Kuge O, Saito K, Nishijima M
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan.
J Biol Chem. 1999 Aug 20;274(34):23844-9. doi: 10.1074/jbc.274.34.23844.
Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-overproducing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO-K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a approximately 4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA-directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer-mediated inhibition of PSS II.
中国仓鼠卵巢（CHO）细胞中的磷脂酰丝氨酸（PtdSer）通过磷脂酰丝氨酸合酶（PSS）I和II的作用合成，它们分别催化L-丝氨酸与磷脂酰胆碱和磷脂酰乙醇胺的碱基部分进行交换。在缺乏PSS I但具有正常PSS II活性的CHO细胞突变体PSA-3中，向培养基中添加PtdSer后，PtdSer的合成几乎完全受到抑制，这与野生型CHO-K1细胞中的情况相同。相反，在CHO-K1的PSS II过量表达稳定转化体K1/wt-pssB中，添加PtdSer后PtdSer的合成仅降低了35%。K1/wt-pssB细胞的膜部分中的丝氨酸交换活性完全不受PtdSer的抑制，而PSA-3和CHO-K1细胞的丝氨酸交换活性则受到>95%的抑制。这些结果表明，PSA-3和CHO-K1细胞中的PSS II活性受到外源性PtdSer的抑制，并且PSS II的过量表达导致外源性PtdSer对PSS II活性的正常控制丧失。尽管K1/wt-pssB细胞中过量表达的PSS II不受外源性PtdSer的正常控制，但在无外源性PtdSer的情况下培养的K1/wt-pssB细胞表现出与CHO-K1细胞相似的正常PtdSer生物合成速率。与K1/wt-pssB细胞相反，CHO-K1的另一个稳定转化体K1/R97K-pssB过量表达R97K突变型PSS II，其PtdSer生物合成速率比CHO-K1细胞高约4倍。这些结果表明，为了维持正常的PtdSer生物合成速率，K1/wt-pssB细胞中过量表达的野生型PSS II的活性通过除外源性PtdSer介导的抑制机制之外的尚未知的翻译后机制而降低，并且PSS II的精氨酸97对于过量表达的PSS II活性的这种降低至关重要。当在CHO-K1细胞的PSS I和PSS II缺陷突变体中以不过量表达的水平表达cDNA指导的野生型和R97K突变型PSS II活性时，突变型PSS II活性的表达而非野生型PSS II活性的表达诱导了对PtdSer耐药的PtdSer生物合成。这表明PSS II的精氨酸97对于外源性PtdSer介导的PSS II抑制也至关重要。
