PROC基因N355S、G392E、T314A突变致蛋白C缺陷的分子机制研究

Molecular Mechanism of Protein C Deficiency Caused by Mutations of PROC Gene N355S,G392E,T314A

目的:研究人蛋白C基因(PROC)N355S、G392E、T314A点突变致蛋白C(PC)功能缺陷的分子机制。方法:构建PROC基因野生型和突变型质粒(PCWT、PCN355S、PCG392E、PCT314A)并瞬时转染至HEK293细胞,进行突变蛋白的体外表达试验,用实时荧光PCR检测野生型和突变型PC转染24 h后的mRNA水平变化。Western blot和ELISA分别检测野生型及突变PC细胞内外蛋白水平变化。将转染24-48 h的细胞上清液进行超滤浓缩,对浓缩液中的蛋白进行定量,并进行PC的激活和酶动力学试验。Clustal Omega多序列比对分析氨基酸突变位点的保守性。利用PyMOL软件分析突变对PC蛋白质结构造成的影响。结果:PCN355S、PCG392E、PCT314A组PROC mRNA相对表达量分别为1.14±0.46、0.96±0.08和1.08±0.17,与PCWT组的1.02±0.24相比均无显著差异(P>0.05)。转染细胞裂解物的Western blot分析结果显示,PCT314A重组蛋白的含量略降低,条带相对较浅。浓缩细胞培养上清液的ELISA结果显示,PCN355S、PCG392E的PC:Ag水平分别为98.8%±2.4%、101.4%±3.1%,与PCWT相比无明显差异,而PCT314A的PC:Ag水平(88.6%±3.2%)则较PCWT有所下降(P<0.05)。酶动力学试验的结果表明,APCN355S(Km=338.3±43.2,V_(max)=2.015±0.12)、APCG392E(Km=292.2±28.4,V_(max)=1.893±0.07)和APCT314A(Km=299.5±24.6,V_(max)=1.775±0.06)与APCWT(Km=238.2±4.58,Vmax=3.205±0.06)相比,均表现为Km值的升高和Vmax的下降。多序列比对提示,3种突变在不同物种中均具有高度保守性。结构模型提示,N355S、G392E、T314A突变的氨基酸替换与周围的氨基酸基团产生碰撞,使周围的结构产生扭曲,对PC的折叠和发挥生物学功能产生不利影响。结论:PROC基因的N355S、G392E和T314A突变通过减弱PC与底物间的结合导致PC的功能缺陷。这3种突变在蛋白结构上产生了严重的空间碰撞,影响了PC的折叠和活性位点的反应性。

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.