摘要
目的:探讨蛋白C(PC)p.Ala333Thr突变导致遗传性蛋白C缺陷症(IPCD)的分子机制。方法:使用氨基酸多序列比对工具T-Coffee评估突变氨基酸位点的保守性,使用突变有害性评分工具PolyPhen-2预测突变对PC结构和功能的危害性。利用定点突变试剂盒QuickMutation^(TM)构建PC基因(PROC)野生型和p.Ala333Thr突变型质粒,并瞬时转染入HEK-293T细胞进行体外表达,RT-qPCR检测突变PC转录24 h后的mRNA水平变化,利用蛋白印迹法(Western blot)和ELISA分别检测突变PC胞内外水平变化,并对细胞内质网、高尔基体PC进行免疫荧光染色,初步探讨p.Ala333Thr突变导致IPCD的分子机制。结果:多序列比对结果显示PC的Ala333位点保守性不高,PolyPhen-2对PROC的p.Ala333Thr突变评分为0.763。成功转染野生型与p.Ala333Thr突变型PROC质粒后,突变型PROC转录水平无明显变化;Western blot结果显示,突变型PROC的PC表达量明显下降;免疫荧光染色结果显示,野生型PC在内质网和高尔基体均有大量分布,而p.Ala333Thr突变型PC主要分布于内质网。结论:PROC p.Ala333Thr突变可能导致了PC的错误折叠和加工障碍,造成胞外PC表达量下降,最终引起了IPCD的发生。
Objective:To investigate the mechanism of inherited protein C deficiency(IPCD)caused by the mutation of protein C(PC)p.Ala333Thr.Methods:Amino acid multiple sequence alignment tool T-Coffee was used to evaluate the conservatism of mutant amino acid sites,and PolyPhen-2 was used to predict the harmfulness of mutations to the structure and the function of PC.The mutant PC plasmid and the wild type PC plasmid were constructed by QuickMutation^(TM) mutation kit and transfected into HEK-293T cells.The mRNA level was detected,using real-time quantitative polymerase chain reaction(RT-qPCR)after 24 h transfection.The intracellular and extracellular protein level of PC was detected by Western blot and ELISA,respectively.Immunofluorescence was used to label the distribution of PC and fluorescent dyes were used to label endoplasmic reticulum(ER)and Golgi apparatus(GA)in HEK-293T cells.Results:The results of multiple sequence alignment showed that the Ala333 locus of PC was not conservative,and the p.Ala333Thr mutation score of PC was 0.763 by PolyPhen-2.The transcriptional level of mutant PROC gene was not significantly different from that of the wild type.The expression level of intracellular and extracellular PC of mutant PROC gene was significantly lower than that of the wild type.Immunofluorescence showed that wild type PC was distributed both in ER and GA,while mutant PC was mainly distributed in ER and only a small amount entered GA.Conclusion:The PC p.Ala333Thr mutation causes the misfolding and processing defect of PC,which induces the low expression of extracellular PC and results in the occurrence of IPCD.
作者
余晓敏
常国林
郑逸旸
陈诚
唐施艺
吴鑫媛
吕佳
林向阳
朱丽青
YU Xiaomin;CHANG Guolin;ZHENG Yiyang;CHEN Cheng;TANG Shiyi;WU Xinyuan;LYU Jia;LIN Xiangyang;ZHU Liqing(Department of Laboratory Medicine,the First Affiliated Hospital of Wenzhou Medical University,Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province,Wenzhou 325015,China;Department of Hematology,Wenzhou Key Laboratory of Hematology,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China;Department of Pathology,the Second Affiliated Hospital of Wenzhou Medical University,Wenzhou 325027,China)
出处
《温州医科大学学报》
2022年第5期352-357,共6页
Journal of Wenzhou Medical University
基金
温州市基础性科研项目(Y20190088)
浙江省医药卫生科技计划项目(2022KY205)
浙江省中医药科技计划项目(2021ZB181)
浙江省自然科学基金项目(LY22H200004)。
关键词
遗传性蛋白C缺陷症
蛋白C
基因突变
胞内降解
inherited protein C deficiency
protein C
gene mutation
intracellular degradation
