Stoll J, Rosenwald A G, Krag S S
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
J Biol Chem. 1988 Aug 5;263(22):10774-82.
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.
先前的结果表明，中国仓鼠卵巢细胞的糖基化突变体F2A8相对于亲代细胞，可用于天冬酰胺连接糖基化反应的磷酸多萜醇量较低。通过用[2-³H]甲羟戊酸孵育F2A8、其亲代细胞系B4-2-1和野生型中国仓鼠卵巢细胞24小时，测定了多聚异戊二烯脂质的稳态量和种类。[³H]甲羟戊酸标记程度最高的中性脂质、泛醌、胆固醇和胆固醇酯，在所有三种细胞类型中的标记情况相同。在野生型和B4-2-1细胞中，甲羟戊酸掺入多萜醇的阴离子糖基化和磷酸化衍生物的量比掺入中性游离多萜醇和多萜醇酯的量高10倍。相比之下，在F2A8细胞中，标记物积累在中性多聚异戊二烯醇脂质中，因此中性脂质与阴离子脂质的比例为1:1，而非1:10。在野生型和B4-2-1细胞中，使用硅胶柱的高压液相色谱显示，以游离醇形式以及磷酸化和糖基化形式存在的多聚异戊二烯主要是多萜醇，即具有饱和末端异戊二烯单元的多聚异戊二烯醇。相比之下，在F2A8细胞中，主要以游离醇形式以及磷酸化和糖基化形式存在的多聚异戊二烯似乎是完全不饱和的多聚戊烯醇。使用反相柱的高压液相色谱测定结果表明，F2A8、B4-2-1和野生型细胞中标记多聚异戊二烯醇的链长分布相同，主要链长为19个异戊二烯单元。这些结果与我们先前对F2A8突变体表型的研究相结合，表明不饱和多聚异戊二烯磷酸衍生物在细胞糖基化反应中的功能不如磷酸多萜醇衍生物。
