Abstract
The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer the oligosaccharide is "processed" by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added. The structure of the oligosaccharide moiety of the lipid-linked precursor has been elucidated in order to further define the steps involved in processing. Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine. Based on endo-beta-N-acetylglucosaminidase CII digestion, alpha-mannosidase digestion, acetolysis, Smith periodate degradation, methylation analysis, and periodate oxidation, we propose the following structure for the oligosaccharide moiety of the lipid-linked oligosaccharide.
Highlights
The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide
“processed” by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added
Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine
Summary
From the Departments of Medicine and Biochemistry, Washington University School of Medicine, St. It is established that the biosynthesis of the asparaginelinked oligosaccharide units of glycoproteins is initiated by the “en bloc” transfer of a preformed oligosaccharide from an oligosaccharide pyrophosphoryldolichol intermediate to an asparagine residue of the nascent polypeptide chain (1) Recent evidence from this laboratory has shown that in virally infected Chinese hamster ovary cells a high molecular weight, mannose-rich, lipid-linked oligosaccharide serves as the immediate precursor of the complex-type oligosaccharide units of the vesicular stomatitis virus G protein and that following transfer to the nascent protein the oligosaccharide is “processed” by the removal of glucose and mannose residues. Its structure was determined by digestions with specific glycosidases, methylation analysis, acetolysis, and Smith periodate degradation with the products being monitored by radioactivity rather than substance Based on these data, a structure for the lipid-linked oligosaccharide is proposed. A similar approach was taken by Spiro et al (5) who reported the partial structure of the lipid-linked oligosaccharide of thyroid tissue
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