Size Of Oligosaccharides Research Articles

The effect of oligosaccharide chains of different sizes on the maturation and physical properties of the G protein of vesicular stomatitis virus.

The envelope glycoprotein, G, of vesicular stomatitis virus (VSV) is initially glycosylated by the en bloc transfer of Glc3Man9GlcNAc2 oligosaccharides to 2 specific asparagine residues in the nascent polypeptide chain. We carried out in vivo and in vitro studies to determine whether the size of the oligosaccharide chains on two related but different G proteins can affect their ability to fold correctly. For the in vivo studies we used a mutant lymphoma cell line, Thy-1-e, which transfers the truncated oligosaccharide, Glc3Man5GlcNAc2, to nascent polypeptides. The growth of VSV in these cells was temperature-sensitive compared to that in parental Thy-1+ cells, and VSV (San Juan) was more affected than VSV (Orsay). These results are congruous with our previous observation that in the absence of glycosylation virus assembly is temperature-sensitive and VSV (San Juan) is inhibited more than VSV (Orsay). To examine the effect of oligosaccharide size on the properties of the G protein in vitro we treated G proteins containing either Man8GlcNAc2 or Man5GlcNAc2 oligosaccharide chains with guanidine hydrochloride and measured their ability to refold using an in vitro aggregation assay. The San Juan G protein with Man5GlcNAc2 oligosaccharides aggregated at 40 degrees C but not at 30 degrees C. The Orsay G protein with Man5GlcNAc2 oligosaccharides and both proteins containing Man8GlcNAc2 oligosaccharides did not aggregate at either temperature. We conclude that the size of the oligosaccharides present on the folding G protein can be crucial in attaining a proper conformation, and the extent of their effect depends on the primary structure of the polypeptide.

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-d-[6-(3)H] glucosamine and guanosine-5'diphospho-d-[U-(14)C]mannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations.Tissue slices from cotyledons at the same stage of development synthesize lipid-linked oligosaccharides from [(3)H]mannose and [(3)H]glucosamine with sizes equivalent to 1, 7, 10, and approximately 15 saccharide units. In pulse-chase experiments, the lipid-linked saccharides with the equivalent of 1 and 10 units rapidly turnover, whereas those with 7 and 15 units do not. Examination of the higher oligosaccharide peaks (10 and 15) by Bio-Gel P-4 gel filtration shows them to comprise 2 distinct subsets of oligosaccharides containing different proportions of glucosamine and mannose units. Tissue slices synthesize products which resemble the completed 7S storage glycoproteins as judged by similarity of molecular weight and precipitation with specific antisera. Analysis of the oligosaccharides obtained by hydrazinolysis of glycoproteins shows the presence of a similar size "high-mannose" type N-linked oligosaccharides as in other glycoproteins from animal and plant cells.

Effects of UDP-glucose addition on the synthesis of mannosyl lipid-linked oligosaccharides by cell-free fibroblast preparations.

The pattern of mannosyl lipid-linked oligosaccharides synthesized by cell-free enzyme preparations from cultured fibroblasts is altered substantially when 0.2 muM UDP-glucose is added to the incubation medium. Inclusion of UDP-glucose results in the appearance of a new labeled oligosaccharide, which is 1 or 2 glycose units larger than the lipid-linked oligosaccharide synthesized in the presence of only GDP-mannose (2 muM) and UDP-N-acetylglucosamine (20 muM). Label from UDP-[3H]glucose is incorporated into the same larger oligosaccharide size class. The results can be explained most easily by assuming that the new mannosyl lipid-linked oligosaccharide contains 1 or 2 glucose residues in addition to 5 to 6 mannose residues. The results are compatible with the recent finding by Spiro et al. (Spiro, M.J., Spiro, R.G., and Bhoyroo, V.D. (1976) Fed Proc. 35, 1375; Spiro M.J. Spiro, R.G., and Bhoyroo, V.D. (1976) J. Biol. Chem. 251, 6400-6408; Spiro, R.G., and Spiro, M.J. AND Bhoyroo, V.D. (1976) J. Biol. Chem. 251, 6409-6419; Spiro, M.J., Spiro R.G., and Bhoyroo, V.D. (1976) J. Biol. Chem. 251, 6420-6425) that naturally occurring mannosyl lipid-linked oligosaccharides contain glucose. In addition to being incorporated into lipid oligosaccharides, glucose residues are also incorporated into endogenous glycoproteins. Incorporation of glucose into glycoproteins that give rise to pronase glycopeptides of the typical asparagine-linked size classes is almost completely dependent on the presence of GDP-mannose.