Cloning of catalase gene and antioxidant genes in Scophthalmus maximus response to metalloprotease of Vibrio anguillarum stress

Metalloproteases represent a class of extracellular proteases found in Vibrio anguillarum that can generate toxic and pathogenic effects in turbot (Scophthalmus maximus). The toxicological effect partly results from oxidative damage due to the production of excessive reactive oxygen species (ROS). Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) are major antioxidant enzymes induced by various oxidative stresses and can scavenge peroxides generated in cells. To evaluate the effects of metalloprotease-induced ROS on the antioxidation defense mechanism of S. maximus head kidney cells, the cDNA of CAT gene (designated as SmCAT) was cloned and characterized. SmCAT comprises a 1 584-bp coding sequence that encodes a protein containing 527 amino acids with a poly(A) tail. Bioinformatics analysis revealed an active site signature sequence, a heme-ligand signature sequence, and three catalytic amino acid residues. The deduced SmCAT amino acid sequence shares a sequence similarity of 66.1%–92.4% with those of other species. Phylogenetic analysis revealed that SmCAT is classified with CAT of other fishes. Quantitative real-time PCR analysis showed that SmCAT was extensively expressed in all tested tissues, especially in blood. The expression of SmCAT, SmMnSOD, and SmGPx were inhibited significantly in head kidney cells treated with metalloprotease from 12 to 24 h. In 6 to 24 h metalloprotease-treated groups compared to that of the untreated group, it was found that the production of ROS was markedly increased, and the mitochondrial membrane potential was decreased considerably. Hoechst 33342 staining revealed the presence of apoptotic bodies when the cells were incubated with 8.0 or 40.0 μg/mL metalloprotease for 12 and 24 h. Hence, the toxic effects of metalloprotease are associated with the down-regulation of antioxidant enzyme expression and increased ROS levels, which trigger the activation of apoptosis in the head kidney cells of turbot. Our findings provide a better understanding on the mechanism of metalloprotease-induced apoptosis in fish.