Carbohydrate structure of vesicular stomatitis virus glycoprotein.

The carbohydrate structure of the membrane glycoprotein of vesicular stomatitis virus (New Jersey serotype) has been determined on material purified from virions grown in spinner cultures of BHKzl cells in the presence of [3H]glucosamine. The intact 3H-glycoprotein contains 6 residues of N-acetyl-n-neuraminic acid, 6 of D-galactose, 6 of D-mannose, 2 of L-fucose, and 10 of N-acetyl-n-glucosamine. Neither D-ghCOSe nor Nacetyl-D-galactosamine was detected. The carbohydrate was not susceptible to fl elimination, and glycopeptides were obtained that yielded 1 mol of aspartic acid/m01 of glycopeptide after acid hydrolysis, both of which indicate that the sugar is linked to the amide nitrogen of asparagine in the glycoprotein. Hydrazinolysis of the glycoprotein yielded a single major oligosaccharide that, after re-N-acetylation, contained 3 residues of N-acetylneuraminic acid, 3 of galactose, 3 of maxmose, 1 of fucose, and 6 of N-acetylglucosamine, suggesting that the glycoprotein contains two identical oligosaccharide chains. The linkages in the hydrazinolysis oligosaccharide were established by methylation analysis. The oligosaccharide was fragmented by partial acid hydrolysis and was specifically depolymerized at de-N-acetylated glucosamine residues by nitrous acid dean&nation, the fragments obtained at each step were purified, and their carbohydrate compositions were determined. A glycopeptide fraction, obtained by digestion of the glycoprotein with trypsin and pronase, was separated on the basis of amino acid differences into two components with identical sugar compositions that are assumed to represent units from the two different sites of glycosylation in the protein. The glycopeptides were subjected to sequential exoglycosidase digestion with a-D-neuraminidase, P-D-galactosidase, F-D-hexosaminidase, a-D-mannosidase, fi-n-mannosidase, and a-Mucosidase. After removal of the N-acetylneuraminic acid, the galactose, and part of the Nacetylglucosamine, the glycopeptides were cleaved with an endo-N-acetyl-/3-D-glucosaminidase. All fragments were reisolated after each digestion step and analyzed for carbohydrate composition or for linkages by methylation analysis. The glycoprotein, glycopeptides, and hydrazinolysis oligosaccharide were oxidized with periodate and subjected to Smith degradation, and the compositions and linkages of the products were determined. The results establish that the glycoprotein contains two identical carbohydrate chains that are composed of three outer chain units, a mannose-rich