Genomic structure, characterization and expression analysis of a manganese superoxide dismutase from pearl oyster Pinctada fucata.

Manganese superoxide dismutase (MnSOD) is a major component of the cellular defense mechanisms against oxidative damage. We cloned and analyzed the expression pattern and genomic structure of the MnSOD gene of pearl oyster Pinctada fucata, hereafter designated as PoMnSOD. The full-length PoMnSOD cDNA was 1080 bp in length and consisted of a 5'-untranslated region (UTR) of 222 bp, a 3'-UTR of 318 bp with a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 540 bp encoding a polypeptide of 180 amino acids with an estimated molecular mass of 20.4 kDa and a predicted pI of 6.72. Sequence analysis showed that PoMnSOD contained MnSOD family signatures F(44)NGGGHLNH(52), I(97)QGSGWGWLA(106) and D(138)VWEHAYY(145), four conserved residues for manganese metal binding (H(4), H(52), D(138) and H(142)), and two potential N-glycosylation sites (N(33) and N(51)). Homology analysis revealed that PoMnSOD shared 47.6-55.9% identity and 57.4-65.6% similarity to the other known PoMnSOD amino acid sequences. PoMnSOD genomic DNA was 5040 bp in length and contained three exons and two introns, which was a tripartite organization and coincided with the consensus GT-AG splicing rule. PoMnSOD promoter contained the various transcription factors associated with the immune modulation and stress responses. Quantitative RT-PCR analysis demonstrated that PoMnSOD was constitutively expressed in all detected tissues, and PoMnSOD mRNA expression was significantly up-regulated in intestine, mantle, gills, digestive gland and haemocytes after Vibrio alginolyticus injection. These results suggested that PoMoSOD was an acute-response protein involved in the innate immune responses of pearl oyster, and provided general information about the mechanisms of innate immune defense against bacterial infection in pearl oyster.

Molecular characterization and mRNA expression of catalase from pearl oyster Pinctada fucata.

Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, a catalase cDNA of peal oyster Pincatada fucata (designated as PoCAT) is cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) methods. PoCAT is 2428 bp long and consists of a 5'-UTR of 140 bp, an unusually long 3'-UTR of 749 bp, and an open reading frame (ORF) of 1539 bp. The ORF of PoCAT encodes a polypeptide of 512 amino acids with molecular weight of 58.1 kDa and the theoretical isoelectric point of 8.4. PoCAT shares 62.3-82.2% identity and 73.0-92.0% similarity to other catalase amino acid sequences. Sequence alignment indicates that PoCAT contains the proximal heme-ligand signature sequence (R³5¹LFSYSDT³58), the proximal active site signature (F6¹NRERIPERVVHAKGGGA78), and the three catalytic amino acid residues (His7², Asn¹45, and Tyr³55). PoCAT has two potential glycosylation sites (N4³6YS4³8 and N478FS48°) and a peroxisome targeting signal (ASL). PoCAT mRNA was ubiquitously expressed in all detected tissues, and the expression level of PoCAT mRNA was higher in intestine and mantle. The expression profile analysis showed that the expression level of PoCAT mRNA in intestine was significantly up-regulated at 2, 4 and 12 h after Vibrio alginolyticus stimulation. These results demonstrated that PoCAT is a typical member of catalase family and might be involved in innate immune responses of pearl oyster.