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

A CHO mutant MI8-5 was found to synthesize Man(9)-GlcNAc(2)-P-P-dolichol ra ther than Glc(3)Man(9)GlcNAc(2)-P-P-dolichol as the oligosaccharide-lipid i ntermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-l abeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells w as analyzed by HPLC and a-mannosidase treatment, and the data were consiste nt with a structure of Man(9)GlcNAc(2). In addition, MI8-5 cells did not in corporate radioactivity into oligosaccharide-lipid during an incubation wit h tritiated galactose, again consistent with MI8-5 cells synthesizing an un glucosylated oligosaccharide-lipid, MI8-5 cells had parental levels of gluc osylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man(9)GlcNAc(2)-P-P-dolichol glucosyltra nsferase activity. MI8-5 cells were found to synthesize glucosylated oligos accharide after they were transfected with Saccharomyces cerevisiae ALG6, t he gene for dolichol-P-Glc:Man(9)GlcNAc(2)-P-P-dolichol glucosyltransferase . MI8-5 cells mere found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.