Molecular Mechanism of Protein C Deficiency Caused by Mutations of PROC Gene N355S,G392E,T314A
10.19746/j.cnki.issn1009-2137.2024.06.030
- VernacularTitle:PROC基因N355S、G392E、T314A突变致蛋白C缺陷的分子机制研究
- Author:
Tian-Yi LI
1
;
Miao JIANG
;
Lu-Lu HUANG
;
Jing-Jing HAN
;
Zhen-Ni MA
;
Xia BAI
;
Li-Jun XIA
Author Information
1. 苏州大学附属第一医院,江苏省血液研究所,国家血液系统疾病临床医学研究中心,国家卫生健康委员会血栓与止血重点实验室,血液学协同创新中心,江苏苏州 215006;放射医学与辐射防护国家重点实验室(苏州大学),江苏苏州 215123
- Keywords:
protein C;
protein C deficiency;
gene mutation;
structural analysis
- From:
Journal of Experimental Hematology
2024;32(6):1834-1840
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To study the molecular mechanism of functional defect of protein C (PC) caused by point mutations of human protein C gene (PROC) N355S,G392E and T314A. Methods:The wild-type and mutant plasmids (PCWT,PCN355S,PCG392E,PCT314A) of PROC gene were constructed and transiently transfected into HEK293 cells. The expression of mutant proteins in vitro were tested. The mRNA level changes of wild-type and mutant PC after 24 h of transfection were detected by real-time PCR. Western blot and ELISA were used to detect the changes of intracellular and extracellular protein levels of wild-type and mutant PC. The supernatant of cells transfected for 24-48 h was concentrated by ultrafiltration. The protein in the concentrated solution was quantified,and PC activation and enzyme kinetics tests were performed. Clustal Omega multiple sequence alignment was used to analyze the conservation of amino acid mutation sites. The effect of mutation on PC protein structure was analyzed by PyMOL software. Results:The relative expression abundances of PROC mRNA in PCN355S,PCG392E and PCT314A groups were 1.14±0.46,0.96±0.08 and 1.08±0.17,respectively,and there were no significant differences compared with 1.02±0.24 in PCWT group (P>0.05). Western blot analysis of the lysates of transfected cells showed that the content of PCT314A recombinant protein slightly decreased and the band became relatively lighter. The ELISA results of the concentrated cell culture supernatants showed that the PC:Ag levels of PCN355S and PCG392E were 98.8％±2.4％ and 101.4％±3.1％,respectively,with no significant differences compared with PCWT,while PCT314A decreased compared with PCWT (PC:Ag:88.6％±3.2％) (P<0.05). The results of enzyme kinetics test showed that APCN355S (Km=338.3±43.2,Vmax=2.015±0.12),APCG392E (Km=292.2±28.4,Vmax=1.893±0.07) and APCT314A (Km=299.5±24.6,Vmax=1.775±0.06) showed an increase in Km and a decrease in Vmax compared with APCWT (Km=238.2±4.58,Vmax=3.205±0.06). Multiple sequence alignment suggested that the three mutations be highly conservative in different species. The structural model suggested that the amino acid substitutions of N355S,G392E and T314A mutations collide with the surrounding amino acid groups,causing distortion of the surrounding structure,which may have adverse effects on the folding and biological function of PC. Conclusion:The N355S,G392E and T314A mutations in the PROC gene cause functional defects in PC by weakening the binding between PC and substrate. These three mutations have caused serious spatial collisions in the protein structure,affecting the folding of PC and the reactivity of active sites.
