Role of cysteine residues in human plasma phospholipid transfer protein.
Role of cysteine residues in human plasma phospholipid transfer protein.
复制标题
半胱氨酸残基在人血浆磷脂转移蛋白中的作用。
DOI：
10.1023/a:1020628006453
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Pownall,HJ
中科院分区：
文献类型：
--
作者：
Qu,SJ;Fan,HZ;Kilinc,C;Pownall,HJ
磷脂转移蛋白（PLTP）属于与小的两亲分子结合的人血浆脂质转移蛋白家族。PLTP在残基5、129、168和318处含有半胱氨酸。杀菌/通透性增加蛋白是同一基因家族的成员，在Cys 135和Cys 175之间含有一个必需的二硫键;这些残基对应于PLTP中的Cys 129和Cys 168，在该基因家族的所有已知成员中是保守的。为了鉴定这些和剩余的半胱氨酸残基对PLTP分泌和活性的重要性，通过定点诱变将每个半胱氨酸残基替换为甘氨酸。将突变体和野生型PLTP cDNA克隆到哺乳动物表达载体pSV·SPORT 1中，转染COS-6细胞进行表达。PLTP Cys 129 → Gly和PLTP Cys 168 → Gly无分泌能力。在细胞裂解物和培养基中均未检测到PLTP质量和活性。相对于野生型PLTP，PLTP Cys 5 → Gly和PLTP Cys 318 → Gly表现出相似的比活性，但部分损害PLTP的合成和分泌。胞内PLTP出现为75和51 kDa的两个条带，对应于糖基化和非糖基化形式的报告分子量。PLTP Cys 5 → Gly和PLTP Cys 318 → Gly的比活性在细胞裂解物和培养基中相似，表明糖基化不影响转移活性。
Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys135and Cys175; these residues, which correspond to Cys129and Cys168in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV·SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys129→ Gly and PLTP Cys168→ Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys5→ Gly and PLTP Cys318→ Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys5→ Gly and PLTP Cys318→ Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.