Chungen Wen

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.

Seasonal variation of a population Unionicola penicillatus (Unionicolidae) from the freshwater bivalve Cristaria plicata (Unionidae) in Poyang Lake, eastern China

Abstract The water mite Unionicola penicillatus (Wen, Gao & Zhu) (Acari: Unionicolidae) was collected in freshwater bivalve Cristaria plicata (Unionidae). The population variation of U. penicillatus was investigated in 504 bivalves during the period from August 2013 to July 2014 in Poyang Lake, East China. The results indicated that the overall prevalence and abundance of U. penicillatus was 9.52% and 1.58±10.38. The highest prevalence was 58.70% in September. The highest abundance levels were observed when the length of bivalves was in 220– 300 mm. The number of U. penicillatus in a host was not correlated with the sex of bivalves. The outer and inner gills of bivalves were the preferred sites of U. penicillatus.

Identification and characterization of two distinct sigma-class glutathione-S-transferase from freshwater bivalve Cristaria plicata

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione, and play an important role in protecting organisms against the toxicity of reactive oxygen species. In this study, two distinct sigma-class GST (CpGSTσ1 and 2) cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full length cDNA of CpGSTσ1 and 2 was 826 bp and 1609 bp, which encoded 213 and 248 amino acid residues, respectively. Their transcripts were expressed in all detected tissues and the highest expression level was in hepatopancreas from C. plicata. The expression level of CpGSTσ1 and 2 in hepatopancreas and hemocytes showed a significantly increased trend after bacterial challenge. The recombinant CpGSTσ1 was successfully expressed as a soluble form in Escherichia coli DE3. The specific activity of recombinase toward CDNB was 46.965 ± 0.082 μmol/min/mg, and its optimum temperature and pH was 37 °C and 9.0, respectively. The recombinant of CpGSTσ1 could bear 6 M urea and 8% SDS, when the concentration of urea was 8 M and its activity was only below 20%. The results might provide a better perspective on the mechanisms of resistance to bacterial infection in molluscs.

Molecular characterization of two distinct Smads gene and their roles in the response to bacteria change and wound healing from Hyriopsis cumingii

Abstract The proteins of Smad family are critical components of the TGF‐&bgr; superfamily signal pathway. In this paper, we cloned two intracellular mediators of TGF‐&bgr; signaling, Smad3 and Smad5, from the pearl mussel Hyriopsis cumingii. The full length cDNA of HcSmad3 and HcSmad5 were 2052 bp and 1908 bp and encoded two polypeptides of 418 and 461amino acid residues, respectively. The deduced amino acid of HcSmad3 and HcSmad5 possessed two putative conserved domains, MH1 and MH2, a conserved phosphorylation motif SSXS at the carboxyl‐terminal. The two Smad genes were detected muscle, mantle, hepatopancreas and gill, but with a very low level in heamocytes. The transcripts of Smad3 and Smad5 were up‐regulated in hemocytes and hepatopancreas after A. hydrophila and PGN stimulation. However, the expression of Smad3 and Smad5 were only up‐regulated in hepatopancreas after A. hydrophila stimulation. The transcripts of Smad3 and Smad5 had a slight change in hepatopancreas after PGN stimulation. The transcripts of HcSmad3 showed very little increase and HcSmad5 mRNA significantly up‐regulated after wounding. HighlightsSmad3 and Smad5 were cloned from Hyriopsis cumingii.The transcripts of Smad3,5 were up‐regulated after A. hydrophila and PGN stimulation.HcSmad5 mRNA significantly up‐regulated after wounding.

Molecular cloning, expression and antioxidative activity of 2-cys-peroxiredoxin from freshwater mussel Cristaria plicata

Abstract Peroxiredoxins (Prxs) play an important role against various oxidative stresses by catalyzing the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides to less harmful form. A 2‐cys peroxiredoxin, designated as CpPrx, was cloned from hemocytes of freshwater mussel Cristaria plicata. The full length cDNA of CpPrx is 1247 bp, which includes an open reading frame (ORF) of 591bp, encoding 196 amino acids. CpPrx possesses two conserved cysteine residues (Cys49, Cys170). The deduced amino acid sequence of CpPrx showed a high level (67–74%) of sequence similarity to 2‐Cys Prxs from other species. The results of real‐time quantitative PCR revealed that CpPrx mRNA was constitutively expressed in tissues, and the highest expression levels were in hepatopancreas and gills. After peptidoglycan (PGN) and Aeromonas hydrophila challenge, the expression levels of CpPrx mRNA were up‐regulated in hemocytes and hepatopancreas. The cDNA of CpPrx was cloned into the plasmid pET‐32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). Comparison with DE3‐pET‐32 and DE3 strain, the cells of DE3‐pET‐32‐CpPrx exhibited resistance to the concentration of 0.4, 0.8 and 1.2 mmoL/L H2O2 in vivo. HighlightsThe full cDNA sequences of CpPrx were cloned.The transcripts of CpPrx were up‐regulated after stimulation.The recombinant had antioxidant activity.

Identification and characterization of two LBP/BPI genes involved in innate immunity from Hyriopsis cumingii

ABSTRACT Lipopolysaccharide‐binding protein and bactericidal permeability‐increasing protein (LBP/BPI) play crucial role in modulating cellular signals in response to Gram‐negative bacteria infection. In the present study, two isoforms of LBP/BPI genes, designated as HcLBP/BPI1 and HcLBP/BPI2, respectively, were cloned from the mussel Hyriopsis cumingii by RACE approach. The full‐length cDNA sequences of HcLBP/BPI1 and HcLBP/BPI2 were 1887 and 2227 bp and encoded two secreted proteins of 501 and 518 amino acid residues, respectively. The deduced amino acid of HcLBP/BPI1 and HcLBP/BPI2 contained several conserved domains, such as signal peptide, two BPI/LBP and one central domain. Phylogentic analysis further supported that HcLBP/BPI1 and HcLBP/BPI2 belonged to new members of invertebrate LBP/BPI family. The mRNA transcripts of HcLBP/BPI1 and HcLBP/BPI2 were ubiquitously expressed in all examined tissues, and the expression level of HcLBP/BPI1 was higher than that of HcLBP/BPI2. The mRNA expression of HcLBP/BPI1 in hepatopancreas and hemocytes was significantly up‐regulate after Aeromonas hydrophila and LPS challenge, and HcLBP/BPI2 in hepatopancreas was only up‐regulated at 6 and 12 h after LPS challenge and at 12 h after A. hydrophila challenge. In addition, the recombinant HcLBP/BPIs displayed antibacterial activity against Gram‐negative bacteria, and the antibacterial index of HcLBP/BPI1 was higher than that of HcLBP/BPI2. These results indicated that HcLBP/BPI1 and HcLBP/BPI2 probably played distinct roles in bacterial mediating immune response in Mollusca. HIGHLIGHTSThe full cDNA sequences of HcLBP/BPI1,2 were cloned.The transcripts of BPI1,2 were up‐regulated after stimulation.The recombinant had antibacterial activity.

Molecular cloning and functional characterization of a novel i-type lysozyme in the freshwater mussel Cristaria plicata

The freshwater bivalve Cristaria plicata, which is widely distributed in Eastern Asia, is a key species in the pearl culture industry. In this study, a novel invertebrate‐type lysozyme, designated as CpLYZ2, was cloned from hemocytes of C. plicata. This lysozyme shares high sequence identity and is homologous to a previously identified lysozyme CpLYZ1 isolated from C. plicata and with HcLyso3 isolated from Hyriopsis cumingii. The full‐length cDNA of CpLYZ2 is 913 bp long, which includes an open reading frame (ORF) of 486 bp, a 3′ untranslated region (UTR) of 389 bp and a 5′ UTR of 38 bp. The ORF encodes a putative polypeptide of 161 amino acids with a predicted molecular mass of 18.2 kDa and a theoretical isoelectric point of 6.56. CpLYZ2 mRNA transcripts can be detected in hemocytes, hepatopancreas, muscle, gills and mantle tissues, the greatest expression being observed in the gills. CpLYZ2 expression in hemocytes, hepatopancreas and gills increased significantly after the mussel was challenged with Aeromonas hydrophila. Furthermore, the optimal pH and temperature for enzyme activity of the recombinant CpLYZ2 were 5.5 and 50°C, respectively. The recombinant lysozyme protein exhibited bacteriolytic activity against Escherichia coli, A. hydrophila, Staphyloccocus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis. The findings of this study help to elucidate immune responses in molluscs and will thus expedite disease management of these key freshwater species, in turn boosting pearl culture in eastern Asia.