Identification and characterization of common carp (Cyprinus carpio L.) granzyme A/K, a cytotoxic cell granule-associated serine protease.
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.
Huang
Rong
R
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Zhong
Shan
S
Liu
Hong
H
Kong
Renqiu
R
Wang
Yaping
Y
Hu
Wei
W
Guo
Qionglin
Q
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002347:Carps / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D005393:Fish Diseases / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D053804:Granzymes / Q000235:genetics* / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011897:Random Allocation; D018353:Rhabdoviridae Infections / Q000201:enzymology / Q000662:veterinary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013950:Thymus Gland / Q000201:enzymology*; D018116:Vesiculovirus / Q000502:physiology
D053804:Granzymes