Processing of high mannose oligosaccharides to form complex type oligosaccharides on the newly synthesized polypeptides of the vesicular stomatitis virus G protein and the IgG heavy chain.

Abstract

The major lipid-linked oligosaccharide of vesicular stomatitis virus infected-Chinese Hamster ovary ICHO) cells has been isolated and partially characterized. The oligosaccharide moiety has a molecular weight of approximately 2080 as determined by gel filtration. The molecule contains 2 N-acetylglucosamine residues in the form of a N,N’-diacetylchitobiose unit at the reducing end and approximately 10 additional monosaccharide residues, of which a minimum of 6 are mannose residues. Jack bean cY-mannosidase releases 3 mannose residues from the oligosaccharide. Pulse-chase experiments with f3Hlmannose indicate that this lipid-linked oligosaccharide is the immediate precursor of the oligosaccharide units of the viral G protein. Within 3 to 5 min of transfer to the protein, processing of the oligosaccharide begins and by 30 min 3 monosaccharide residues have been excised. This is followed by the rapid removal of 4 more monosaccharides which results in a core with the composition (Man),(GlcNAc),Asn. The sugar residues which comprise the outer branches of the oligosaccharide are then transferred to the molecule. A variant line of CHO cells which is deficient in UDP-GlcNAc:glycoprotein N-acetylglucosaminyltransferase activity appears to have a processing defect in that the oligosaccharide units of the mature viral G protein have the composition (Man),(GlcNAc)2. This suggests that the removal of the final 2 mannose residues during processing may be linked to the particular UDP-GlcNAc glycosyltransferase which transfers N-acetylglucosamine residues to the mannose residues in the oligosaccharide core. Experiments performed with mouse LPCl cells which secrete IgG demonstrated a similar sequence of processing of the oligosaccharide units of this immunoglobulin, indicating that processing is probably a general phenomenon.