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

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 (PC_WT, PC_N355S, PC_G392E, PC_T314A) 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 PC_N355S, PC_G392E and PC_T314A 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 PC_WT group (P >0.05). Western blot analysis of the lysates of transfected cells showed that the content of PC_T314A 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 PC_N355S and PC_G392E were 98.8%±2.4% and 101.4%±3.1%, respectively, with no significant differences compared with PC_WT, while PC_T314A decreased compared with PC_WT (PC:Ag: 88.6%±3.2%) (P < 0.05). The results of enzyme kinetics test showed that APC_N355S (K_m=338.3±43.2, V_max=2.015±0.12), APC_G392E (K_m=292.2±28.4, V_max=1.893±0.07) and APC_T314A (K_m=299.5±24.6, V_max=1.775±0.06) showed an increase in K_m and a decrease in V_max compared with APC_WT (K_m=238.2±4.58, V_max=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.