Thesis (M.Sc.)--Chulalongkorn University, 2007

Many studies suggest that apoptosis occurs following white spot syndrome virus (WSSV) infection in Penaeus monodon and might be the cause of death in viral infection. Among the molecules involved in apoptosis process, cathepsins have been reported to act as proapoptotic mediators of apoptosis in a variety of vertebrate species. In shrimp, cathepsin L mRNA was up-regulated in lymphoid organs after infection with WSSV. Since cathepsins seem to be interesting molecules, this study was aimed to verify the involvement of apoptosis function of cathepsin L in WSSV infected shrimp. Western blotting of the crude protein from haemocyte, heart, hepatopancreas and lymphoid organ revealed a single 28 kDa protein band of mature cathepsin L in hepatopancreas. Immunohistochemistry analysis in cephalothorax of Penaeus monodon found that the cathepsin L was detected mainly in B cells of hepatopancreas and in spheroid of lymphoid organ. Moreover, the cathepsin L level in lymphoid organ was found to be decreased at 24 h when compared with the control LHM-injected shrimp. The involvement of cathepsin L in apoptosis was further elucidated in the primary cell culture of lymphoid organ. The cells treated with actinomycin D and caspase-3 inhibitor or actinomycin D and cathepsin L inhibitor showed lower percentage of dead cells compared with the cells treated with actinomycin D only. The appropriate concentrations of caspase-3 and cathepsin L inhibitors for successful inhibition of apoptosis were 0.5 and 1 µM, respectively. However, when the DNA laddering assay was performed to detect apoptosis, the characteristic apoptotic DNA ladder of approximately 200 bp was seen in all 40 nM actinomycin D treated cells with or without inhibitor as well as the control (untreated cells). As a result of cell culture assay, the study reveals an involvement in apoptosis of cathepsin L. Furthermore, in order to verify the function of cathepsin L, the procathepsin L was expressed in E. coli BL21(DE3) expression system. However, auto activation of the recombinant procathepsin L in acidic condition was unsuccessful, thus, the active cathepsin L was not obtained.