The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X
The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X
复制标题
蛋白酶结构域中 Cys22 和 Cys27 之间的二硫键调节凝血因子 X 的凝血活性
DOI：
10.1055/s-0039-1683442
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发表时间：
2019-06-01
影响因子：
6.7
通讯作者：
Wu, Wenman
中科院分区：
文献类型：
--
作者：
Li, Fang;Chen, Changming;Wu, Wenman
凝血因子X（FX）蛋白酶结构域的Cys22 - Cys27二硫键在各凝血因子中并不保守，其对生理性止血的作用以及在止血和血栓形成相关疾病发病机制中的意义仍有待阐明。在一个先天性FX缺乏症家系中鉴定出了p.Cys27Ser突变，对瞬时转染的HEK293细胞进行的荧光标记研究显示，FX p.Cys27Ser在细胞内积聚，这表明分泌功能异常是导致FX缺乏的部分原因。FX p.Cys27Ser的凝血活性降至野生型的约90%，而用重组FXa突变体测定的酰胺水解活性和凝血酶原酶活性（kcat/Km）分别降低至野生型的1.33倍和4.77倍。分子动力学模拟显示，FXa p.Cys27Ser与野生型FXa的整体结构无重大变化；然而，由于缺乏Cys22 - Cys27二硫键，激活裂解后催化结构域新形成的N端的插入受到阻碍，从而干扰了从酶原到酶的构象转变。FXa的晶体结构表明，该二硫键可与溶剂接触，这表明其稳定性可能受氧化/还原平衡的影响。正如本文中FX p.Cys27Ser所证明的，Cys22 - Cys27二硫键可能调节FX的凝血活性，FX活性降低则促凝活性减弱。
Abstract The Cys22-Cys27 disulfide bond of factor X (FX) protease domain is not conserved among coagulation factors and its contribution to the physiological haemostasis and implication in the pathogenesis of haemostatic and thrombotic disorders remain to be elucidated. Mutation p.Cys27Ser was identified in a pedigree of congenital FX deficiency and fluorescence labelling study of transiently transfected HEK293 cells showed accumulation of FX p.Cys27Ser within cell, indicating incompetent secretion partially responsible for the FX deficiency. The clotting activity of FX p.Cys27Ser was decreased to about 90% of wild-type, while amidolytic and pro-thrombinase activities (kcat/Km) determined with recombinant FXa mutant were 1.33- and 4.77-fold lower. Molecular dynamic simulations revealed no major change in global structure between FXa p.Cys27Ser and wild-type FXa; however, without the Cys22-Cys27 disulfide bond, the insertion of newly formed N terminal of catalytic domain after the activation cleavage is hindered, perturbing the conformation transition from zymogen to enzyme. The crystal structure of FXa shows that this disulfide bond is solvent accessible, indicating that its stability might be subject to the oxidation/reduction balance. As demonstrated with FX p.Cys27Ser here, Cys22-Cys27 disulfide bond may modulate FX clotting activity, with reduced FX pertaining less pro-coagulant activity.