Peroxiredoxin-1 aggravates lipopolysaccharide-induced septic shock via promoting inflammation.

Septic shock induced by lipopolysaccharide (LPS) is characterized by serious systemic inflammatory response and robust production of pro-inflammatory cytokines from activated macrophages. Damage-associated molecular patterns (DAMPs) secreted by activated macrophages are key contributors to septic shock. However, the current knowledge on those DAMPs that promote inflammatory response under LPS-induced septic shock remains poorly understood. Here, we report that Peroxiredoxin 1 (Prdx1) plays a detrimental role in LPS-induced septic shock. Intraperitoneal injection of LPS elicited a progressive course of septic shock in mice, which was characterized by significant lethality along with robust production of cytokines (IL-1ß, IL-6 and TNF-α). Removal of Prdx1 strongly protected mice from LPS-induced death, and decreased IL-1ß, IL-6 and TNF-α productions. Additionally, primary macrophages deficient in Prdx1 are less able to produce much more IL-1ß, IL-6 and TNF-α. Collectively, we provide a demonstration for Prdx1 contributing to LPS-induced septic shock likely via promoting inflammation.

Novel 2-Cys Peroxiredoxin gene confers biotic and abiotic stress resistance in Penaeus monodon.

Peroxiredoxins (Prxs) are crucial antioxidant proteins that protect against biotic and abiotic stresses in many organisms, ranging from bacteria to mammals. In the present work, a novel 2-Cys Peroxiredoxin gene (PmPrxn), which contains a 153 bp 5'-terminal untranslated region (5'-UTR), a 636 bp open reading frame encoding a protein with 211 amino acids, and an 898 bp 3'-UTR, was successfully identified and characterized in the black tiger shrimp, Penaeus monodon. Tissue-specific expression analysis revealed that the PmPrxn mRNA was ubiquitously expressed and was comparatively highly expressed in the hepatopancreas. To explore the immunity-related and anti-stress roles of PmPrxn, the gills and hepatopancreas were chosen as target tissues in P. monodon and challenged with Vibrio harveyi, Streptococcus agalactiae, and toxic environmental stressors. The results indicate that PmPrxn might play a vital role in response to biotic and abiotic stresses. Furthermore, the antimicrobial and heavy metal toxicity stress-resistance properties of PmPrxn were evaluated and investigated in vitro using a prokaryotic expression system. These results provide useful information that will help further understand the functional mechanisms of PmPrxn in the defense against bacterial pathogens and environmental acute stresses in shrimp.

Transcriptional modifications and the cytoprotective, DNA protective, and wound healing effects of peroxiredoxin-1 from Sebastes schlegelii.

Peroxiredoxins are a group of thiol-specific antioxidant proteins that take six isoforms in vertebrates and allow the innate immune system to sense and detoxify reactive oxygen species. In this study, we identified and characterized the perxiredoxin-1 (SsPrdx1) cDNA sequence from the rockfish, Sebastes schlegelii. In silico analysis revealed that SsPrdx1 contained a 594 bp long open reading frame (ORF) encoding a protein of 198 amino acids, with a predicted molecular weight and theoretical isoelectric point of 21.97 kDa and 6.30, respectively. The SsPrdx1 gene comprised six exons linked by five introns, while peroxiredoxin signature motifs were found in the highly conserved third, fourth, and fifth exons. Phylogenetic analysis and sequence alignment suggested that SsPrdx1 is evolutionarily conserved and that its most closely related counterpart is Salarias fasciatus. Recombinant SsPrdx1 (rSsPrdx1) displayed supercoiled DNA protection and insulin disulfide reduction activities in a concentration-dependent manner, while cells transiently transfected with pcDNA3.1 (+)/SsPrdx1 exhibited significant cytoprotective effects under oxidative stress and wound healing activity. SsPrdx1 transcripts were constitutively expressed under normal physiological conditions, with the highest expression observed in the blood. Moreover, SsPrdx1 expression increased in the blood, spleen, and liver following immune provocation by LPS, poly I:C, and Streptococcus iniae injection. Thus, this study provides insights into the role of SsPrdx1 in rockfish immune protection.

Evaluation of toxoplasmosis in pregnant women using dot-immunogold-silver staining with recombinant Toxoplasma gondii peroxiredoxin protein.

BACKGROUND: Toxoplasma gondii infection endangers human health and affects animal husbandry. Serological detection is the main method used for epidemiological investigations and diagnosis of toxoplasmosis. The key to effective diagnosis of toxoplasmosis is the use of a standardized antigen and a specific and sensitive detection method. Peroxiredoxin is an antigenic protein and vaccine candidate antigen of T. gondii that has not yet been exploited for diagnostic application. METHODS: In this study, recombinant T. gondii peroxiredoxin protein (rTgPrx) was prepared and used in dot-immunogold-silver staining (Dot-IGSS) to detect IgG antibodies in serum from mice and pregnant women. The rTgPrx-Dot-IGSS method was established and optimized using mouse serum. Furthermore, serum samples from pregnant women were analyzed by rTgPrx-Dot-IGSS. RESULTS: Forty serum samples from mice infected with T. gondii and twenty negative serum samples were analyzed. The sensitivity and specificity of rTgPrx-Dot-IGSS were 97.5 and 100%, respectively, equivalent to those of a commercial ELISA kit for anti-Toxoplasma IgG antibody. Furthermore, 540 serum samples from pregnant women were screened with a commercial ELISA kit. Eighty-three positive and 60 negative serum samples were analyzed by rTgPrx-Dot-IGSS. The positive rate was 95.18%, comparable to that obtained with the commercial ELISA kit. CONCLUSIONS: The Dot-IGSS method with rTgPrx as an antigen might be useful for diagnosing T. gondii infection in individuals.

The biological role of peroxiredoxins in innate immune responses of aquatic invertebrates.

Peroxiredoxins (Prxs) are a widespread and greatly transcribed family of antioxidant proteins, which rapidly detoxify peroxynitrite, hydrogen peroxide and organic hydroperoxides. The Prxs family members also modulate various physiological functions, including cell growth, differentiation, embryonic development, immune response, apoptosis, lipid metabolism, and cellular homeostasis. In mammals, the physiological functions of Prxs have extensively been studied; however, the knowledge is scanty in their counterpart, aquatic invertebrates. In recent years, substantial progress has been made in our knowledge of Prxs physiological functions in aquatic invertebrates, which has raised interest in defining the contribution of immune responses and removal of reactive oxygen species. In this review, we describe the recent knowledge on the Prxs physiological function in immune responses and DNA protection activity in aquatic invertebrates.

Molecular characterization and functional activity of Prx1 in grass carp (Ctenopharyngodon idella).

Peroxiredoxin (Prx) family are known as an important antioxidant enzyme as the first line of defense against oxidative damage, and also involved in immune responses following viral and bacterial infection. Here, a full-length Prx1 cDNA sequence (CiPrx1) was cloned from grass carp (Ctenopharyngodon idella), which was 1029 bp, including a 5'-terminal untranslated region (UTR) of 121 bp, a 3'-UTR of 272 bp, an open reading frame of 600 bp encoding 199 amino acids with molecular weight of 22.21 kDa and isoelectric point of 6.30. CiPrx1 shares 80.8-99% protein sequence similarity with Prx1 of other fishes. The conserved peroxidase catalytic center "FYPLDFTFVCPTEI" and "GEVCPA" were observed in the sequence of CiPrx1; this indicated that it was a member of 2-Cys Prx. Subcellular localization of CiPrx1 was only strongly distributed in the cytoplasm. Quantitative real-time PCR (RT-qPCR) assays revealed that CiPrx1 mRNA was ubiquitously detected in all tested tissues, and the expression was comparatively high in liver, gill and spleen. Further, the expression of CiPrx1 can be induced by grass carp reovirus (GCRV), lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly I:C) infection in the different tissues. Moreover, the recombinant CiPrx1 (rCiPrx1) protein was found a potential antioxidant enzyme, that could inhibit DNA damage from oxidants. Altogether, our results imply that CiPrx1 is associated with defending against virus and bacteria pathogens and oxidants in grass carp.

Cloning and functional characterization of rockfish peroxiredoxin 4 homolog with its innate immune responses.

The fourth member of the typical 2-cysteine peroxiredoxin (Prx4) is a well-known antioxidant enzyme, which reduces different peroxides in their catalytic process. The present study reports the identification of the rockfish Sebastes schlegelii Prx4 (SsPrx4) at a genomic level, as well as the characterization of its structural and functional features. SsPrx4 harbors a complete ORF of 786 bp encoding a polypeptide (29 kDa) of 262 amino acids (aa) with an isoelectric point of 6.2. Thioredoxin 2 domain was prominent in the SsPrx4 sequence, which has a signal peptide (31 bp) at the N-terminus. Hence, the SsPrx4 may be functionally active in the cytoplasm of rockfish cells. Moreover, two VCP motifs and three catalytic triad residues (112T, 115C, 191R) were identified in the SsPrx4 protein sequence. A peroxidatic cysteine (115CP) and resolving cysteines (236CR) were detected at the VCP motifs. The rockfish Prx4 genome consists of seven exons, which are similar to the architecture of other Prx4 orthologs. The deduced amino acid sequence of SsPrx4 shares a relatively high amino acid sequence identity (91.6%) and close evolutionary relationship with Miichthys miiuy and Stegastes partitus Prx4. The potential for scavenging extracellular H2O2 was evidenced by the purified recombinant SsPrx4 protein (rSsPrx4) in vitro system. Moreover, rSsPrx4 may protect the plasmid DNA in a metal-catalyzed oxidation system and catalyze the reduction of an insulin disulfide bond. Quantitative real-time PCR revealed that SsPrx4 mRNA was ubiquitously expressed in fourteen different tissues, with the highest expression observed in the liver followed by the ovary, and kidney tissues. Transcriptional modulations were observed in liver and spleen tissues of rockfish after injecting them with bacterial stimuli, including Streptococcus iniae, LPS, and a viral mimic of poly I:C. Together, the results suggest that SsPrx4 may play an important role in both the antioxidant and innate immune defense of black rockfish. These findings provide structural and functional insights into the SsPrx4 of the teleost.

Identification and molecular characterization of peroxiredoxin 2 in grass carp (Ctenopharyngodon idella).

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5'-untranslated region (UTR) of 52 bp, a 3'-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H2O2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses.

Cloning and molecular characterization of thiol-specific antioxidant gene of Leishmania tropica Turkey isolate

Background/aim: Thiol-specific antioxidant (TSA) protein is one of the most promising molecules among candidates for vaccine against cutaneous leishmaniasis. It was found to be significantly protective against different Leishmania species. In this study, cloning and molecular characterization of thiol-specific antioxidant gene of L. tropica Turkey isolate (LtTSA) were aimed. Materials and methods: LtTSA was amplified by PCR using the specific primers of TSA gene and cloned into the pcDNA3.1 vector. The cloning was confirmed by PCR screening, restriction enzyme reactions, and DNA sequence analysis. Finally, three-dimensional structure and antigenic properties of the protein encoded by the LtTSA were determined Results: Six hundred base pair bands belonging to LtTSA were shown with electrophoresis. It was found that LtTSA and its encoded protein have high similarity with different Leishmania species. LtTSA protein consisting of 199 amino acids was found to have 7 different antigenic regions. Conclusion: LtTSA and its encoded TSA protein were found to be highly immunogenic and similar to TSA proteins previously tested as a vaccine candidate.

Molecular cloning and characterization of two genes encoding peroxiredoxins from freshwater bivalve Anodonta woodiana: Antioxidative effect and immune defense.

Members of Prx family function as an important players in host defense against oxidative stress, and modulate immune responses. In the current study, two complete Prx sequences were isolated from bivalve Anodonta woodiana and respectively named AwPrx4a and AwPrx4b. Regulative characterizations of AwPrx4a and AwPrx4b derived from perfluorooctanesulfonate (PFOS), perfluoroocanoic acid (PFOA), lipopolysaccharide (LPS) and polyinosinic:polycytidylic (Poly I:C) challenge in hepatopancreas, gill and hemocytes were measured by quantitative real-time PCR, respectively. The full-length cDNA of AwPrx4a had an open reading frame ORF of 588 bp encoding 196 amino acids. Two highly conserved Prxs signature motifs were observed in deduced amino acid sequence, one was FYPLDFTFACPTEI, and the other was GEVCPA. Complete cDNA sequence of AwPrx4b was comprised of a 5' untranslated region (UTR) of 120 nucleotides, a 426 bp ORF which was encoded 142 amino acids, and a long 3'-UTR of 412 nucleotides. Expressions of AwPrx4a and AwPrx4b showed a significant up-regulation pattern in groups at lower concentration treatment of PFOS and PFOA, a biphasic profile in groups with a higher concentration treatment. Compared with that of control group, expressions of AwPrx4a and AwPrx4b were significantly induced by LPS and Poly I:C treatment in the hepatopancreas, gill and hemocytes. These results indicate up-regulations of AwPrx4a and AwPrx4b expression are associated with eliminating oxidative stress derived from PFOS and PFOA administration as well as enhancing immune defense against LPS and Poly I:C challenge.

Essential role of the peroxiredoxin 4 in Procambarus clarkii antioxidant defense and immune responses.

Peroxiredoxin (Prx) family members play a key role in host defense against oxidative stress, and modulate immune responses following microbial infection. Here, we cloned and characterized Procambarus clarkii Prx4 (Peroxiredoxin 4) cDNA, a regulator of oxidative stress and its expression analysis upon lipopolysaccharide (LPS) and polyriboinosinic polyribocytidylic acid (Poly I:C) infection. The cDNA fragment of PcPrx4 was 744 bp in length, encoding a putative protein of 248 amino acid residues. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the PcPrx4 was expressed in all the examined tissues, and it was highest in the hepatopancreas followed by the hemocytes and gill. The challenge with LPS and Poly I:C significantly up-regulated the expression of PcPrx4 in hepatopancreas, hemocytes and gill when compared with the control. Recombinant PcPrx4 protein was used to investigate the antioxidant function in vitro by mixed-function oxidase assay. The results demonstrated a dose-dependent inhibition of DNA damage by rPcPrx4 protein. Altogether, our results imply that PcPrx4 is implicated in defense against microbial pathogens and oxidants in P. clarkii.

Peroxiredoxin 1 Contributes to Host Defenses against Mycobacterium tuberculosis.

Peroxiredoxin (PRDX)1 is an antioxidant that detoxifies hydrogen peroxide and peroxinitrite. Compared with wild-type (WT) mice, Prdx1-deficient (Prdx1-/-) mice showed increased susceptibility to Mycobacterium tuberculosis and lower levels of IFN-γ and IFN-γ-producing CD4+ T cells in the lungs after M. tuberculosis infection. IL-12 production, c-Rel induction, and p38 MAPK activation levels were lower in Prdx1-/- than in WT bone marrow-derived macrophages (BMDMs). IFN-γ-activated Prdx1-/- BMDMs did not kill M. tubercuosis effectively. NO production levels were lower, and arginase activity and arginase 1 (Arg1) expression levels were higher, in IFN-γ-activated Prdx1-/- than in WT BMDMs after M. tuberculosis infection. An arginase inhibitor, Nω-hydroxy-nor-arginine, restored antimicrobial activity and NO production in IFN-γ-activated Prdx1-/- BMDMs after M. tuberculosis infection. These results suggest that PRDX1 contributes to host defenses against M. tuberculosis PRDX1 positively regulates IL-12 production by inducing c-Rel and activating p38 MAPK, and it positively regulates NO production by suppressing Arg1 expression in macrophages infected with M. tuberculosis.

Potential of recombinant 2-Cys peroxiredoxin protein as a vaccine for Fasciola gigantica infection.

Helminth 2-cys peroxiredoxin (Prx) is a major antioxidant enzyme that protects parasites against hydrogen peroxide-generating oxidative stress from the hosts' immune responses. This enzyme has been found in all stages of the tropical liver fluke, Fasciola gigantica. To investigate the potential of the recombinant F. gigantica Prx-2 (rFgPrx-2) as a vaccine candidate, vaccine trials in mice were carried out. In this study, the ICR mice were immunized with rFgPrx-2 combined with Freund's adjuvant and infected with F. gigantica metacercariae. The vaccine efficacy was estimated by quantitate fluke recovery, antibody levels and liver function. The protection by rFgPrx-2 against F. gigantica infection was achieved at 43-46% compared with adjuvant-infected and non-immunized-infected control groups, respectively. The vaccine elicited both Th1 and Th2 humoral immune responses with predominance of Th2 as indicated by the higher level of IgG1 in sera of immunized mice. However, the levels of liver damage markers, serum glutamate oxalic transaminase (SGOT) and serum glutamic pyruvate transaminase (SGPT) in rFgPrx-2 immunized group did not show significant difference in comparison with the controls. This study suggested that rFgPrx-2 may have a potential as a vaccine against tropical fasciolosis.

The multiple roles of peroxiredoxins in tick blood feeding.

Hydrogen peroxide (H2O2) and hydroxyl radicals (HO·) are generated through partial reduction of oxygen. The HO· are the most reactive and have a shorter half-life than H2O2, they are produced from comparatively stable H2O2 through Fenton reaction. Although controlling HO· is important and biologically advantageous for organisms, it may be difficult. Ticks are obligate hematophagous arthropods that need blood feeding for development. Ticks feed on vertebrate blood containing high levels of iron. Ticks also concentrate iron-containing host blood, leading to high levels of iron in ticks. Host-derived iron may react with oxygen in the tick body, resulting in high concentrations of H2O2. On the other hand, ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), to scavenge H2O2. Gene silencing of Prxs in ticks affects their blood feeding, oviposition, and H2O2 concentration. Therefore, Prxs could play important roles in ticks' blood feeding and oviposition through the regulation of the H2O2 concentration. This review discusses the current knowledge of Prxs in hard ticks. Tick Prxs are also multifunctional molecules related to antioxidants and immunity like other organisms. In addition, tick Prxs play a role in regulating the host immune response for ticks' survival in the host body. Tick Prx also can induce Th2 immune response in the host. Thus, this review would contribute to the further understanding of the tick's antioxidant responses during blood feeding and the search for a candidate target for tick control.

Evaluation of vaccine potential of 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis.

Ticks require blood feeding on vertebrate animals throughout their life cycle, and also concentrate the iron-containing blood, resulting in a high concentration of hydrogen peroxide (H2O2). High concentrations of H2O2 are harmful to organisms, due to their serious damage of macromolecules. Ticks have antioxidant enzymes, such as peroxiredoxins (Prxs), that scavenge H2O2. Prxs may have important roles in regulating the H2O2 concentration in ticks during blood feeding and oviposition. Moreover, Prxs are considered potential vaccine candidates in other parasites, such as Leishmania and Fasciola. In the present study, the efficacy of a tick Prx (HlPrx2) as a vaccine candidate antigen was evaluated. First, recombinant HlPrx2 (rHlPrx2) was expressed in Escherichia coli, and then, its purity and endotoxin levels were confirmed prior to administration. The rHlPrx2 proteins were of high purity with acceptably low endotoxin levels. Second, the ability of rHlPrx2 administration to stimulate mouse immunity was evaluated. The rHlPrx2 protein, with or without an adjuvant, could stimulate immunity in mice, especially the IgG1 of Th2 immune response. Using Western blot analysis, we also observed whether rHlPrx2-immunized mice sera could recognize native HlPrx2 protein in crude tick midgut proteins. Western blot analysis demonstrated that rHlPrx2-administrated mouse sera could detect the native HlPrx2. Finally, the effects of rHlPrx2 immunization in mice were studied using nymphal ticks. Although the challenged ticks were not affected by rHlPrx2 immunization, rHlPrx2 still might be considered as a vaccine candidate against ticks because of its high immunogenicity.

The role of peroxiredoxin 4 in inflammatory response and aging.

In prior studies, we determined that the moderate overexpression of the Drosophila endoplasmic reticulum (ER)-localized peroxiredoxin (Prx), dPrx4, reduced oxidative damage and conferred beneficial effects on life span, while a high-level expression increased the incidence of tissue-specific apoptosis and dramatically shortened longevity. The detrimental pro-apoptotic and life-shortening effects were attributed to aberrant localization of dPrx4 and the apparent ER stress elicited by dPrx4 overexpression. In addition, the activation of both the NF-κB- and the JAK/STAT-mediated stress responses was detected, although it was not clear whether these served as functional alarm signals. Here we extend these findings to show that the activation of the NF-κB-dependent immunity-related/inflammatory genes, associated with life span shortening effects, is dependent on the activity of a Drosophila NF-κB ortholog, Relish. In the absence of Relish, the pro-inflammatory effects typically elicited by dPrx4 overexpression were not detected. The absence of Relish not only prevented the hyperactivation of the immunity-related genes but also significantly rescued the severe shortening of life span normally observed in dPrx4 overexpressors. The overactivation of the immune/inflammatory responses was also lessened by JAK/STAT signaling. In addition, we found that cellular immune/pro-inflammatory responses provoked by the oxidant paraquat but not bacteria are mediated via dPrx4 activity in the ER, as the upregulation of the immune-related genes was eliminated in flies underexpressing dPrx4, whereas immune responses triggered by bacteria were unaffected. Finally, efforts to reveal critical tissues where dPrx4 modulates longevity showed that broad targeting of dPrx4 to neuronal tissue had strong beneficial effects, while targeting expression to the fat body had deleterious effects.

Mitochondrial peroxiredoxins are essential in regulating the relationship between Drosophila immunity and aging.

Previously, we have shown that flies under-expressing the two mitochondrial peroxiredoxins (Prxs), dPrx3 and dPrx5, display increases in tissue-specific apoptosis and dramatically shortened life span, associated with a redox crisis, manifested as changes in GSH:GSSG and accumulation of protein mixed disulfides. To identify specific pathways responsible for the observed biological effects, we performed a transcriptome analysis. Functional clustering revealed a prominent group enriched for immunity-related genes, including a considerable number of NF-kB-dependent antimicrobial peptides (AMP) that are up-regulated in the Prx double mutant. Using qRT-PCR analysis we determined that the age-dependent changes in AMP levels in mutant flies were similar to those observed in controls when scaled to percentage of life span. To further clarify the role of Prx-dependent mitochondrial signaling, we expressed different forms of dPrx5, which unlike the uniquely mitochondrial dPrx3 is found in multiple subcellular compartments, including mitochondrion, nucleus and cytosol. Ectopic expression of dPrx5 in mitochondria but not nucleus or cytosol partially extended longevity under normal or oxidative stress conditions while complete restoration of life span occurred when all three forms of dPrx5 were expressed from the wild type dPrx5 transgene. When dPrx5 was expressed in mitochondria or in all three compartments, it substantially delayed the development of hyperactive immunity while expression of cytosolic or nuclear forms had no effect on the immune phenotype. The data suggest a critical role of mitochondria in development of chronic activation of the immune response triggered by impaired redox control.

Peroxiredoxin 1 from cuttlefish (Sepiella maindroni): Molecular characterization of development and its immune response against Vibrio alginolyticus.

The aim of this work was constructive to understand the function of peroxiredoxin (PRDX) family member Peroxiredoxin 1 in Sepiella maindroni (SmPrx1) through molecular mechanisms of reproduction, embryonic development and immune responses to Vibrio alginolyticus. The full-length cDNA of SmPrx1 was of 1062 bp, contains a 5' untranslated region (UTR) of 79bp, a 3' UTR of 359 bp, an open reading frame of 624 bp encoding 207 amino acids. The conserved peroxidase catalytic center "FYPLDFTFVCPTEI" and "GEVCPA" were observed in the sequence of SmPrx1; this indicated that it was a member of 2-Cys Prx. Quantitative real-time (qRT)-PCR assays revealed that SmPrx1 was ubiquitously expressed in all examined tissues, muscle, ink sac, liver, ovary, testis, intestine, gill and totally blood cells, and showed high levels in testis. SmPrx1 mRNA was ubiquitously detected in all tested tissues, and the expression was comparatively high in testis, hemocyte, liver and ovary. Moreover, the SmPrx1 gene transcript was detected at all five stages of embryonic development phases that were respectively the zygote stage, the pre-embryonic stage, the organogenesis stage, the morphological integrity stage, the pre-hatching stage. The general tendency of expression was gradually increased and rapidly decreased. High expressed in progenitive tissues and embryonic development exhibit the proliferation-associated protein characterization like in mammal. The expression levels of SmPrx1 in liver and hemocytes grew swiftly and quickly reached peak value after Vibrio alginolyticus challenge. As hours passed by, the expression level began to reduce and resumed to normal levels after 48 h. The antioxidant activity and peroxidase activity of SmPrx1 were 6.17 U/mg. The results showed that the recombined protein of SmPrx1 had antioxidant activity and was the importance part of the antioxidant system in Sepiella maindroni. This study provides useful information to help further understand the functional mechanism of Prx 1 in marine cephalopod immunity.

Identification and characterization of atypical 2-cysteine peroxiredoxins from mud crab Scylla paramamosain: The first evidence of two peroxiredoxin 5 genes in non-primate species and their involvement in immune defense against pathogen infection.

Peroxiredoxin 5 (Prx5) belongs to a novel family of evolutionarily conserved antioxidant proteins that protect cells against various oxidative stresses. Generally, no more than one Prx5 transcript had been reported in non-primate species. In this study, two Prx5 genes (coined as SpPrx5-1 and SpPrx5-2) were firstly isolated from the mud crab, Scylla paramamosain, through RT-PCR and RACE methods. The open reading frame of SpPrx5-1 and SpPrx5-2 were 561 bp and 429 bp in length, encoding 186 and 142 amino acids polypeptide, respectively. Both the conserved signatures of peroxiredoxin catalytic center and Prx5-specific domain were identified in SpPrx5-1 and SpPrx5-2. Phylogenetic analysis indicated that both SpPrx5 clustered together with other animal Prx proteins and were classified into Prx5 subfamily. Tissue-specific expression analysis revealed that both SpPrx5-1 and SpPrx5-2 were ubiquitously expressed, highest in hepatopancreas, and showed remarkably similar transcription patterns. Quantitative RT-PCR analysis exhibited that both SpPrx5 genes changed dramatically in hepatopancreas, although showing different expression profiles, after virus-analog poly (I:C) or Vibrio alginolyticus challenge. The expression levels of both SpPrx5s were significantly enhanced in hepatopancreas after poly (I:C) stimulation, while SpPrx5-2 exhibited a more prompt response than SpPrx5-1. Nevertheless, the expression levels of both SpPrx5s were significantly reduced in hepatopancreas after Vibrio alginolyticus challenge in which SpPrx5-1 showed a more prompt response than SpPrx5-2. These results suggested the involvement of SpPrx5s in responses against viral and bacterial infections and further highlighted their functional importance in the immune system of Scylla paramamosain.

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

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.