Cathepsin L in the orange-spotted grouper, Epinephelus coioides: molecular cloning and gene expression after a Vibrio anguillarum challenge

Abstract

The orange-spotted grouper, Epinephelus coioides, is an important fish maricultured in many Asian countries. In the present study, the full-length cDNA of cathepsin L, an immunity related gene of fishes, was isolated from E. coioides using rapid amplification of cDNA ends (RACE). It is 1,443 bp in length, including an open reading frame (ORF) of 1,011 bp. The open reading frame encoded a preproprotein of 336 amino acids (aa), which consisted of a signal peptide of 16 aa, a proregion peptide of 98 aa and a mature peptide of 222 aa. The preproprotein contained an oxyanion hole (Gln), a catalytic triad formed by Cys, His and Asn, and the conserved ERWNIN, GNFD and GCNGG motifs, all characteristic of cathepsin L. Homology analysis revealed that the deduced amino acid sequence of E. coioides cathepsin L shared 80.1-94.8 % identity with those of reported fishes. Tissue-dependent mRNA expression analysis showed that the cathepsin L transcript was expressed in all the examined tissues of the healthy E. coioides, being highest in the liver and moderate in the heart, gonad and intestine. After Vibrio anguillarum stimulation, the mRNA expression of cathepsin L in E. coioides was significantly increased in the skin, fin, gills, liver, blood, spleen, head kidney and intestine, with the highest observed in the spleen (10.6-fold) at 12 h post-injection and the next in blood (7.5-fold) at 8 h post-injection. These results provided initial information for further studies on the physiological and immunological roles of the cathepsin L gene in the orange-spotted grouper.