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Identification and purification of a serine protease involved in cell‐mediated immunity in fish
Author(s) -
Matsuura Yuta,
Yabu Takeshi,
Shiba Hajime,
Nakanishi Teruyuki
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1083.14
Subject(s) - serine protease , proteases , protease , cytotoxic t cell , biology , crucian carp , masp1 , cysteine protease , effector , cytotoxicity , antigen , microbiology and biotechnology , biochemistry , enzyme , immunology , in vitro , fishery , fish <actinopterygii>
Cell‐mediated immunity is crucial immune response against non‐self antigen such as allograft, tumor and virus infected cells. Non‐self antigens could be eliminated by effector cells (e.g. cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells) with secretory and/or non‐secretory cell‐death pathways in mammal. Also in fish, cytotoxic effector cells have been characterized in lymphocyte subsets level; however, 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 the study on 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 leukocytes of ginbuna crucian carp. Cytotoxicity was significantly inhibited by serine protease inhibitor, 3,4‐Dichloroisocoumarin: “DCI”. By contrast the cytotoxicity was partially inhibited by cysteine protease inhibitor. Then we tried to explore a substrate of the serine protease involved in killing activity, and we found that a protease activity against a peptide substrate “z‐Gly‐Pro‐Arg‐4‐Methylcoumaryl‐7‐amide (MCA) was inhibited by “DCI”. In addition, z‐Gly‐Pro‐Arg‐MCA cleaving activity was significantly enhanced by allo‐antigen stimulation. These results suggest that the serine protease which cleaved z‐Gly‐Pro‐Arg‐MCA substrate was involved in killing against allogeneic cells. In order to analyze the enzymatic function of the serine protease, we tried to purify the protease. Purification was sequentially performed on two steps of benzamidine‐sepharose and SP‐sepharose chromatography. The purified protease was visualized as a single protein band and a molecular mass was estimated to be approximately 27,000 Da by using SDS‐PAGE. The protease was certainly serine protease because it was not inhibited by cysteine, metallo‐ and aspartic protease inhibitors but serine protease inhibitor. In conclusion, we succeeded to purify the serine protease involved in the killing against allogeneic cells. Now we initiate proceeding to examine a function and to find endogenous substrate of the protease with the purified protease. Support or Funding Information Sasakawa Scientific Research Grant from the Japan Science Society