Molecular cloning, identification and functional characterization of a novel intracellular Cu-Zn superoxide dismutase from the freshwater mussel Cristaria plicata.
Superoxide dismutases (SODs, EC 1.15.1.1) are one family of important antioxidant metalloenzymes involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular CuZnSOD gene of Cristaria plicata (Cp-icCuZnSOD) was identified from hemocytes by homology cloning and the rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of Cp-icCuZnSOD consisted of 891 nucleotides with a canonical polyadenylation signal sequence ATTAAA, a poly (A) tail, and an open-reading frame of 468 bp encoding 155 amino acids. The deduced amino acids of CpSOD shared high similarity with the known icCuZnSODs from other species, and several highly conserved motifs including Cu/Zn ions binding sites (His-46, His-48, His-63, His-120 for Cu(2+) binding, and His-63, His-71, His-80, Asp-83 for Zn(2+) binding), intracellular disulfide bond and two CuZnSOD family signatures were also identified in CpSOD. Furthermore, the recombinant Cp-icCuZnSOD with high enzyme activity was induced to be expressed as a soluble form by IPTG supplemented with Cu/Zn ions at 20 degrees C for 8 h, and then was purified by using the native Ni(2+) affinity chromatography. The specific activity of the purified rCp-icCuZnSOD enzyme was 5368 U/mg, which is 2.6-fold higher than that of zebrafish Danio rerio rZSOD and 5.3-fold higher than that of bay scallop Argopecten irradians rAi-icCuZnSOD. The enzyme stability assay showed that the purified rCp-icCuZnSOD enzyme maintained more than 80% activity at temperature up to 60 degrees C, at pH 2.0-9.0, and was resistant to 8 mol/L urea or 8% SDS. In addition, the addition of active rCp-icCuZnSOD enzmye could protect hepatocyte L02 cells from oxidative damage as assessed using an alcohol-injured human liver cell model.
Xu
Huan-Huan
HH
School of Life Sciences and Food Engineering, Nanchang University, Nanchang 330031, China.
Ma
Hui
H
Hu
Bao-Qing
BQ
Lowrie
Douglas B
DB
Fan
Xiao-Yong
XY
Wen
Chun-Gen
CG
D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001483:Base Sequence; D049872:Bivalvia / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
D000975:Antioxidants; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase