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 deficient in the glucosylation of oligosaccharide-lipid. Despite these properties, protein glycosylation in B211 cells proceeds at a level similar to (50% of) parental cells. We asked whether the near wild-type level of glycosylation was due to the transfer of alternative oligosaccharide structures to protein in B211 cells. The aberrant size of ( 35 S)methionine-labeled VSV G protein and the increased percentage of endoglycosidase H-resistant tryptic peptides as compared to parental cells supported this hypothesis. B211 cells were labeled with (2- 3 H)mannose either for 1 min or for 1 h in the presence of glycoprotein-processing inhibitors so that the oligosaccharides initially transferred to protein could be analyzed. In addition to Glc 3 Man 9 GlcNAc 2 , a second, endoglycosidase H-resistant oligosaccharide was transferred whose structure was determined by α-mannosidase digestion, gel filtration 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-lipid was also a phenotype seen in another glycosylation mutant, Lec9, we analyzed the long-chain prenol in B211 cells. B211 cells synthesized and utilized polyprenol rather than dolichol for all N-linked glycosylation intermediates as determined by HPLC analysis of ( 3 H)mevalonate-labeled lipids. Cell fusions analyzed by similar techniques indicated that B211, originally isolated as a concanavalin A-resistant cell line, is in the Lec9 complementation group.
Archives of Biochemistry and Biophysics – Elsevier
Published: Oct 15, 1998
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