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 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.
Quellhorst
G J
GJ
Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street,Baltimore, MD 21205, USA.
O'Rear
J L
JL
Cacan
R
R
Verbert
A
A
Krag
S S
SS
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D015870:Gene Expression; D005964:Glucosyltransferases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008358:Mannose / Q000378:metabolism*; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000096:biosynthesis; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014162:Transfection
D009844:Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; D011506:Proteins; C076885:Man(9)(GlcNAc)(2)-diphosphate-dolichol; D005964:Glucosyltransferases; C054676:dolichol phosphate glucose-dolichol diphosphate oligosaccharide glucosyltransferase; D008358:Mannose