Identification and characterization of common carp ( Cyprinus carpio L.) granzyme A/K, a cytotoxic cell granule-associated serine protease

Abstract

Granzyme (Gzm) is an important member of serine protease family, and key component in the specific and non-specific cell-mediated cytotoxicity. Partial GzmA/K cDNA sequence of common carp ( Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp GzmA/K was obtained by means of 3′ RACE and 5′ RACE, respectively. The full length cDNA of carp GzmA/K was 1053 bp, consisting of a 5′-terminal untranslated region (UTR) of 65 bp, a 3′-terminal UTR of 214 bp, and an open reading frame of 774 bp. Amino acid sequence analysis indicated the existence of a signal peptide, eight consensus cysteine residues, one conserved IIGG motif and three conserved residues as central elements of the GzmA/K active site. Carp GzmA/K shared 36% and 39% amino acid identity to human GzmA and K, respectively, and was phylogenetically related to the granzyme A and K subgroups. Then, a genomic DNA, which covers the promoter region and entire coding region of carp GzmA/K, was obtained by PCR. In the 5.4 k-long genomic sequence, five exons and four introns were identified. Real-time RT-PCR analysis showed that carp GzmA/K transcript was predominantly detected in the immune-related tissues, and after SVCV infection, was up-regulated in most immune-related tissues in a time-dependent manner. Real-time RT-PCR results also showed that carp GzmA/K transcript was up-regulated in thymus tissue of GH transgenic carp. These results will help to understand the molecular characterization and the potential role of teleost GzmA/K, a cytotoxic cell granule-associated serine protease.