Molecular characterization, expression and immune functions of cystatin B in Japanese flounder (Paralichthys olivaceus)

Cystatin B is an intracellular inhibitor that regulates the activities of cysteine proteases. In this study, cystatin B in Japanese flounder (Paralichthys olivaceus) was characterized and its immune function was analyzed. This gene had a high similarity with the sequence of cystatin B in other fish species, and the derived peptide shared typical features of cystatin proteins including the QXVXG motif. The results of quantitative real-time PCR showed that cystatin B mRNA was constitutively expressed in all examined tissues, with the highest level in gill. The stimulations of lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid effectively increased the expression level of cystatin B mRNA. Functional analysis implied that the recombinant P. olivaceus cystatin B purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Furthermore, we found that P. olivaceus cystatin B had no effects on the expression of inflammatory factors cytokines tumor necrosis factor α, interleukin 10, interleukin 1β and interferon γ. These results indicate that cystatin B of P. olivaceus is potentially involved in immune responses against invading microbial pathogens, and provide a better understanding of the immune mechanisms of cystatins in teleosts.