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

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol ratherthan Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediatein N-glycosylation of proteins. MI8-5 cells were incubated with labeledmevalonate, and the prenol was found to be dolichol. The mannose-labeledoligosaccharide released from oligosaccharide-lipid of MI8-5 cells wasanalyzed by HPLC and alpha-mannosidase treatment, and the data wereconsistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells didnot incorporate radioactivity into oligosaccharide- lipid during anincubation with tritiated galactose, again consistent with MI8-5 cellssynthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells hadparental 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 cellswere found to synthesize glucosylated oligosaccharide after they weretransfected with Saccharomyces cerevisiae ALG 6, the gene fordolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cellswere found to incorporate mannose into protein 2-fold slower than parentalcells and to approximately a 2-fold lesser extent.