Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity.
Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383 bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6 h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83 % after 10 min incubation at 10-50 °C, and more than 87 % after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.
Key Laboratory of Aquatic Genetic Resources and Aqua-cultural Ecosystem Certified by the Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China.
Xianggang
X
Gao
Chongbo
C
He
Hong
H
Liu
Hongjun
H
Li
Dan
D
Zhu
Shengli
S
Cai
Ying
Y
Xia
Ying
Y
Wang
Zhe
Z
Yu
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004795:Enzyme Stability; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000201:enzymology*; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D012333:RNA, Messenger; D013482:Superoxide Dismutase