Molecular cloning and functional characterization of rat plasma protein S.
Molecular cloning and functional characterization of rat plasma protein S.
复制标题
大鼠血浆蛋白S的分子克隆和功能表征。
DOI：
10.1093/jb/117.2.374
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发表时间：
1995
影响因子：
2.7
通讯作者：
K. Suzuki
中科院分区：
文献类型：
--
作者：
F. Yasuda;T. Hayashi;K. Tanitame;J. Nishioka;K. Suzuki
血浆蛋白S是活化蛋白C调节凝血系统的辅因子。从大鼠血浆中纯化的大鼠S蛋白对大鼠APC具有辅因子活性，而对人APC则不具有辅因子活性。用凝胶层析法将大鼠血浆蛋白S分离为游离型和C4BP络合型两种形式。在5 mM Ca~(2+)存在下，大鼠蛋白S与大鼠和人C4BP形成固相络合物，表观解离常数分别为6.7×10~(-8)M和1.2×10~(-8)M。人蛋白S还与人和大鼠C4BP固相结合，KDS分别为6.3×10(-9)和2.7×10(-8)M。人C4BP对人和大鼠S蛋白的APC辅因子活性均有较强的抑制作用，而大鼠C4BP仅有微弱的抑制作用。C4BP的抑制活性程度似乎取决于S蛋白与C4BP的亲和力。为了研究大鼠蛋白S的结构与功能关系，测定了大鼠蛋白S的全长基因序列。该基因全长3,315个碱基，包括一个103个碱基的5‘非编码区，一个2,028个碱基的编码区，编码一个41个氨基酸的前体序列，一个由634个氨基酸组成的成熟蛋白S和一个终止密码子，以及一个1,184个碱基的3’非编码区。大鼠成熟蛋白S由与人蛋白S功能相似的结构域组成，具有两个潜在的天冬氨酸连接的糖基化位点。大鼠成熟蛋白S与人、牛和兔成熟蛋白S的氨基酸序列同源性分别为80.4%、78.7%和79.7%。这些结果表明，尽管大鼠蛋白S的APC辅因子活性具有物种特异性，但它在结构上与人类蛋白S非常相似。RNA印迹分析表明，大鼠蛋白S不仅在肝脏中表达，而且在大鼠的肺、脾、睾丸和子宫中也有表达。
Plasma protein S is a cofactor of activated protein C (APC) in the regulation of the blood coagulation system. Rat protein S homogeneously purified from plasma showed cofactor activity for rat APC, but not for human APC when the APC cofactor activity was assayed using protein S- and C4b-binding protein (C4BP)-depleted human plasma. Rat plasma protein S was separated by gel chromatography into two forms, a free form and a form complexed with C4BP. Rat protein S forms complexes with rat and human C4BP in a solid-phase model with apparent dissociation constants (Kds) of 6.7 x 10(-8) and 1.2 x 10(-8) M, respectively, in the presence of 5 mM Ca2+. Human protein S also forms a complex with solid-phase human and rat C4BP with Kds of 6.3 x 10(-9) and 2.7 x 10(-8) M, respectively. Human C4BP strongly inhibited the APC cofactor activity of both human and rat protein S, whereas rat C4BP was only weakly inhibitory. The degree of the inhibitory activity of C4BP appears to depend on the affinity between protein S and C4BP. In order to evaluate the structure-function relationship of the rat protein S, the complete cDNA sequence of rat protein S was determined. This cDNA of 3,315 bp was composed of a 103-bp 5'-noncoding region, a 2,028-bp coding region that encodes a preprosequence of 41 amino acids, a mature protein S of 634 amino acids and a stop codon, and a 1,184-bp 3'-noncoding region. The rat mature protein S consisted of domains with distinct functions similar to those of human protein S, and with two potential Asn-linked glycosylation sites. The amino acid sequence of the mature form of rat protein S showed 80.4, 78.7, and 79.7% identity with those of human, bovine, and rabbit mature protein S, respectively. These findings suggest that despite the species-specificity of the APC cofactor activity of rat protein S, it is structurally very similar to human protein S. Expression of rat protein S mRNA (approximately 3.5 kb) was demonstrated by RNA blot analysis not only in the liver, but also in the lung, spleen, testis, and uterus of rats.