蛋白C基因C64W和F139V突变致蛋白C缺陷的分子机制研究  

Molecular mechanisms of protein C deficiency caused by C64W and F139V mutations

作　　者：周荣富[1] 蔡晓红[1] 谢爽[1] 王文斌[1] 戴菁[1] 丁秋兰[1] 方怡[1] 谢飞[1] 王学锋[1] 王鸿利[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院、上海血液学研究所,200025

出　　处：《中华血液学杂志》2007年第3期156-159,共4页Chinese Journal of Hematology

摘　　要：目的研究蛋白C（PC）基因C64W、F139V和K150缺失突变（K150d）致PC缺陷症的分子机制。方法构建PC基因野生型和突变体表达质粒（PCwt、PC C64W、PC F139V、PC K150d）并瞬时转染至COS-7细胞或CHO细胞，进行体外表达试验和细胞免疫荧光染色；用荧光实时PCR（real-time PCR）检测转染细胞PC mRNA表达量的改变；蛋白降解抑制实验检测突变蛋白在细胞内的降解途径；内糖苷酶-H（Endo H）酶切试验检测PC翻译后侧链糖化修饰。结果PC C64W未从转染细胞内分泌且在细胞内逐渐降解，PC F139V仅部分从细胞内分泌，大部分未分泌并在细胞内逐渐降解，PC K150d突变体蛋白的分泌未受突变的明显影响。real-time PCR结果显示，与野生型PC mRNA相比，突变体PC mRNA不降低；蛋白降解抑制实验显示，PC C64W和PC F139V通过蛋白酶体途径进行细胞内降解。Endo H酶切和转染细胞荧光染色显示，PC C64W和PC F139V主要位于前高尔基体。结论分泌障碍和细胞内降解是PC C64W和PC F139V导致PC缺陷症的原因，PC K150d对突变体蛋白的分泌无明显影响。Objective To study the molecular mechanisms of protein C （PC） deficiency caused by PC gene mutations of C64W, F139V and K150 deletion （K150d）. Methods Wild-type and mutant PC eDNA expression plasmids （PCwt, PC C64W, PC F139V, PC K15Od） were constructed and transfected into COS-7 cells or CHO cells respectively for in vitro expression study and immunofluorescent assay. Fluorescent real-time PCR was used to detect the expression of PC mRNA, protein degradation inhibition and endo-β-Nacetylglucosaminidase H （Endo H） digestion experiments to explain the mutant protein degradation pathway and its lecalizations inside the cells. Results PC C64W was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC F139V, most of the protein molecule was not secreted and degraded intracellularly. Mutant PC K150d was secreted normally from the cells. Fluorescent real-time PCR analysis of total mRNA from transfected cells showed no reduction of the mutant PC mRNA expression compared with that of wild-type PC mRNA. Protein degradation inhibition experiments showed that mutants PC C64W and PC F139V were degraded intracellularly through the proteasome pathway. Endo H digestion experiments and immunofluorescence results suggested that mutant PC molecules were located mainly in pre-Golgi apparatus. Conclusions Impaired secretion and degradation intracellularly of the mutants might be the molecular mechanisms of PC deficiency caused by C64W and F139V mutations. K150 deletion mutation might not affect the secretion of the mutant.

关 键 词：基因 蛋白C 基因突变 蛋白C缺陷症 

分 类 号：R686[医药卫生—骨科学]