Kasthuri Saranya Revathy

A novel molluscan sigma-like glutathione S-transferase from Manila clam, Ruditapes philippinarum: Cloning, characterization and transcriptional profiling

Glutathione S-transferases (GSTs) are versatile enzymes, act as primary intracellular detoxifiers and contribute to a broad range of physiological processes including cellular defense. In this study, a full-length cDNA representing a novel sigma-like GST was identified from Manila clam, Ruditapes philippinarum (RpGSTσ). RpGSTσ (884 bp) was found to possess an open reading frame of 609 bp. The encoded polypeptide (203 amino acids) had a predicted molecular mass of 23.21 kDa and an isoelectric point of 7.64. Sequence analysis revealed two conserved GST domain profiles in N- and C-termini. Alignment studies revealed that the identity between deduced peptides of RpGSTσ and known GSTσ members was relatively low (<35%), except a previously identified Manila clam GSTσ isoform (87.2%). Phylogenetic analysis indicated that RpGSTσ clustered together with molluscan GSTσ homologs, which were closely related to insect GSTσs. The RpGSTσ was subsequently cloned and expressed as recombinant protein, in order to characterize its biological activity. The recombinant RpGSTσ exhibited characteristic glutathione conjugating catalytic activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloronitrobenzene and ethacrynic acid. It had an optimal pH and temperature of 8.0 and 35 °C, respectively. Expression profiles under normal conditions and in response to lipopolysaccharide-, poly I:C- and Vibrio tapetis-challenges were also investigated. RpGSTσ demonstrated a differential tissue distribution with robust transcription in gills of normal animals. We explored potential association of GSTσ in cellular defense during bacterial infection and found that in challenged clams, RpGSTσ gene was significantly induced in internal and external tissues, in conjunction with manganese- as well as copper-zinc superoxide dismutase (MnSOD and CuZnSOD) genes. Moreover, the induction was remarkably higher in hemocytes than in gill. Collectively, our findings suggested that RpGSTσ could play a significant role in cellular defense against oxidative stress caused by bacteria, in conjunction with other antioxidant enzymes, such as SODs.

A manganese superoxide dismutase with potent antioxidant activity identified from Oplegnathus fasciatus: Genomic structure and transcriptional characterization

In this study, we describe the identification and characterization of manganese superoxide dismutase, an important antioxidant enzyme acting as the chief reactive oxygen species (ROS) scavenger, from rock bream Oplegnathus fasciatus (Of-mMnSOD) at genomic- and transcriptional-levels as well as the biological activity of recombinant protein. The Of-mMnSOD protein portrayed distinct MnSOD family features including signature motifs, metal association sites and the typical active site topology. It was also predicted to be localized in mitochondrial matrix. The Of-mMnSOD had a quinquepartite genome organization encompassing five exons interrupted by four introns. Comparison of its sequence and gene structure with that of other lineages emphasized its strong conservation among different vertebrates. The Of-mMnSOD was ubiquitously transcribed in different rock bream tissues with higher levels in blood cells and metabolically active tissues. Transcription of Of-mMnSOD was kinetically modulated in response to investigational challenges using mitogens (lipopolysaccharide and poly I:C) and live-pathogens (Edwardsiella tarda and rock bream irido virus) in blood cells and liver tissue. The purified recombinant Of-mMnSOD possessed potential antioxidant capacity and actively survived over a range of pH (7.5-11) and temperature (15-40 °C) conditions. Collectively, findings of this study suggest that Of-mMnSOD combats against oxidative stress and cellular damages induced by mitogen/pathogen-mediated inflammation, by detoxifying harmful ROS (O(2)(●-)) in rock bream.

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.

Cytosolic thioredoxin from Ruditapes philippinarum: Molecular cloning, characterization, expression and DNA protection activity of the recombinant protein

Thioredoxin (TRx) is a small redox protein that plays significant roles in protection against oxidative stress and in cell homeostasis by maintaining oxidized proteins in a reduced state. Here, we describe the isolation and characterization of a full-length TRx cDNA sequence from manila clam, Ruditapes philippinarum and named it as RpTRx. The full length sequence consists of 1416 bp with an open reading frame of 318 bp encoding for 106 amino acids. RpTRx protein harbors evolutionarily-conserved TRx active site (32)WCGPC(36). Phylogenetic analysis revealed a close proximity of RpTRx with the orthologue in Japanese scallop, Chlamys farreri. RpTRx was found to be constitutively expressed in hemocyte, gill, mantle, foot and siphon indicating a general role in physiological processes in various tissues. With regard to a potential role in immune responses, the RpTRx mRNA was found to be up-regulated in hemocytes after bacterial (Vibrio tapetis) and lipopolysaccharide (LPS) challenge at 3h post-infection (p.i.); a wavering increase was observed up to 96 h p.i. for LPS challenge and 48 h p.i. for bacterial challenge. Thus, RpTRx may function as an intracellular antioxidant to protect the cells against ROS induced by LPS and bacterial challenges. Indeed, when recombinant RpTRx protein (rRpTRx) was over-expressed in Escherichiacoli Rosetta gami(TM) (DE3) cells, it was able to scavenge free radicals and protect super-coiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. In summary, RpTRx plays an essential role in cellular defense and maintenance of homeostasis in the manila clam.

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.

Mitochondrial thioredoxin-2 from Manila clam (Ruditapes philippinarum) is a potent antioxidant enzyme involved in antibacterial response.

Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561 bp) consists of a 498 bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic (89)WCGPC(93) catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams.

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 teleostean angiotensinogen from Oplegnathus fasciatus responses to immune and injury challenges.

Angiotensinogen (AGT) is the precursor of the renin-angiotensin system and contributes to osmoregulation, acute-phase and immune responses. A full-length cDNA of the AGT (2004 bp with a 1389 bp coding region) was isolated from rock bream (Rb), Oplegnathus fasciatus. The encoded polypeptide of 463 amino acids had a predicted molecular mass of 51.6 kDa. RbAGT possessed a deduced signal peptide of 22 residues upstream of a putative angiotensin I sequence ((23)NRVYVHPFHL(32)). RbAGT possessed a specific domain profile and a signature motif which are characteristics of the serpin family. Sequence homology and phylogenetic analysis indicated that RbAGT was evolutionarily closest to AGT of Rhabdosargus sarba. The mRNA expression profile of RbAGT was determined by quantitative RT-PCR and it demonstrated a constitutive and tissue-specific expression with the highest transcript level in the liver. Significantly up-regulated RbAGT expression was elicited by systemic injection of a lipopolysaccharide, rock bream iridovirus (RBIV) and bacteria (Edwardsiella tarda and Streptococcus iniae), revealing its pathogen inducibility. RbAGT manifested a down-regulated response to systemic injury, contemporaneously with two other serpins, protease nexin-1 (PN-1), and heparin cofactor II (HCII). In addition, a synchronized expression pattern was elicited by RbAGT and RbTNF-α in response to injury, suggesting that TNF-α might be a potential modulator of AGT transcription.

Genomic identification and molecular characterization of a non-mammalian TNFAIP8L2 gene from Oplegnathus fasciatus.

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a newly described negative immune regulator, whose enigmatic biological functions are not clearly understood. In the present study, the TNFAIP8L2 homolog of rock bream (Oplegnathus fasciatus) was identified and characterized. The genomic composition of rock bream TNFAIP8L2 (~6.7 kb) represented a tripartite arrangement in which three exons are interrupted by two introns. The rock bream TNFAIP8L2 transcript (1974 bp) possessed a coding sequence of 561 bp encoding a peptide of 186 amino acids. The predicted rock bream TNFAIP8L2 protein was 21.1kDa and revealed the typical features of known TNFAIP8L2 members including the DED-like domain. Rock bream TNFAIP8L2 was composed of six α-helices and demonstrated a distinct folding pattern of the TNFAIP8L2 family. It showed a certain degree of homology and phylogenetic relationship with the corresponding tilapia counterpart. Based on an interspecies genomic organizational comparison of TNFAIP8L2 orthologs, they could be classified into two classes, with teleost and non-teleost origin respectively. While teleost TNFAIP8L2s manifest a tripartite arrangement, non-teleost counterparts demonstrate a dipartite structure suggesting the loss of an intron during the post-piscine speciation. Promoter proximal region of rock bream TNFAIP8L2 consisted of multiple immune responsive cis-regulatory elements. Analysis of basal transcription in eleven tissues revealed its constitutive expression in examined tissues with highest magnitude in the head kidney. The modulated temporal mRNA expression of rock bream TNFAIP8L2 in head kidney post-challenges with stimulants (LPS and poly I:C) and pathogens (Streptococcus iniae and irido virus) was stimulant-specific. Additionally, a drastic down-regulation of rock bream TNFAIP8L2 mRNA level occurred in blood cells collected from experimentally injured animals, and it was accompanied by a contemporaneous down-regulation of cytokines, TNF-α and TGFβ3. All these findings imply that rock bream TNFAIP8L2 is potentially responsible for immune and inflammatory modulation in rock bream.