Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells.
Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.
Wellner
R B
RB
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Ghosh
P C
PC
Roecklein
B
B
Wu
H C
HC
D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D005260:Female; D037102:Lectins; D008565:Membrane Proteins / Q000096:biosynthesis*; D008970:Molecular Weight; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D010053:Ovary; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010641:Phenotype; D014316:Tritium
D037102:Lectins; D008565:Membrane Proteins; D009227:Myristic Acids; D009844:Oligosaccharides; D010169:Palmitic Acids; D019814:Myristic Acid; D014316:Tritium; D019308:Palmitic Acid