Identification of a novel fish granzyme involved in cell-mediated immunity

Granzymes (Gzms) are serine proteases released from cytoplasmic granules within cytotoxic T lymphocytes and natural killer (NK) cells. Gzms induce apoptosis within virus-infected and transformed cells. In fish as well as mammals, Gzms appear to play a major role in inducing target cell death. However, information on the function of fish Gzms is limited, although Gzm-like genes have been reported in several species. We identified and characterized a fish Gzm (termed gcGzm) in ginbuna crucian carp, Carassius auratus langsdorfii. The primary structure of gcGzm resembled mammalian GzmB, and gcGzm clustered with mammalian GzmB by phylogenetic tree analysis. gcGzm was secreted from HEK293T cells transfected with gcgzm cDNA and was predominantly expressed in CD8(+) T cells, as in mammals. Expression of gcgzm mRNA was greatly enhanced by allo-sensitization and infection with the intracellular pathogen Edwardsiella tarda, indicating that gcGzm is involved in cell-mediated immunity. However, its enzymatic activity was different from mammalian Gzms because gcGzm did not cleave the known substrates for mammalian Gzms. Thus we conclude that the newly discovered gcGzm is a novel secretory serine protease involved in cell-mediated immunity in fish, with similar structure to human GzmB but different substrate specificity.