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

背景资料：我们以前分离的CHO细胞突变体（命名为PSA-3），需要磷脂酰丝氨酸（PS）的细胞生长，并表明突变体PSA-3是惊人的缺陷PS生物合成。对突变体PSA-3的表征表明，至少存在两种PS酶，突变体PSA-3在CHO细胞中具有两种不同的表达载体（分别命名为PSS I和PSS II），并且突变体PSA-3在其中一种磷酸酶PSS I中是缺陷的。（分别命名为pssA和pssC），其能够赋予突变体PSA-3以PS原养型。1）使用针对合成的pssA肽产生的抗体，我们表明pssA基因编码PSS I。2）pssC基因显示编码线粒体磷脂酰丝氨酸脱羧酶，其通过三个翻译后加工步骤成熟。3)从CHO细胞中分离到一个与pssA cDNA序列相似的cDNA（命名为pssB），它编码PSS Ⅱ，由该cDNA序列推断PSS Ⅱ是一个55 kD的蛋白质，含有9个潜在的跨膜结构域。4)从突变体PSA-3中分离出PSS I和II缺陷的CHO细胞突变体（命名为#16）。突变体#16的表征表明PSS II催化磷脂酰乙醇胺转化为PS。5）我们发现PSS I携带一个调节自身催化活性的结构域，并鉴定了一个参与这种调节的氨基酸残基。6)为了纯化PSS I和II，我们开发了在昆虫细胞中过表达PSS I和II的方法。

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.