this effect late in the culture process. These data indicate that the addition of Mn2+ has complex effects on stressed batch fed cultures. Biotechnol. Bioeng. 2007;96: 538,549. © 2006 Wiley Periodicals, Inc. [source] Enhancing the secretion of recombinant proteins by engineering N-glycosylation sitesBIOTECHNOLOGY PROGRESS, Issue 5 2009Yan Liu Abstract N - glycosylation is important for the folding and quality control of membrane and secretory proteins. We used mutagenesis to introduce N - glycosylation sequons in recombinant proteins to improve their secretion in HEK293 cells. Seven recombinant proteins, with or without endogenous N - glycosylation sequons, were tested by this method. Our results indicate that N - glycosylation sequons located at the N - or C-terminal are glycosylated at high rates and thus the N - and C-terminal may be convenient sites for effectively attaching oligosaccharide chains. More importantly, introduction of oligosaccharide chains at such positions has been found to improve the secretion levels for the majority of the recombinant proteins in our studies, regardless of endogenous N - glycosylation, presumably by improving their folding in the endoplasmic reticulum. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | |||||||||||||