Molecular cloning, immunohistochemical localization, characterization and expression analysis of caspase-8 from the blunt snout bream (Megalobrama amblycephala) exposed to ammonia.
Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this study, we isolated the caspase-8 gene from Megalobrama amblycephala, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The 2034 bp full-length M. amblycephala caspase-8 cDNA sequence contained an ORF of 1467 bp encoding a polypeptide of 489 amino acid residues, a 5'-UTR of 102 bp and a 3'-UTR of 462 bp. The caspase-8 amino acid sequences contained two highly conservative death effector domains (DEDs) at N-terminal, the caspase family domains P20 and P10, caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. Phylogenetic analysis revealed that M. amblycephala caspase-8 were clustered with the caspase-8 from other vertebrate. Real-time quantitative PCR analysis revealed that caspase-8 transcripts were detected in liver after exposure to ammonia. Meanwhile using Western blot analysis, caspase-8 cleaved fragment was detected and significant alteration of procaspase-8 level was found with the same ammonia treatment condition. Furthermore, the result of immunohistochemical detection showed that remarkable changes of immunopositive staining were observed after ammonia treatment. Accordingly, the results signify that caspase-8 of fish may play an essential role in ammonia induced apoptosis.
Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, PR China.
Shengming
S
Sun
Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, PR China. Electronic address: gexp@ffrc.cn.
Xianping
X
Ge
Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, PR China.
Jian
J
Zhu
Wuxi Fishery College Nanjing Agricultural University, Wuxi 214081, PR China.
Wuxiao
W
Zhang
Wuxi Fishery College Nanjing Agricultural University, Wuxi 214081, PR China.
Qiong
Q
Zhang
D000595:Amino Acid Sequence; D000641:Ammonia / Q000633:toxicity*; D000818:Animals; D001483:Base Sequence; D053181:Caspase 8 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003530:Cyprinidae / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D009928:Organ Specificity; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment / Q000662:veterinary; D013312:Stress, Physiological; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D018076:DNA, Complementary; D029941:Fish Proteins; D012333:RNA, Messenger; D014874:Water Pollutants, Chemical; D000641:Ammonia; D053181:Caspase 8
Ammonia; Apoptosis; Caspase-8; Megalobrama amblycephala; Oxidative stress