The carbohydrate components of arterial basement-membrane-like material. Studies on rabbit aortic myomedial cells in culture.
ABSTRACT: The carbohydrate composition of arterial basement-membrane-like material was investigated. Basement-membrane-like material was isolated from cultures of aortic myomedial cells by a sonication/differential-centrifugation technique. Purified basement-membrane-like material contained a total of 5% sugars, comprising glucose, galactose, mannose, fucose, sialic acid, glucosamine and galactosamine in the approximate molar proportions 3.2:3.5:3.4:3.2:1:5.5:3.1. In addition, small amounts of xylose were found. Analyses for uronic acid showed that glycosaminoglycans comprised about 1% of isolated basement-membrane-like material. The carbohydrate composition indicated the presence of complex-type oligosaccharides in addition to hydroxylysine-linked disaccharides. [3H]Glucosamine-labelled glycopeptides obtained by proteinase digestion and gel filtration were resistant to endo-beta-N-acetylglucosaminidase D, but more than 10% were susceptible to alpha-mannosidase, demonstrating the presence of high-mannose-type oligosaccharides. The distribution of carbohydrates among peptides of basement-membrane-like material on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was investigated after labelling with [3H]mannose, [3H]fucose, [3H]galactose and [3H]glucosamine. Among peptides that appeared to carry carbohydrates were a proteoglycan(s) and seven glycoproteins in the molecular-weight range 120 000-700 000.
Project description:The asparagine-linked oligosaccharides of the complex acidic-type from [3H]mannose-, [3H]glucosamine- or [3H]galactose-labelled membrane glycoproteins of BHK21 cells and Rous-sarcoma virus were analysed by gel filtration combined with extensive digestion with endo- and exo-glycosidases from bacterial and eukaryotic sources. The neutral products from the digestion with a mixture of exoglycosidases and endo-beta-N-acetylglucosaminidase D from Diplococcus pneumoniae included a series of [3H]mannose- and [3H]glucosamine-labelled neutral oligosaccharides that were all converted by digestion with eukaryotic beta-N-acetylglucosaminidases into free N-acetylglucosamine and a small oligomannosyl core containing two alpha-linked mannose residues and a third mannose residue beta-linked to N-acetylglucosamine. These studies suggested that the complex acidic-type oligosaccharides from cellular and viral membrane glycoproteins contained a common oligomannosyl core region (Man2 alpha leads to Man beta leads to GlcNAc2), with heterogeneity in the number and/or linkage of outer branch N-acetylglucosamine residues resulting in partial resistance to beta-N-acetylglucosaminidase from a bacterial source.
Project description:The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.
Project description:Hepatic apolipoprotein (apo) B-100 isolated from human plasma is known to contain N-linked oligosaccharides of high-mannose-type and complex-type structures. Sequencing data have revealed that apo B-48 of small-intestinal origin, which represents about 48% of apo B-100 polypeptide from the N-terminus, possesses six potential sites for N-linked oligosaccharides, of which five are likely to be glycosylated. The characterization of the carbohydrate moiety of apo B-48 is the focus of this study. Apo B-48 was labelled with L-[35S]methionine and D-[3H]glucosamine in organ culture of human small-intestinal explants. N-Glycanase treatment resulted in loss of radioactivity from D-[3H]glucosamine-labelled but not L-[35S]methionine-labelled apo B-48 secreted into the medium, and caused no distinct change in mobility of apo B-48 upon electrophoresis on 5% polyacrylamide gel. Analysis of monosaccharide content revealed the presence of 16.8, 17.8, 13.4, 3.4, 2.4 and 2.3 residues of N-acetylglucosamine, mannose, galactose, fucose, xylose and N-acetylgalactosamine respectively. Small-intestinal apo B-48 from human lymph chylomicrons bound to [14C]concanavalin A, and the binding could be inhibited with methyl alpha-D-mannoside. In addition, wheat-germ, peanut, Limulus, soya-bean and Ulex lectins bound apo B-48 specifically. To characterize the carbohydrate moiety further, N-linked oligosaccharides were released by N-Glycanase treatment and reduced with NaB3H4. Labelled oligosaccharides were separated on a concanavalin A-Sepharose column. The majority (78%) were biantennary complex-type structures, 16% were high-mannose type and 6% (not retained by the column) most probably represented higher-branched oligosaccharides. These results suggest the presence of one high-mannose-type and four biantennary complex-type oligosaccharides, as well as probable O-linked sugars in apo B-48. By the use of h.p.l.c., exoglycosidase treatments and ion-exchange chromatography, a mixture of high-mannose-type species with predominant Man8GlcNAc2 as well as monosialylated, desialylated and fucosylated forms of complex-type oligosaccharides were detected.
Project description:Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of D-[2-3H]mannose, D-[6-3H]galactose, N-acetyl-D-[1-3H]glucosamine and L-[5,6-3H]fucose by 2-15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.
Project description:Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.
Project description:A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.
Project description:Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.
Project description:Incorporations of radioactive mannose, galactose and fucose into MOPC 104E mouse plasma-cell tumour suspensions suggest a stepwise addition of carbohydrate residues to immunoglobulin M (IgM) during the process of secretion. Mannose and glucosamine residues are added at an early stage, whereas galactose and fucose are added just before, or at the time that, IgM leaves the cell. Free light chains secreted in excess by the same tumour cells incubated with mannose, galactose or fucose contained barely detectable amounts of radioactivity.
Project description:The carbohydrate-derived lipoic acid derivatives were studied as protein and cell resistant biomaterials. Six types of carbohydrates were examined for their abilities to reduce nonspecific adsorption of human serum and Hela cell using quartz crystal microbalance. Our data suggested that the structures of carbohydrates play an important role in resisting nonspecific binding. Specifically, the resistance was found to increase in the order lipoic fucose < lipoic mannose < lipoic N-acetyl glucosamine < lipoic glucose < lipoic sialic acid < lipoic galactose, where lipoic galactose derivative resisted most nonspecific adsorption. Furthermore, the combination of lipoic galactose and BSA was the most effective in reducing the adsorption of even undiluted human serum and the attachment of Hela cells while allowing specific binding. Several control experiments have demonstrated that the resistant-ability of mixed lipoic galactose and BSA was comparable to the best known system for decreasing nonspecific adsorption.
Project description:1. Electron microscope autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were both incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. 2. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. 3. Epidermis was separated from dermis using CaCl2, and was extracted with 8 M-urea/5% (w/v) sodium dodecyl sulphate and was then analysed by gel electrophoresis. The major component labelled with D-[3H]glucosamine had an apparent molecular weight in excess of 200 000. This material was not labelled with L-[3H]fucose. Lower molecular-weight components were labelled to a similar extent with both L-[3H]fucose and D-[3H]glucosamine. 4. The high molecular-weight material labelled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was either surface-associated or was extracellular. It was also labelled with D-[14C]glucuronic acid, 35SO4(2-) and to a small extent with 14C-labelled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans. This was confirmed by the fact that it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is therefore only very loosely bound at the cell surface or is present in the extracellular spaces. 5. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent mol.wt. range 70 000--150 000. [3H]Glycoproteins in this molecular-weight range were also major components of a plasma-membrane-enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues, since they were degraded by neuraminidase.