Document Detail

Transfer of two oligosaccharides to protein in a Chinese hamster ovary cell B211 which utilizes polyprenol for its N-linked glycosylation intermediates.
MedLine Citation:
PMID:  9784244     Owner:  NLM     Status:  MEDLINE    
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.
A Kaiden; A G Rosenwald; R Cacan; A Verbert; S S Krag
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Archives of biochemistry and biophysics     Volume:  358     ISSN:  0003-9861     ISO Abbreviation:  Arch. Biochem. Biophys.     Publication Date:  1998 Oct 
Date Detail:
Created Date:  1998-11-13     Completed Date:  1998-11-13     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0372430     Medline TA:  Arch Biochem Biophys     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  303-12     Citation Subset:  IM    
Copyright Information:
Copyright 1998 Academic Press.
School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland, 21205, USA.
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MeSH Terms
CHO Cells
Dolichol / biosynthesis
Fatty Alcohols / metabolism*
Glucose / metabolism
Lipid Metabolism
Lipids / biosynthesis
Mannose / metabolism
Molecular Sequence Data
Oligosaccharides / metabolism*
Proteins / metabolism*
Grant Support
Reg. No./Substance:
0/Fatty Alcohols; 0/Lipids; 0/Oligosaccharides; 0/Proteins; 10028-17-8/Tritium; 2067-66-5/Dolichol; 31103-86-3/Mannose; 50-99-7/Glucose

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