Mutations in the shutter region of antithrombin result in formation of disulfide-linked dimers and severe venous thrombosis
Mutations in the shutter region of antithrombin result in formation of disulfide-linked dimers and severe venous thrombosis
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DOI：
10.1111/j.1538-7836.2004.00749.x
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发表时间：
2004-06-01
影响因子：
10.4
通讯作者：
Carrell, RW
中科院分区：
文献类型：
--
作者：
Corral, J;Huntington, JA;Carrell, RW
背景：导致蛇类构象改变的错义突变可能是与人类疾病相关的蛋白质缺乏的原因。然而，关于影响主要止血蛇毒蛋白抗凝血酶的起始作用的构象结果的数据很少。目的：研究影响抗凝血酶快门区突变的构象和临床效应。患者和方法：我们确定了两个循环抗凝血酶显著降低的家系，表现为早期和严重的静脉血栓形成，经常与妊娠或感染相关。突变用标准分子方法测定。对血浆样本进行生化研究。通过肝素亲和层析和凝胶过滤纯化了一个突变体(P80S)，并对其进行了蛋白质组学分析。最后，我们对突变二聚体的结构进行了建模。结果：我们发现了两个影响抗凝血酶快门区域的错义突变：P80S和G424R。这两种突变的携带者都出现了类似的异常抗凝血酶的痕迹，支持低效表达的变异体而不是不表达的变异体。从P80S携带者中提纯的异常抗凝血酶是一种突变的抗凝血酶的二硫键连接二聚体，其性质与反应环的头对头插入一致。结论：我们的数据支持这样的结论，即影响蛇类快门区域的错义突变具有特定的构象效应，从而导致突变寡聚体的形成。随之而来的分泌效率低下解释了伴随而来的功能缺陷和丧失，但与这些启动相关的血栓形成的严重程度表明，寡聚体还具有新的和未明确的病理特性，这些特性可能在怀孕或感染时加剧。
Background: Missense Mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences Of Initiations affecting antithrombin, the main hemostatic serpin. objectives: To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin. Patients and methods: We identified two families with significant reduction Of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparinaffinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer. Results: We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal anti thrombin, supporting inefficiently expressed rather than non-expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide-linked dimer of mutant antithrombin whose properties are consistent with head-to-head insertion of the reactive loop. Conclusions: Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these Initiations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated bt pregnancy or infection.