Identification and characterization of novel granzyme in fish (971.1)

Abstract

Granzymes are serine proteases released by cytoplasmic granules within cytotoxic T lymphocytes and natural killer (NK) cells. Granzymes induce apoptosis within virus‐infected and transformed cells. Information on the function of fish granzymes is limited, although granzyme‐like genes have been reported in several fishes. Here, we report the identification and characterization of a granzyme in ginbuna crucian carp, Carassius auratus langsdorfii, focusing on substrate specificity and gene expression after allo‐sensitization and bacterial infection.A full‐length granzyme cDNA, termed gcGranzyme was identified from ginbuna crucian carp. gcGranzyme molecule has trypsin‐like catalytic triad: His66, Asp109, and Ser201 residues, and active center: Ser201 residue. Phylogenetic analysis of deduced amino acids sequence revealed that gcGranzyme formed a cluster with human and mouse granzyme B (separate from the other members of the granzyme). gcGranzyme was secreted from the HeLa cells transfected with gcGranzyme as in mammals. Enzyme assay using recombinant gcGranzyme indicated that the enzyme hydrolyzed Bz‐Arg‐4Methyl‐Coumaryl‐7‐amide, suggesting that the gcGranzyme was different from mammalian granzyme B in substrate specificity.To investigate the role of gcGranzyme in cell‐mediated immunity in fish, we analyzed the gcGranzyme mRNA expression in allo‐sensitized fish. Expression level of gcGranzyme mRNA from CD8+ T cells was greatly elevated by allo‐sensitization. Furthermore, gcGranzyme mRNA expression in CD8+ T cells was also enhanced by the in vivo infection with intracellular bacteria, Edwardsiella tarda.These results suggest that newly found gcGranzyme is a novel secretory serine protease involved in cell‐mediated immunity in fish with similar structure to human granzyme B but different substrate specificity.