Geng J P, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Thromb Haemost. 1997 May;77(5):926-33.
A chimeric cDNA, encoding residues 1-46 (the gamma-carboxyglutamic acid module and its trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419 of human anticoagulant protein C (PC), was constructed and expressed. The resulting protein, r-[delta GD-HSPC/[symbol: see text] GD-HSfVII]PC, was properly processed with regard to signal/propeptide release, cleavage of the K156R dipeptide, Gla and Hya contents, and the presence of glycosylation. The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to acidic phospholipid vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for the Ca(2+)-induced conformation of the Gla domain (GD) of PC, but did react with another MAb directed in part to the Ca(2+)-dependent conformation of the GD of fVII. Further, this chimeric protein possessed similar steady state constants as wild-type r-PC toward activation by thrombin and thrombin/thrombomodulin. The activated form of the chimera was very similar to that of its wild-type counterpart in its whole plasma anticoagulant activity, as well as its activity toward inactivation of coagulation factor VIII. The chimeric protein did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region of fVII is insufficient for that particular interaction. The results demonstrate that the GD/HS of fVII, when present in the PC and APC background, serves to maintain the Ca2+/PL-related functions of these latter proteins, and suggest that the Ca2+ and PL-dependent interactions of the GD-HS of PC are sufficiently general in nature such that the GD-HS regions of other proteins of this type can satisfy most of the requirements of PC and APC. The data presented also offer support for the independent nature of the domain unit consisting of the GD/HS module.
构建并表达了一种嵌合cDNA，其编码人凝血因子（f）VII的1 - 46位残基（γ-羧基谷氨酸模块及其后续螺旋堆叠），并与抗凝血蛋白C（PC）的47 - 419位残基相连。所得蛋白质r - [δGD - HSPC / [符号：见原文] GD - HSfVII] PC在信号/前肽释放、K156R二肽的切割、Gla和Hya含量以及糖基化存在方面得到了正确处理。该突变蛋白在采用其金属离子依赖性构象以及与酸性磷脂囊泡结合方面对Ca2 +表现出正常依赖性。该嵌合体无法识别针对PC的Gla结构域（GD）的Ca(2 +)诱导构象的单克隆抗体（MAb），但确实与另一种部分针对fVII的GD的Ca(2 +)依赖性构象的MAb发生反应。此外，这种嵌合蛋白与野生型r - PC在凝血酶和凝血酶/血栓调节蛋白激活方面具有相似的稳态常数。该嵌合体的活化形式在其全血浆抗凝血活性以及对凝血因子VIII失活的活性方面与其野生型对应物非常相似。该嵌合蛋白不与fVII辅因子组织因子结合，表明fVII的GD / HS结构域区域不足以进行该特定相互作用。结果表明，当fVII的GD / HS存在于PC和APC背景中时，可维持这些后者蛋白质的Ca2 + / PL相关功能，并表明PC的GD - HS的Ca2 +和PL依赖性相互作用在本质上足够普遍，以至于这种类型的其他蛋白质的GD - HS区域可以满足PC和APC的大多数要求。所呈现的数据也支持由GD / HS模块组成的结构域单元的独立性。
