Construction and Characterization of Thrombin-resistant Variants of Recombinant Human Protein S
Construction and Characterization of Thrombin-resistant Variants of Recombinant Human Protein S
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重组人蛋白 S 的凝血酶抗性变体的构建和表征
DOI：
10.1055/s-0038-1648944
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发表时间：
1994
影响因子：
6.7
通讯作者：
B. Bouma
中科院分区：
文献类型：
--
作者：
G. Chang;Leonie Aaldering;T. Hackeng;P. Reitsma;R. Bertina;B. Bouma
蛋白S是一种维生素K依赖性血浆蛋白，在凝血因子Va和Villa的失活中作为活化蛋白C（APC）的辅因子发挥作用。蛋白S在还原SDS聚丙烯酰胺凝胶电泳上以双峰形式迁移。这种分子量的异质性可以用蛋白S的有限蛋白水解来解释。人蛋白S在Arg-49、Arg-60和Arg-70处含有三个潜在的切割位点。切割是否发生在所有三个位点尚不清楚。为了研究这些精氨酸残基在人类蛋白S中的作用，我们用亮氨酸或异亮氨酸取代了它们。构建了所有七种可能的变体：三种具有单突变的变体（R49 L、R60 L、R70 I），三种具有双突变的变体（R49 L/R60 L、R60 L/R70 I、R49 L/R70 I）和一种具有三突变的变体（R49 L/R60 L/R70 I）。在还原型SDS聚丙烯酰胺凝胶上，单和双变体像野生型蛋白S一样以双峰形式迁移。三重变体作为单一条带迁移，分子量对应于双峰的上部条带。单、双变异体的上带可通过凝血酶处理转变为下带，但三变异体的上带不能。所有变体在凝血测定中显示出对APC的辅因子活性。凝血酶处理后，单变体（R49 L、R60 L、R70 I）和双变体（R49 L/R60 L、R60 L/R70 I、R49 L/R70 I）的辅因子活性消失，而在几种浓度下测试的三变体（R49 L/R60 L/R70 I）完全保留了其辅因子活性，表明对凝血酶具有抗性。这表明凝血酶可以在所有三个精氨酸位点处切割，并且在这些位点中的每一个处的切割导致APC辅因子活性的丧失。最后，所有变体以与野生型重组分子相似的亲和力结合C4 b结合蛋白。
Summary Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulation factors Va and Villa. Protein S, migrates as a doublet on reduced SDS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleavage sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants were constructed: three variants with single mutations (R49L, R60L, R70I), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R70I) and one variant with a triple mutation (R49L/R60L/R70I). On reduced SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper band of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by thrombin treatment. All variants showed cofactor activity to APC in a clotting assay. After thrombin treatment, this cofactor activity was abolished for the single (R49L, R60L, R70I) and double variants (R49L/R60L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) tested at several concentrations, retained its cofactor activity completely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity. Finally, all variants bind to C4b-binding protein with an affinity similar as the wild type recombinant molecule.
影响因子：
15.9
作者：
COMP, PC;NIXON, RR;ESMON, CT
通讯作者：
ESMON, CT