1.Department of Laboratory Medicine, the First Affiliated Hospital of Wenzhou Medical University,Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou325015, China; 2.Department of Hematology, Wenzhou Key Laboratory of Hematology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China; 3.Department of Pathology, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325027, China
YU Xiaomin,CHANG Guolin,ZHENG Yiyang, et al. The pathogenic mechanism of inherited protein C deficiency caused by Ala333Thr mutation[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2022, 52(5): 352-357.
Abstract：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 QuickMutationTM 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.
