Molecular cloning, characterization, and the response of manganese superoxide dismutase from the Antarctic bivalve Laternula elliptica to PCB exposure

摘要

Manganese superoxide dismutase (leMnSOD) cDNA was cloned from the Antarctic bivalve Laternula elliptica. The full-length cDNA of leMnSOD is 1238 bp in length and contains an open reading frame of 681 bp encoding 226 amino acid residues including a putative mitochondrial targeting peptide of 26 amino acids in the N-terminal region. The calculated molecular mass is 24.8 kDa with an estimated isoelectric point of 6.75. leMnSOD signatures from 185 to 192 (DVWEHAYY) and four conserved amino acids (H52, H11, D185, and H192) responsible for binding manganese were observed. Sequence comparison showed that leMnSOD had high levels of identity with MnSOD from Haliotis discus discus, Mizuhopecten yessoensis, and Crassostrea gigas (68%, 66%, and 59%, respectively). RT-PCR analysis revealed the presence of leMnSOD transcripts in all tissues examined. Quantitative real-time RT-PCR assay indicated that treatment with polychlorinated biphenyls (PCBs) significantly increased leMnSOD mRNA expression in an organ-, time-, and dose-dependent manner. The mRNA expression with exposure to PCBs at 0.1 and 10 ppb reached the highest level at 6 h and then recovered slightly from 6 to 48 h in the gill. In contrast, the expression of leMnSOD mRNA showed a different expression pattern related to PCB concentration in the digestive gland. The mRNA expression at 0.1 ppb PCBs increased up to 12 h and then decreased by 48 h, but increased immediately at 10 ppb PCBs. The leMnSOD was overproduced in Escherichia coli and purified. The recombinant leMnSOD showed maximum activity at pH 9.0, and it retained more than 50% of its original activity after incubation for 30 min at 40 super(o)C.