A potential antioxidant enzyme belonging to the atypical 2-Cys peroxiredoxin subfamily characterized from rock bream, Oplegnathus fasciatus.

Peroxiredoxins (Prxs), a diverse family of antioxidant enzymes, exert their antioxidant function through which different peroxide species are detoxified. This study describes both structural and functional characterization of a mitochondrial Prx identified in rock bream, Oplegnathus fasciatus (RbPrx5). The ORF (573 bp) of RbPrx5 encoded a protein of 190 amino acids (20 kDa) containing a putative mitochondrial targeting sequence (residues 1-20) and a thioredoxin-2 motif (residues 31-190) and three conserved Cys residues. Homology assessment and phylogenetic analysis clearly disclosed relatively higher amino acid sequence similarities and a closer evolutionary position of RbPrx5 with those of other teleost homologs. The ORF of RbPrx5 was distributed among six exons as found in other vertebrates, but it possessed an additional exon in its 5'-UTR. In silico examination of RbPrx5 gene's putative promoter region revealed the presence of several cis-elements which may be important in its transcriptional regulation. Constitutive expression of RbPrx5 was detected in eleven tissues with the highest level in the heart. Modulation of RbPrx5 transcription was evidenced from varying mRNA levels in head kidney post in vivo LPS-, poly I:C-, Edwardsiella tarda bacterial- and rock bream iridoviral-challenges. The antioxidant function of RbPrx5 was investigated using recombinant RbPrx5 protein. Results of an in vitro mixed-function oxidase assay demonstrated a dose-dependent inhibition of DNA damage by rRbPrx5. A H2O2 tolerance assay showed that in vivo overexpression of rRbPrx5 increased the bacterial survival under H2O2-mediated oxidative stress condition. These findings provide an overall insight into the structural, expressional and functional aspects of RbPrx5.

A mu class glutathione S-transferase from Manila clam Ruditapes philippinarum (RpGSTµ): cloning, mRNA expression, and conjugation assays.

Glutathione S-transferases (GSTs) are enzymes that catalyze xenobiotic metabolism in the phase II detoxification process. GSTs have a potential for use as indicators or biomarkers to assess the presence of organic and inorganic contaminants in aquatic environments. In this study, a full-length cDNA of a mu (µ) class GST (RpGSTµ) was identified from Manila clam (Ruditapes philippinarum) and biochemically characterized. The 1356 bp of the cDNA included an open reading frame of 651 bp encoding a polypeptide of 217 amino acid residues with a molecular mass of 25.04 kDa and an estimated pI of 6.34. Sequence analysis revealed that the RpGSTµ possessed several characteristic features of µ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione (GSH), a C-terminal domain containing substrate binding sites, and a µ loop. The recombinant RpGSTµ (rRpGSTµ) protein exhibited GSH-conjugating catalytic activity towards several substrates, and significantly strong activity was detected against 4-nitrophenethyl bromide (5.77 ± 0.55) and 1-chloro-2,4-dinitrobenzene (CDNB, 3.19 ± 0.05). Kinetic analysis as a function of GSH and CDNB concentrations revealed relatively low Km values of 1.03 ± 0.46 mM and 0.56 ± 0.20 mM, respectively, thereby indicating a GSH-conjugation attributed with high rates. The optimum pH and temperature for the catalytic activity of the rRpGSTµ protein were 7.7 and 37°C, respectively. The effect of two inhibitors, Cibacron blue and hematin, on the activity of rRpGSTµ was evaluated and the IC50 values of 0.65 µM and 9 µM, respectively, were obtained. While RpGSTµ transcripts were highly expressed in gills and hemocytes, a significant elevation in mRNA levels was detected in these tissues after lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C) and live bacterial (Vibrio tapetis) challenges. These findings collectively suggest that RpGSTµ functions as a potent detoxifier of xenobiotic toxicants present in the aquatic environment, and that its mRNA expression could be modulated by pathogenic stress signal(s).

Identification and in silico analysis of a novel troponin C like gene from Ruditapes philippinarum (Bivalvia: Veneridae) and its transcriptional response for calcium challenge.

Troponin C (TnC) is one of the subunits composing the troponin complex, which is primarily expressed in muscle tissue and plays a major role in regulating contractility. We have identified a novel TnC-like gene (RpTnC) from the Ruditapes philippinarum Manila clam. Sequence analysis indicated that RpTnC has a 450bp coding sequence, encoding a 150 amino acid protein with a molecular mass of 17.4 kDa. The RpTnC protein consisted of four EF-hand motifs (I-IV), each with a Ca2+-binding site. In silico comparative analysis of protein sequence showed that only site IV, demonstrating a conserved stretch (DxDxSx6E), is functionally active for Ca2+-coordination. Moreover, RpTnC was homologically (61.3% identity) and phylogenetically closest to Japanese flying squid TnC. The mRNA expression analysis using quantitative real-time PCR revealed a differential basal-expression of RpTnC transcripts in six different clam tissues, with higher levels in adductor muscle and mantle. Intramuscular administration of CaCl2 caused a prominent upregulation of RpTnC transcripts in adductor muscle (~5 fold). Collectively, our findings suggest that the TnC homolog of Manila clam identified in this study may be involved in important role(s) in clam physiology, mainly in its muscle tissues, and its transcription could be significantly influenced by increased Ca2+ levels.

A novel acute phase reactant, serum amyloid A-like 1, from Oplegnathus fasciatus: genomic and molecular characterization and transcriptional expression analysis.

Acute phase response is a significant component of innate immunity, playing a vital role in the signaling processes and elimination of invading pathogens. Acute phase proteins are synthesized in liver and secreted into the blood for transportation to an infection site, where the defense function is exerted. Serum amyloid A (SAA) and C-reactive proteins are the major positive acute phase proteins. In this study, we have identified and characterized a novel SAA related gene from rock bream (Oplegnathus fasciatus), designated OfSAAL1. Genomic characterization revealed the presence of 13 exons and 12 introns, similar to SAAL1 in zebrafish. Multiple protein sequence alignment revealed high conservation with other SAAL1 homologues. Phylogenetic analysis showed that OfSAAL1 clustered with another fish homologue, and pairwise alignment revealed highest identity and similarity at the amino acid level with zebrafish SAAL1. Promoter region analysis revealed the presence of immunologically significant transcription factor binding sites. Tissue distribution profiling to indicate physiological relevance showed the highest levels occur in blood, followed by liver, suggesting a positive immune role in rock bream. Transcriptional analysis by reverse transcription polymerase chain reaction to understand OfSAAL1 responsiveness to immune challenge with poly I:C, Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus, revealed a significant level of elevation from 12h to 48 h post-infection in blood, spleen, head kidney, and liver. To our knowledge, OfSAAL1 is the first characterized SAAL1 homologue from teleosts. We anticipate that its identification will prove inspiring for further studies of SAAL1 homologues as biomarkers of the acute phase response.

First molluscan theta-class Glutathione S-Transferase: identification, cloning, characterization and transcriptional analysis post immune challenges.

Glutathione S-Transferases (GSTs) are multifunctional cytosolic isoenzymes, distinctly known as phase II detoxification enzymes. GSTs play a significant role in cellular defense against toxicity and have been identified in nearly all organisms studied to date, from bacteria to mammals. In this study, we have identified a full-length cDNA of the theta class GST from Ruditapes philippinarum (RpGSTθ), an important commercial edible molluscan species. RpGSTθ was cloned and the recombinant protein expressed, in order to study its biochemical characteristics and determine its physiological activities. The cDNA comprised an ORF of 693 bp, encoding 231 amino acids with a predicted molecular mass of 27 kDa and an isoelectric point of 8.2. Sequence analysis revealed that RpGSTθ possessed characteristic conserved domains of the GST_N family, Class Theta subfamily (PSSM: cd03050) and GST_C_family Super family (PSSM: cl02776). Phylogenetic analysis showed that RpGSTθ evolutionarily linked with other theta class homologues. The recombinant protein was expressed in Escherichia coli BL21(DE3) cells and the purified enzyme showed high activity with GST substrates like CDNB and 4-NBC. Glutathione dependent peroxidase activity of GST, investigated with cumene hydroperoxide as substrate affirmed the antioxidant property of rRpGSTθ. By quantitative PCR, RpGSTθ was found to be ubiquitously expressed in all tissues examined, with the highest levels occurring in gills, mantle, and hemocytes. Since GSTs may act as detoxification enzymes to mediate immune defense, the effects of pathogen associated molecular pattern, lipopolysaccharide and intact Vibrio tapetis bacteria challenge on RpGSTθ gene transcription were studied. Furthermore, the RpGSTθ expression changes induced by immune challenges were similar to those of the antioxidant defense enzyme manganese superoxide dismutase (RpMnSOD). To our knowledge, RpGSTθ is the first molluscan theta class GST reported, and its immune-related role in Manila clam may provide insights into potential therapeutic targets for protecting this important aquaculture species.