Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain.
Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain.
复制标题
通过 γ-羧基谷氨酸结构域的定点诱变增强人类 C 蛋白功能。
DOI：
10.1074/jbc.273.47.31086
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发表时间：
1998
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;Dahlbäck,B;Nelsestuen,GL
本研究报告的人蛋白C的定点突变体，显示增强的钙和/或膜结合特性的属性。含有S11 G修饰的突变体在饱和钙浓度下均显示出对膜的亲和力增加。Ser-11是人类蛋白C所特有的，而所有其他维生素K依赖性蛋白质都含有甘氨酸。该位点位于蛋白质的紧凑区域，靠近建议的膜接触位点。H10 Q或S12 N的额外变化导致蛋白质具有较低的膜接触钙需求，但在饱和钙时膜亲和力没有进一步增加。突变Q32 E和N33 D本身并不显著改变膜亲和力。这些突变包括在其他突变蛋白中，并可能在一定程度上有助于这些突变体的更高功能。这个突变体家族有助于区分蛋白质-膜结合所必需的事件。这些包括钙结合到游离蛋白质和随后的蛋白质-膜接触。根据所用测定的条件，突变体显示相应的活化蛋白C（APC）衍生物的活性增加。活性增强的程度（高达10倍）取决于测定中使用的磷脂浓度和磷脂质量（±磷脂酰乙醇胺）。这是预期的，因为APC在其膜相关形式中是活跃的，这可以通过蛋白质或磷脂的变化来调节。正如预期的那样，突变体的最大影响发生在低磷脂浓度和磷脂酰乙醇胺的情况下。所有蛋白质的抗凝活性被蛋白S刺激，对增强突变体的影响最大。而血浆含有因子V：R506 Q是部分抵抗所有形式的APC，增强的变体比正常的APC更活跃。具有增强功能的蛋白C变体为凝血研究提供了新的试剂，并可能为生物医学应用提供改进的材料。
This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (± phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.