A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions.
Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.
Stoll
J
J
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
Rosenwald
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AG
Krag
S S
SS
D000818:Animals; D001216:Asparagine; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004224:Diterpenes / Q000378:metabolism*; D004288:Dolichol Phosphates / Q000378:metabolism; D004286:Dolichols / Q000378:metabolism*; D005260:Female; D006031:Glycosylation; D008798:Mevalonic Acid / Q000378:metabolism; D009154:Mutation; D010053:Ovary; D011105:Polyisoprenyl Phosphate Sugars / Q000096:biosynthesis* / Q000302:isolation & purification; D014316:Tritium
D004224:Diterpenes; D004288:Dolichol Phosphates; D004286:Dolichols; D011105:Polyisoprenyl Phosphate Sugars; D014316:Tritium; D001216:Asparagine; D008798:Mevalonic Acid