TRANSFER OF 2 OLIGOSACCHARIDES TO PROTEIN IN A CHINESE-HAMSTER OVARY CELL B211 WHICH UTILIZES POLYPRENOL FOR ITS N-LINKED GLYCOSYLATION INTERMEDIATES

B211, a glycosylation mutant isolated from Chinese hamster ovary cells , synthesizes 10- to 15-fold less Glc(3)Man(9)GlcNAc(2)-P-P-lipid, the substrate used by the oligosaccharide transferase in the synthesis of asparagine-linked glycoproteins. B211 cells are also 10- to 15-fold d eficient in the glucosylation of oligosaccharide-lipid. Despite these properties, protein glycosylation in B211 cells proceeds at a level si milar to (50% of) parental cells, We asked whether the near wild-type level of glycosylation was due to the transfer of alternative oligosac charide structures to protein in B211 cells. The aberrant size of [S-3 5] methionine-labeled VSV G; protein and the increased percentage of e ndoglycosidase II-resistant tryptic peptides as compared to parental c ells supported this hypothesis, B211 cells were labeled with [2-H-3]ma nnose either for 1 min or for 1 h in the presence of glycoprotein-proc essing inhibitors so that the oligosaccharides initially transferred t o protein could be analyzed. In addition to Glc(3)Man(9)GlcNAc(2) a se cond, endoglycosidase Ii-resistant oligosaccharide was transferred who se structure was determined by alpha-mannosidase digestion, gel filtra tion chromatography, and HPLC to be Glc(0,1)Man(5)GlcNAc(2). Finally, since the synthesis of reduced amounts of Glc(3)Man(9)GlcNAc(2)-P-P-li pid was also a phenotype seen in another glycosylation mutant, Lec9, w e analyzed the long-chain prenol in B211 cells. B211 cells synthesized and utilized polyprenol rather than dolichol for all N-linked glycosy lation intermediates as determined by HPLC analysis of [H-3]mevalonate -labeled lipids. Cell fusions analyzed by similar techniques indicated that B211, originally isolated as a concanavalin A-resistant cell lin e, is in the Lec9 complementation group, (C) 1998 Academic Press.