Exploring the Serine Protease Involved in Cell‐mediated Immunity in Fish

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Viral and intracellular bacterial diseases are big problem in aquaculture. Defense against these diseases is largely dependent on cell‐mediated immunity in which cytotoxic T lymphocytes and NK cells play major roles. These cells kill cellular targets via either of two pathways, i.e. secretary (e.g. perforin/granzyme) and non‐secretary (e.g. Fas/FasL) one. In fish, biochemical mechanisms of the killing remain unknown. Here we tried to identify the protease involved in the killing adopting ginbuna crucian carp which has been used as a nice model for cell‐mediated immunity in fish. In order to identify the protease involved in the killing of allogeneic target cells, we performed cytotoxicity assays in the presence of serine or cysteine protease inhibitors using lymphocytes of ginbuna. Cytotoxicity was completely inhibited by the serine protease inhibitor “DCI”. By contrast cytotoxicity was partially inhibited with cysteine protease inhibitor, E‐64d. These findings suggest that serine proteases play a major role in the killing of target cells. We further analyzed the substrate specificity of the protease involved in cytotoxicity. Protease activity of lymphocyte lysate was enhanced by allo‐sensitization when Ac‐IETD‐MCA, Bz‐R‐MCA, Z‐GPR‐MCA, K‐MCA, and Suc‐AAPF‐MCA were used as substrates. To classify the group of protease involved in the cytotoxicity, protease inhibitor was added in enzyme assay. Serine protease inhibitor “AEBSF” completely inhibited enzyme activity only when Z‐GPR‐MCA was used. Furthermore, there was no inhibition with cysteine protease inhibitor “E‐64”. These results suggest that the serine protease which cleaves Z‐GPR‐MCA is involved in cytotoxicity.