Transfer of two 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 Glc3Man9GlcNAc2-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 [35S]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 [2-3H]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 Glc3Man9GlcNAc2, a second, endoglycosidase H-resistant oligosaccharide was transferred whose structure was determined by alpha-mannosidase digestion, gel filtration chromatography, and HPLC to be Glc0,1Man5GlcNAc2. Finally, since the synthesis of reduced amounts of Glc3Man9GlcNAc2-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 [3H]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.
Kaiden
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A
School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland, 21205, USA.
Rosenwald
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AG
Cacan
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R
Verbert
A
A
Krag
S S
SS
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004286:Dolichols / Q000096:biosynthesis; D005233:Fatty Alcohols / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D050356:Lipid Metabolism; D008055:Lipids / Q000096:biosynthesis; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D014316:Tritium
D004286:Dolichols; D005233:Fatty Alcohols; D008055:Lipids; D009844:Oligosaccharides; D011506:Proteins; D014316:Tritium; D005947:Glucose; D008358:Mannose