Cloning of a Chinese hamster ovary (CHO) cDNA encoding phosphatidylserine synthase (PSS) II, overexpression of which suppresses the phosphatidylserine biosynthetic defect of a PSS I-lacking mutant of CHO-K1 cells
Cloning of a Chinese hamster ovary (CHO) cDNA encoding phosphatidylserine synthase (PSS) II, overexpression of which suppresses the phosphatidylserine biosynthetic defect of a PSS I-lacking mutant of CHO-K1 cells
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DOI：
10.1074/jbc.272.31.19133
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发表时间：
1997-08-01
影响因子：
4.8
通讯作者：
Nishijima, M
中科院分区：
文献类型：
--
作者：
Kuge, O;Saito, K;Nishijima, M
哺乳动物细胞中的磷脂酰丝氨酸（PtdSer）是通过将游离L-丝氨酸交换为预先存在的磷脂的极性头基（碱基）来合成的。我们先前显示了在中国仓鼠卵巢（CHO）细胞中存在催化丝氨酸碱基交换的两种不同的酶，并分离了其中一种酶的cDNA，PtdSer合成酶（PSS）I，在本研究中，我们克隆了CHO cDNA，命名为pssB，其编码的蛋白与CHO PSS I具有32%的氨基酸序列同一性。将pssB基因导入CHO-K1细胞后，丝氨酸和乙醇胺的碱基交换活性均显著增加，而pssB基因不能显著增加胆碱的碱基交换活性。发现pssB cDNA可将PtdSer营养缺陷型PSS I缺失突变体转化为PtdSer原养型，在无外源PtdSer的情况下生长2天的所得突变体的PtdSer含量是突变体的4倍，并且与CHO-K1细胞的PtdSer含量相似，表明pssB cDNA补充了PSS I缺失突变体的PtdSer生物合成缺陷，这些结果表明，pssB cDNA编码的第二PtdSer合酶PSS II，催化丝氨酸和乙醇胺碱交换，但不胆碱碱交换。
Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free L-serine for the polar head group (base) of preexisting phospholipid, We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(s) for ethanolamine and choline, In this study, we cloned a CHO cDNA, designated as pssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssB cDNA into CHO-K1 cells resulted in striking increases in both the serine and ethanolamine base exchange activities, In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity, The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy, The PtdSer content of the resultant transformant grown without exogenous PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant, These results suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanol amine base exchange, but not the choline base exchange.