ABSTRACT
Microcystin (MC) is a cyclic heptapeptide toxin. Nuclear factor erythocyte 2-related factor 2 (Nrf2) can enhance cellular survival by mediating phase 2 detoxification and antioxidant genes. In this study, CpNrf2 cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full-length CpNrf2 cDNA sequence was 4259 bp, and its homology was the highest with Mizuhopecten yessoensis, reaching 46%. CpNrf2 transcription levels were examined in all tested tissues, and the highest level was in hepatopancreas from C. plicata. The recombinant protein pET32-CpNrf2 was purified with the content of 1.375 mg/mL. The expression levels of CpNrf2 mRNA were raised in hepatopancreas after MC stimulation. After CpNrf2 knockdown, CpNrf2 mRNA levels were significantly down-regulated after 24 h. Compared with control group, the expression levels of ARE-driven enzymes (CpMnSOD, CpCuZnSOD, CpTRX, CpPrx, CpSe-GPx and Cpsigma-GST) were significantly increased, and those enzyme activities were also significantly up-regulated in MC-stimulated group. However, in CpNrf2-iRNA group, they were significantly down-regulated. The results revealed that Nrf2/ARE pathway is very crucial to protect molluscs from MC.
Subject(s)
Antioxidants/metabolism , Gene Expression/drug effects , Microcystins/toxicity , NF-E2-Related Factor 2/genetics , Unionidae/drug effects , Water Pollutants, Chemical/toxicity , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Phylogeny , Recombinant Proteins/genetics , Unionidae/enzymology , Unionidae/geneticsABSTRACT
Selenoprotein W (SelW) is a selenocysteine containing protein with redox activity involved in the antioxidant response. In this study, a selenoprotein W was cloned from pearl mussel Cristaria plicata (designated as CpSelW), and the expression patterns were characterized in tissues after Aeromonas hydrophila challenged. The full-length cDNA of cpSelW was of 858bp, containing a 5' untranslated region (UTR) of 145bp, a 3' UTR of 455bp with a poly (A) tail, and an open reading frame (ORF) of 258bp encoding a polypeptide of 85 amino acids with the predicted molecular mass of 9.277kDa, which shared 61% identity with SelW from Gallus gallus. A tertiary structure model generated for the CpSelW displayed a ß-α-ß-ß-ß-α secondary structure pattern, which was similar to mouse SelW protein 3D structure. The mRNA of CpSelW was constitutively expressed in tested tissues of healthy mussel, including mantle, gill, hemocytes, muscle, and hepatopancreas, and it was highly expressed in hepatopancreas. After mussels were stimulated by A. hydrophila, the mRNA expression of CpSelW in hemocytes at 6, 12 and 24h, in gill at 12h and in hepatopancreas at 24h was significantly down-regulated.
Subject(s)
Bivalvia/genetics , Gene Expression Regulation , Selenoprotein W/genetics , Aeromonas hydrophila/physiology , Amino Acid Sequence , Animals , Base Sequence , Bivalvia/microbiology , Cattle , Cloning, Molecular , DNA, Complementary/genetics , Humans , Mice , Models, Molecular , Molecular Sequence Data , Organ Specificity , Phylogeny , Protein Structure, Secondary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Selenoprotein W/chemistry , Sequence HomologyABSTRACT
The phagocyte NADPH oxidase plays a crucial role in host defense against invading microorganisms by catalyzing the formation of reactive oxygen species, which is the precursor of a variety of microbicidal oxidants such as hydrogen peroxide (H2O2). In the present study, full-length cDNAs of three regulatory subunits of NADPH oxidase, including p40phox, p47phox, p67phox were cloned from head kidney of mandarin fish utilizing the reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Sequence analysis showed that the full length cDNA of p40phox is 1 406 nt, containing a 1 050 nt open reading frames that encodes a 349 amino acid protein, the full length cDNA of p47phox is 1 686 nt, containing a 1 209 nt open reading frames that encodes a 402 amino acid protein, the full length cDNA of p40phox is 2 185 nt, containing a 1 488 nt open reading frames that encodes a 495 amino acid protein. Semi-quantitative RT-PCR analyses from various tissues indicated that mRNAs of the three subunits can be detected in the blood, brain, heart, spleen, kidney and thymus, but their expression intensity are different in tissues. Stimulating the mandarin fish with formalin killed Flavobacterium columnare G4 significantly up-regulated the expression of p40phox in blood and head kidney; and p47phox in head kidney and spleen; and p67phox in blood, head kidney and spleen. The results suggested that mandarin NADPH oxidase was involved in the immune responses against bacteria.
Subject(s)
DNA, Complementary/metabolism , NADPH Oxidases/genetics , Perciformes/genetics , Protein Subunits/physiology , Reverse Transcriptase Polymerase Chain Reaction/methods , Amino Acid Sequence , Animals , Cloning, Molecular/methods , DNA, Complementary/genetics , Gene Expression Regulation , Hydrogen Peroxide/chemistry , Molecular Sequence Data , Phosphoproteins/genetics , Phosphoproteins/metabolismABSTRACT
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.
