Molecular characterization and organs expression of cytochrome P450 1B1 from Japanese eel (Anguilla japonica)

Abstract

The CYP1 family, one of the gene families of the CYP superfamily, has four subfamilies deposited in the GenBank/EMBL so far; CYP1A, CYP1B, CYP1C, and the newly identified CYP1D. The metabolic activation and elimination of polyaromatic hydrocarbons, polychlorinated biphenyls, and aryl amines from fish body is largely mediated by the CYP enzymes. A new cDNA of the CYP1B subfamily encoding CYP1B1 was isolated from Japanese eel liver after a single intraperitoneal injection of β-naphthoflavone (BNF). The full-length cDNA obtained was 2985 bp and contained a 5' noncoding region of 294 bp, an open reading frame of 1626 bp coding for 541 amino acids and a stop codon and a 3' noncoding region of 1065 bp. The predicted molecular weight of the protein was approximately 61.27 kDa. The deduced amino acid sequence of Japanese eel CYP1B1 showed 62% similarity to three-spined stickleback CYP1B1 and zebrafish CYP1B1. It exhibited similarities of 66% with that of killifish, Indian medaka and our previously reported carp CYP1B1 and -1B2 while the higher similarities (67 and 69%) of the deduced amino acids was observed with that of Nile tilapia CYP1B1 and rainbow trout respectively. The percent identities of Japanese eel CYP1B1 cDNA showed similarities with those of the reported CYP1Bs of mammals of 57, 57, and 56% for human, rat, and mouse CYP1B1, respectively. Japanese eel CYP1B1 was aligned with the CYP1 sequences and has been deposited in the GenBank/EMBL data bank with the accession number AY518340. The phylogenetic tree constructed using the previously reported CYP1B sequences of mammals and fish suggested the closer relationship of the newly identified Japanese eel CYP1B1 to rainbow trout CYP1B1. QRT-PCR analysis of liver, kidney, gills and intestine revealed a distinct induced expression in liver, kidney and gills (71.93, 3.87 and 539.56 respectively) while the constitutive expression (0.062) was observed in intestine.