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Genetic and Biochemical Study of Phosphatidylserine biosynthesis and its regulation in animal cells

动物细胞磷脂酰丝氨酸生物合成及其调控的遗传与生化研究

基本信息

批准号：
07672411
负责人：
KUGE Osamu
金额：
$ 1.47万
依托单位：
National Institute of Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672411/
关键词：
Phospholipid Phosphatidylserine ホスファチジルセリン

项目摘要

Background : We previously isolated a CHO cell mutant (designated as PSA-3) which requires phosphatidylserine (PS) for cell growth and showed that mutant PSA-3 is strikingly defective in PS biosynthesis. On characterization of mutant PSA-3, it was shown that there are at least two PS synthases (designated as PSS I and II,respectively) in CHO cells and that the mutant PSA-3 is defective in one of the synthases, PSS I.Furthermore, we isolated two different CHO cDNAs (designated as pssA and pssC,respectively) which are capable of conferring PS-prototrophy on mutant PSA-3.Research Results : 1) Using antibodies raised against synthetic pssA peptides, we showed that the pssA gene encodes PSS I.2) The pssC gene was shown to encode a mitochondrial phosphatidylserine decarboxylase that matures through three steps of post-translational processing. 3) A CHO cDNA (designated as pssB) similar in sequence to the pssA cDNA was isolated and shown to encode PSS II.PSS II deduced from the cDNA sequence is a 55kD protein containing nine potential membrane-spanning domains. 4) A CHO cell mutant (designated as #16) defective in both PSS I and II was isolated from mutant PSA-3. The characterization of mutant #16 showed that PSS II catalyzes the conversion of phosphatidylethanolamine to PS.5) We found that PSS I carries a domain which regulates own catalytic activity, and identified an amino acid residues involved in this regulation. 6) In order to purify PSS I and II,we developed methods for overexpression of PSS I and II in insect cells.

背景：我们以前分离到一个CHO细胞突变体(命名为PSA-3)，它需要细胞生长所需的磷脂酰丝氨酸(PS)，并表明突变体PSA-3在PS的生物合成方面存在显著缺陷。在突变体PSA-3的鉴定方面，我们发现在CHO细胞中至少存在两种PS合成酶(分别命名为PSS I和PSS II)，并且突变体PSA-3在其中一种合成酶PSS I中存在缺陷。此外，我们分离到了两种不同的CHO cDNAs(分别命名为PSSA和PSSC)，它们能够赋予突变体PSA-3 PS原生性。研究结果：1)利用合成的PSSA多肽抗体，我们证明了PSSA基因编码PSS I2)PSSC基因编码线粒体磷脂酰丝氨酸脱羧酶，该酶通过三步翻译后加工成熟。3)克隆了一个与PSSA基因序列相似的CHO基因，命名为PsB，编码PSS II。PSS II是一个55kD的蛋白，含有9个潜在的跨膜结构域。4)从突变体PSA-3中分离到一株PSS I和PSS II均有缺陷的CHO细胞突变体(编号#16)。突变体#16的鉴定表明，PSS II催化磷脂酰乙醇胺转化为PS。5)我们发现PSS I带有一个调节自身催化活性的结构域，并确定了参与这一调节的氨基酸残基。6)为了纯化PSS I和PSS II，我们建立了在昆虫细胞中过表达PSS I和PSS II的方法。