Purification and characterization of a fish granzymeA involved in cell-mediated immunity

• We purified and characterized fish homolog of granzymeA in ginbuna crucian carp.
• GranzymeA-like serine protease was involved in cytotoxicity against allogeneic cells.
• The purified enzyme was trypsin-like serine protease and showed granzymeA-like substrate specificity.
• The molecular weight of the granzymeA was estimated to be 26,900 by SDS-PAGE analysis.
• Kinetics analysis of purified granzymeA showed a Km of 220 μM, a Kcat of 21.7 sec−1 and a Kcat/Km of 98,796 sec−1 M−1.

Granzymes are serine proteases involved in the induction of cell death against non-self cells. The enzymes differ in their primary substrate specificity and have one of four hydrolysis activities: tryptase, Asp-ase, Met-ase and chymase. Although granzyme genes have been isolated from several fishes, evidence for their involvement in cytotoxicity has not yet been reported. In the present study, we attempted to purify and characterize a fish granzyme involved in cytotoxicity using ginbuna crucian carp. The cytotoxicity of leukocytes was significantly inhibited by the serine protease inhibitor ‘‘3, 4-dichloroisocoumarin’’. In addition, we found that granzymeA-like activity (hydrolysis of Z-GPR-MCA) was inhibited by the same inhibitor and significantly enhanced by allo-antigen stimulation in vivo. Proteins from leukocyte extracts were subjected to two steps of chromatographic purification using benzamidine-Sepharose and SP-Sepharose. The molecular weight of the purified enzyme was estimated to be 26,900 Da by SDS-PAGE analysis. The purified enzyme displayed a Km of 220 μM, a Kcat of 21.7 sec−1 and a Kcat/Km of 98,796 sec−1 M−1 with an optimal pH of 9.5 for the Z-GPR-MCA substrate. The protease was totally inhibited by serine protease inhibitors and showed granzymeA-like substrate specificity. Therefore, we conclude that the purified enzyme belongs to the mammalian granzymeA (EC 3.4.21.78) and appears to be involved in cytotoxicity in fish.