Serum glycoprotein-type sequence of monosaccharides in membrane glycoproteins of Semliki Forest virus

Abstract

Semliki Forest virus was grown in BHK-21 cells and labelled in vivo with radioactive monosaccharides. The virus was disrupted with sodium dodecyl sulphate and the polypeptides were hydrolyzed with pronase. A mixture of type A glycopeptides (for nomenclature, see Johnson and Clamp (1971) Biochem. J. 123, 739–745) of the membrane glycoproteins E 1 and E 3 was isolated by gel filtration and subjected to sequential degradation with exo-glycosidases. The reduction in the apparent molecular weight and the cleavage of radioactive monosaccharides were monitored with gel filtration. The results suggest that the type A oligosaccharides have similar average structures and contain at the non-reducing terminus 3.4 mol of α- d-sialic acid and 0.7 mol of α- l-fucose, followed by 3.1 mol of β- d-galactose, 4.2 mol of N- acetyl-β- d-glucosamine , 0.7–1.5 mol of α- d-mannose, 0.5 mol of β- d-mannose and 0.6–2.2 mol of N- acetyl-β- d-glucosamine attached to 1.0 mol of N-acetylglucosamine resistant to N- acetyl-β- d-glucosaminiase . This innermost monosaccharide unit, therefore, appears to be attached to the peptide. The peptides attached to this N-acetyl-glucosamine had an apparent molecular weight of 720 ± 100. We propose the following average structure, compatible with most of our data, for the type A glycopeptides of Semliki Forest virus: ▪