A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather
than Glc3Man9GlcNAc2-P-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 oligosaccharide-lipid of MI8-5 cells was
analyzed by HPLC and alpha-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 oligosaccharide-lipid. 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 ALG 6, 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.
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