PUBLICATION
Molecular cloning, identification and functional characterization of a novel intracellular Cu-Zn superoxide dismutase from the freshwater mussel Cristaria plicata
- Authors
- Xu, H.H., Ma, H., Hu, B.Q., Lowrie, D.B., Fan, X.Y., and Wen, C.G.
- ID
- ZDB-PUB-100628-1
- Date
- 2010
- Source
- Fish & shellfish immunology 29(4): 615-622 (Journal)
- Registered Authors
- Ma, Hui
- Keywords
- Cristaria plicata, Superoxide dismutase (SOD), Enzyme activity, L02 hepatocyte, Oxidative damage
- MeSH Terms
-
- Antioxidants/metabolism
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/metabolism
- Amino Acid Sequence
- Cloning, Molecular
- DNA, Complementary/chemistry
- DNA, Complementary/genetics
- Cell Line
- Superoxide Dismutase/chemistry
- Superoxide Dismutase/genetics*
- Superoxide Dismutase/metabolism*
- Polymerase Chain Reaction
- Animals
- Hydrogen-Ion Concentration
- Molecular Sequence Data
- Protein Denaturation
- Bivalvia/genetics*
- Bivalvia/metabolism*
- Temperature
- Sequence Alignment
- Humans
- Base Sequence
- PubMed
- 20561586 Full text @ Fish Shellfish Immunol.
Citation
Xu, H.H., Ma, H., Hu, B.Q., Lowrie, D.B., Fan, X.Y., and Wen, C.G. (2010) Molecular cloning, identification and functional characterization of a novel intracellular Cu-Zn superoxide dismutase from the freshwater mussel Cristaria plicata. Fish & shellfish immunology. 29(4):615-622.
Abstract
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 468bp 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 8h, and then was purified by using the native Ni(2+) affinity chromatography. The specific activity of the purified rCp-icCuZnSOD enzyme was 5,368 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 8mol/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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping