Cloning, characterization, sequence analysis and expression patterns in vivo of testicular 20β-hydroxysteroid...

We have cloned a full-length cDNA for testicular 20β-HSD in yellow catfish. The validated 20β-HSD cDNA full-length sequence, 1141 bp in length, contained a 108 bp 5'-untranslated region (UTR), a 202 bp 3'-UTR with an AATAAAA frame, and an 831 bp open reading frame (ORF) which encoded a propeptide of 277 amino acid residues. The enzyme shows the highest structural homology with that of zebrafish, and rainbow trout. Quantitative real-time PCR revealed that 20β-HSD has widespread tissue distribution, with expression being abundant in tissues with high metabolic rates like gonads, liver, intestine, stomach and gill. In vivo experiments showed that expression level was highest at testicular mature stage indicating that 20β-HSD could play an important role in testicular developmental maturation in yellow catfish. During testicular mature stage, 20β-HSD related metabolism was regulated by GnRH and LH. Moreover, structural analysis showed that the predicted 20β-HSD contained 7 functional motifs of SDR superfamily of enzymes, including the putative coenzyme binding domain (Rossmann fold), GlyXXXGlyIleuGly, and the region responsible for nucleophilic attack of the substrate pocket, TyrXXXLys. These motifs are strictly conserved in yellow catfish 20β-HSD. Comprehensive functional analysis revealed that this enzyme has multiple functions, such as xenobiotic metabolism, and steroid conversion. Catfish 20β-HSD contains multiple potential post-translational modification sites. Its subcellular location, theoretical isoelectric point and molecular weight were also investigated. Furthermore, we constructed its phylogenetic tree and secondary structure. All results provided basic information for further studies of its structure, functions and properties.