Transcriptional up-regulation of disk abalone selenium dependent glutathione peroxidase by H(2)O(2) oxidative stress and Vibrio alginolyticus bacterial infection.
ABSTRACT Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant in the cellular defence system. We have identified Se-GPx full length cDNA from disk abalone (Haliotis discus discus) designated as AbSe-GPx. It has a characteristic codon at (223)TGA(225) that corresponds to selenocysteine (Sec) amino acid as U(75). The full length cDNA consists of 675 bp, an open reading frame encoding 225 amino acids. Sequence characterization revealed that AbSe-GPx contains a characteristic GPx signature motif 2 ((97)LGFPCNQF(104)), an active site motif ((183)WNFEKF(188)) and essential residues for the enzymatic function. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) is conserved in the 3' UTR. The AbSe-GPx amino acid sequence exhibited the highest level of identity (46%) with insect (Ixodes scapularis) GPx, and shares 41% with bivalve (Unio tumidus) Se-GPx. The RT-PCR analysis revealed that AbSe-GPx mRNA was expressed constitutively in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes in a tissue specific manner. AbSe-GPx mRNA expression was significantly up-regulated in gill and digestive tract tissues after H(2)O(2) injection and Vibrio alginolyticus infection. However, AbSe-GPx expression was not up-regulated after Aroclor 1,254 injection. These results indicate that AbSe-GPx mRNA is expressed at a basal level in abalone tissues, which can be up-regulated transcriptionally by H(2)O(2) oxidative stress and Vibrio alginolyticus infection. Therefore, AbSe-GPx may be involved in a protective role against H(2)O(2) oxidative stress and immune defence against bacterial infection.
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ABSTRACT: In this study, we have reported a full length of peroxiredoxin (designated MrPrdx) gene, identified from the transcriptome of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrPrdx is 940 base pairs in length, and encodes 186 amino acids. MrPrdx contains a long thioredoxin domain in the amino acid sequence between 34 and 186. The gene expressions of MrPrdx in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction. MrPrdx is highly expressed in all the other tissues of M. rosenbergii considered for analysis and the highest in gills. The expression is strongly up-regulated in gills after IHHNV infection. To understand MrPrdx functional properties, the recombinant MrPrdx protein was expressed in Escherichia coli BL21 (DE3) and purified. A peroxidise activity assay was conducted using recombinant MrPrdx protein at different concentrations. This peroxidises activity showed that the recombinant MrPrdx is a thiol-dependant protein. Additionally, this result showed that recombinant MrPrdx protein, as a secretory protein can remove H₂O₂ and protect DNA damage. This finding leads a possible way to propose the recombinant MrPrdx protein as an effective medicine for reactive oxygen species (ROS) related diseases.Fish & Shellfish Immunology 04/2012; 33(1):121-9. · 2.96 Impact Factor
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ABSTRACT: 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 β-α-β-β-β-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.Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 10/2013; · 1.61 Impact Factor
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ABSTRACT: A selenium-dependent glutathione peroxidase cDNA was obtained from green mud crab Scylla paramamosain (SpGPx) by homology PCR technique and rapid amplification of cDNA ends (RACE) methods. The 1135 bp full-length cDNA contains a 9 bp 5′-untranslated region (UTR), an open reading frame (ORF) of 564 bp encoded a deduced protein of 187 amino acids (aa), and a 562 bp 3′-UTR with a 100 bp conserved eukaryotic selenocysteine insertion sequence (SECIS). It involves a putative selenocysteine (Sec40, or U40) residue which is encoded by an opal codon, 127TGA129, and forms an active site with residues Q74 and W142. Sequence characterization revealed that SpGPx contain a characteristic GPx signature motif 2 (64LAFPCNQF71), an active site motif (152WNFEKF157), a potential N-glycosylation site (76NTT78), and two residues (R90 and R168) which contribute to the electrostatic architecture by directing the glutathione donor substrate. Multiple sequence alignment and phylogenetic analysis showed that SpGPx share a high level of identities and closer relationship with other selected invertebrate GPxs and vertebrate GPx1 and GPx2. Molecular modelling analysis results also supported these observations. Real time quantitative PCR analysis revealed that SpGPx was constitutively expressed in 10 selected tissues, and its expression level in gill and testis was higher than that in the other tissues (p < 0.05). The SpGPx expression increased and then declined during ovarian and testicular development implying thatnscrpits yowed that SpGPx might play an important role in gonad development by protecting them from oxidative stress. The expression of SpGPx mRNA was induced by lipopolysaccharide (LPS) and hydrogen peroxide (H2O2) in hepatopancreas and haemocytes. The results suggested that SpGPx was implicated in the immune response induced by LPS and H2O2.Fish & Shellfish Immunology 09/2012; 33(3):532–542. · 2.96 Impact Factor