In vitro characterization of missense mutations associated with quantitative protein S deficiency
In vitro characterization of missense mutations associated with quantitative protein S deficiency
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DOI：
10.1111/j.1538-7836.2006.02061.x
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发表时间：
2006-09-01
影响因子：
10.4
通讯作者：
Kojima, T.
中科院分区：
文献类型：
--
作者：
Okada, H.;Yamazaki, T.;Kojima, T.
目的：为了阐明遗传性蛋白S（PS）缺陷的分子后果，我们研究了PS错义突变体在COS-1细胞中的体外合成及其活化的蛋白C（APC）辅因子活性。患者：4例患有静脉血栓形成的定量PS缺乏症患者进行了检查。结果如下：我们发现了三个不同的新的错义突变，R275 C，P375 Q和D455 Y，和两个以前报道的错义突变，C80 Y和R314 H。在1例患者中发现P375Q和D455Y突变，并观察到在同一等位基因上连锁。R314 H突变体表现出最低水平的表达（32.7%），而C80 Y、P375 Q + D455 Y和R275 C突变体表现出中度表达损伤，即分别为野生型的43.8%、49.5%和72.3%。此外，脉冲追踪实验表明，所有突变体显示出受损的分泌和更长的半衰期在细胞中比野生型PS。在APC辅因子测定中，C80Y突变体显示没有辅因子活性，R275C突变体显示活性降低，为野生型PS的62.3%，而R314H和P375Q + D455Y突变体显示正常辅因子活性。结论：这些数据表明，C80 Y和R275 C突变影响PS分子的分泌和功能，R314 H和P375 Q + D455 Y突变仅负责分泌缺陷，导致在患者中观察到的定量PS缺陷的表型。
Objective: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. Patients: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. Results: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. Conclusion: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.