Identification and characterization of two selenium-dependent glutathione peroxidase 1 isoforms from Larimichthys crocea

Abstract

Glutathione peroxidases, a vital family of antioxidant enzymes in oxybiotic organisms, are involved in anti-pathogen immune response. In this study, two complete selenium-dependent glutathione peroxidase 1 cDNAs (designated as LcGPx1a and LcGPx1b) were obtained from the large yellow croaker Larimichthys crocea by rapid amplification of cDNA ends. The full-length sequence of LcGPx1a was 917 bp with a 5′-untranslated region (UTR) of 52 bp, a 3′-UTR of 289 bp, and an open reading frame of 576 bp encoding 191 amino acid (aa) polypeptides. The cDNA of LcGPx1b was composed of 884 bp with a 5′-UTR of 59 bp, a 3′-UTR of 258 bp, and an open reading frame of 567 bp encoding 188 aa polypeptides. The conserved selenocysteine insertion sequence was detected in the 3′-UTR of both isoforms, which can classify types I and II. Protein sequence analysis revealed that both isoforms included a selenocysteine encoded by an opal codon (TGA) and formed the functioning tetrad site with glutamine, tryptophan, and asparagine. Three conservative motifs, including one active site motif (“GKVVLIENVASLUGTT”) and two signature site motifs (“LVILGVPCNQFGHQENC” and “V(A/S)WNFEKFLI”), were conserved both in sequence and location. Multiple alignments revealed that they exhibited a high level of identities with GPx1 from other organisms, especially in the abovementioned conserved amino acid sequence motifs. Tissue expression analysis indicated that LcGPx1a and LcGPx1b had a wide distribution in nine tissues with various abundances. The transcript level of LcGPx1a was not significantly different among the nine tissues, whereas that of LcGPx1b was higher in the kidney and head kidney than in the other tissues. After Vibrio parahaemolyticus stimulation, the expression levels of LcGPx1a and LcGPx1b were unanimously altered in the liver, spleen, kidney, and head kidney but with different magnitudes and response time. LcGPx1a and LcGPx1b showed distinct expression trends in the liver, where LcGPx1b was induced and LcGPx1a was depressed in response to pathogen infection. These results indicate that LcGPx1a and LcGPx1b display functional diversities and play crucial roles in mediating the immune response of fish.