Journal Article

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

George J. Quellhorst, Jessica L. O'Rear, René Cacan, André Verbert and Sharon S. Krag

in Glycobiology

Published on behalf of Society for Glycobiology

Volume 9, issue 1, pages 65-72
Published in print January 1999 | ISSN: 0959-6658
Published online January 1999 | e-ISSN: 1460-2423 | DOI: https://dx.doi.org/10.1093/glycob/9.1.65
Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

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A CHO mutant MI8-5 was found to synthesize Man9GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharidelipid of MI8-5 cells was analyzed by HPLC and α-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide-lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharidelipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.

Keywords: ALG6; CHO cells; glucosyl transferase; N-linked glycosylation

Journal Article.  4617 words.  Illustrated.

Subjects: Carbohydrates

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