Vibrio vulnificus ATCC29307 균주에서 분비되는 단백질분해효소에 의한 procaspase-3의 활성화에 관한 연구

Abstract

Vibrio vulnificus (V. vulnificus) is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhage complications. The metalloprotease (vEP) from V. vulnificus sp. strain ATCC29307 has previously been shown to have prothrombin activation activity. vEP also exhibits broad substrate specificity, enabling it to cleave a numbers of plasma proteins that are associated with blood coagulation, as well as those not associated with blood coagulation such as BSA and g-globulin. In this study, the ability of vEP to activate another zymogen, procaspase-3(D3A), was investigated. Procaspase-3 has been chosen for this study because it is an important enzyme that mediates the final step of the apoptosis cascade. A procaspase-3 mutant (D3A) in which the native cleavage sites essential for activation have been abolished was used as a substrate for vEP. Procaspase-3(D3A) exhibited some activity towards the caspase-3-specific chromgenic substrate, Ac-DEVD-pNA. After being activated by vEP through proteolytic cleavage, its activity increased approximately 3 fold. Similar to the activation of prothrombin by vEP whereby the formation of thrombin is transient, the formation of mature enzyme from procaspase-3(D3A) by cleavage with vEP was also transient, with the activity increasing with time and then decreasing upon further incubation in the presence of vEP. Western blot analysis of the time-dependent activation of procapase-3(D3A) by vEP showed a band corresponding to the size of the large subunit of mature caspase-3 when monoclonal antibody raised against caspase-3 large subunit. The activated enzyme displayed similar substrate specificity to native caspase-3 in that it cleaved poly(ADP-ribose) polymerase (PARP) in a cell-free system prepared from NIH3T3 cells and was sensitive to Ac-DEVD-CHO, a highly specific caspase-3 inhibitor. Taken together, the results obtained from this study suggest that vEP could act as a procaspase-3(D3A) activator.