Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10.
Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10.
复制标题
通过诱变增强蛋白 C 的活性以改善膜结合位点：与脯氨酸 10 相关的研究。
DOI：
10.1021/bi971730v
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Nelsestuen,GL
中科院分区：
文献类型：
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作者：
Shen,L;Shah,AM;Dahlback,B;Nelsestuen,GL
牛和人蛋白C在其GLA结构域（氨基末端44个残基）的氨基酸上显示出高度的同源性，尽管人蛋白C的膜亲和力高出约10倍。一项膜接触位点和机制的研究表明，这种差异主要是由于牛蛋白C第10位的脯氨酸与人蛋白C第10位的组氨酸的存在[McDonald， J. F., Shah， A. M., Schwalbe， R. A., Kisiel， W., Dahlback， B.，和Nelsestuen， G. L.（1997）生物化学36,5120−5127]。本研究检测了用组氨酸取代牛蛋白C中的脯氨酸-10的影响，以及人蛋白C的相反变化。在两种情况下，含有脯氨酸-10的蛋白质表现出较低的膜亲和力，牛蛋白C的约低10倍，人蛋白C的低5倍。正如预期的那样，在10号位置含有脯氨酸的活化人蛋白C （hAPC）的活性比野生型hAPC低2.4 - 3.5倍，具体取决于所使用的测定方法。最有趣的是，含有组氨酸-10的牛APC的活性比野生型bAPC高15倍。这证明了通过突变改善膜接触和活性的能力。这一总体策略应该适用于其他依赖维生素k的蛋白质，为研究功能以及生产可能在病理状态下用作血液凝固促进剂和抑制剂的蛋白质提供了机会。
Bovine and human protein C show high homology in the amino acids of their GLA domains (amino-terminal 44 residues), despite the about 10-fold higher membrane affinity of the human protein. A proposed membrane contact site and mechanism suggested that this difference was largely due to the presence of proline at position 10 of bovine protein C versus histidine at position 10 of human protein C [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997)Biochemistry 36, 5120−5127]. This study examined the impact of replacing proline-10 in bovine protein C with histidine, and the reverse change in human protein C. In both cases, the protein containing proline-10 showed lower membrane affinity, about 10-fold lower for bovine protein C and 5-fold lower for human protein C. As expected, activated human protein C (hAPC) containing proline at position 10 showed 2.4−3.5-fold lower activity than wild type hAPC, depending on the assay used. Most interesting was that bovine APC containing histidine-10 displayed up to 15-fold higher activity than wild type bAPC. This demonstrated the ability to improve both membrane contact and activity by mutation. This general strategy should be applicable to other vitamin K-dependent proteins, providing opportunities to study function as well as to produce proteins that may find use as promoters and inhibitors of blood coagulation in pathological states.