Full length article
Mitochondrial peroxiredoxin 3 (Prx3) from rock bream (Oplegnathus fasciatus): Immune responses and role of recombinant Prx3 in protecting cells from hydrogen peroxide induced oxidative stress

https://doi.org/10.1016/j.fsi.2014.12.011Get rights and content

Highlights

  • PRx3 is important for antioxidant defense and redox signaling.

  • RbPrx3 is categorized into 2-Cys Prx.

  • Constitutive expression of RbPrx3 is shown in wider array of rock bream tissues.

  • Immune challenge activates the transcription of Prx3 in liver.

  • Recombinant Prx6 can protect cells from oxidative stress by reducing H2O2.

Abstract

Pathogenic infections and environmental factors cause a variety of stresses in fish including oxidative stress by rapid elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Transcriptional activation and expression of antioxidant enzymes are essential for reducing the oxidative stress. In this study, we present the molecular characterization, immune responses and ROS scavenging activity of mitochondrial peroxiredoxin 3 from Oplegnathus fasciatus (RbPrx3). Coding sequence (CDS) of RbPrx3 contains 248 amino acids polypeptide which consists of highly conserved peroxiredoxin super family domain and two cysteine residues. Pairwise sequence comparison revealed that RbPrx3 has the greatest identity (94.8%) to Sparus aurata Prx3. Transcriptional analysis of RbPrx3 indicated the ubiquitously expressed mRNA in wide array of organs showing the highest expression in the liver of rock bream. Upon immune challenge of Edwardsiella tarda, Streptococcus iniae, rock bream iridovirus (RBIV) and lipopolysaccharide (LPS), RbPrx3 mRNA level was up-regulated in immunocompetent liver tissues compared to unchallenged fish. Purified recombinant RbPrx3 treated THP-1 cells showed higher survival rate against H2O2 induced oxidative stress and significantly reduced the level of intracellular ROS. Overall results from our study suggest that RbPrx3 may be involved in broader functions such as regulating oxidative stresses by scavenging ROS and activating immune responses in rock bream.

Introduction

ROS and RNS are elevated in metabolic cells when exposed to environmental stresses [1]. They are considered as mediators of intracellular signaling cascades, immune reactions, cell proliferation, and inflammation [2], [3], [4]. Besides that, continuous exposure and or excessive levels of ROS may lead to protein oxidation, lipid peroxidation, DNA damage, loss of cellular function, and apoptosis [5]. It is well understood that antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), thioredoxin peroxidase (TPx) and peroxiredoxins (Prxs) act as defensive molecules to reduce the excessive amounts of ROS/RNS and regulate their mediatory role [3]. However, catalytic efficiency of Prxs is comparatively lower than that of CAT or GPx [6].

Prxs are non-selenium dependent enzymes that catalyze/reduce the cytokine induced peroxides and which activity is regulated by thioredoxin [7]. More importantly thioredoxin is utilized as an electron donor in order to succeed the peroxiredoxin activity [8]. Prxs are also referred as thioredoxin peroxidases [9] and alkylhydroperoxide reductase C22 proteins [10] which are initially identified from yeast [11]. Members of Prxs shows protective antioxidant role (ROOH + 2e → ROH + H2O) against ROS/RNS: hydrogen peroxide, peroxynitrite and organic hydroperoxides (ROOH) through their peroxidase activity [1], [12]. Six isoforms of Prxs have been reported in variety of prokaryotes and eukaryotes [12], [13]. All Prxs share conserved cysteine (Cys) residues and classified into three sub-groups namely typical 2-Cys Prxs (Prx1 ∼ Prx4), atypical 2-Cys Prx (Prx5) and 1-Cys Prx (Prx6). Among them, Prx 1–5 have catalytically active Cys surrounded by conserved residues, though only N-terminal Cys is directly involved in primary site of oxidation using peroxide substrate. Prx6 has single conserved catalytically active Cys residue [7], [14], [15]. Peroxiredoxins can be regulated by phosphorylation, redox and possibly oligomerization states [16]. These enzymes share the same basic catalytic mechanism, in which a redox-active cysteine (peroxidatic cysteine) in the active site is oxidized to a sulfenic acid by the peroxide substrate. In biology, members of 2-Cys peroxiredoxins are reduced by thiols such as glutathione, while the 1-Cys enzymes may be reduced by ascorbic acid or glutathione [17]. Prxs are localized in various cellular compartments such as cytosol (Prx1, Prx2, Prx5, Prx6), endoplsmic reticulum (Prx4), mitochondria (Prx3, Prx5), peroxisomes (Prx5) [7], [14], [18] in order to protect every part of the cell. Functionally Prx3 involves in reducing intracellular H2O2 and it acts as a key regulator of apoptosis as well [7], [9], [19], [20], [21]. Prx3 has been reported from few fish species such as Oryzias latipes [22] and Sparus aurata [7]. To date, there is no report on immune response and functional characterization of Prx 3 in rock bream. Considering disease prevention and health aspects of fish, it would be useful to investigate the role of Prx3 under fish immune system and associated functions. The rock bream is a commercially important fish species in East Asian mariculture. Currently several infectious diseases (Edwardsiellosis, iridovirus, scuticociellosis) have been reported from rock bream [23]. Thus, study of RbPrx3 can lead to a better understanding of immune and oxidative stress responses in fish.

In this study, full-length cDNA of RbPrx3 was identified from rock bream transcriptome data. In silico analysis of RbPrx3 was conducted to determine the primary structure and evolutional relationship. Constitutive expression in different organs and expression profiles in response to Edwardsiella tarda, Streptococcus iniae, rock bream iridovirus (RBIV) and lipopolysaccharide (LPS) were determined by quantitative real-time PCR (qPCR). Functional role of RbPrx3 was investigated (in vitro) using recombinant RbPrx3 as an antioxidant enzyme to reduce the H2O2 induced oxidative stress in THP-1 cells.

Section snippets

Rock bream transcriptome and identification of RbPrx3

Rock bream transcriptome database was established after sequencing of normalized cDNA using Roche 454 genome sequencer FLX systems (DNA Link, Republic of Korea) as previously described [24]. The RbPrx3 gene was identified by homology screening and comparative analysis of known Prx family members using the BLAST program available at NCBI [25].

In silico sequence profiling

CDS of RbPrx3 was translated into corresponding putative amino acid sequence using DNAssist (v 2.2) software. The characteristic domain architecture was

Sequence profiling of RbPrx3

RbPrx3 homolog was selected from rock bream transcriptome data and identified as a member of Prx3 family by blasting the sequence with NCBI database. The nucleotide sequence of RbPrx3 bears 913 bp of full length cDNA and 747 bp of CDS (including stop codon). The CDS of RbPrx3 was coded into a putative peptide of 248 amino acids. Previously reported teleostean Prx3 homologs from gilthead sea bream Prx3 [7] has very similar and identical amino acids composition (247 amino acids) to that of RbPrx3

Acknowledgments

This research was supported by the project titled ‘Development of Fish Vaccines and Human Resource Training’, funded by the Ministry of Oceans and Fisheries, Korea.

References (47)

  • G. Leyens et al.

    Cloning of bovine peroxiredoxins-gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins

    Com Biochem Phys B

    (2003)
  • Y. Manevich et al.

    Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism

    Free Radic Biol Med

    (2005)
  • T.S. Chang et al.

    Peroxiredoxin III, a mitochondrion-specific peroxidase, regulates apoptotic signaling by mitochondria

    J Biol Chem

    (2004)
  • N. Umasuthan et al.

    Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): molecular characterization, cloning and expression analysis

    Fish Shellfish Immun

    (2011)
  • S.F. Altschul et al.

    Basic local alignment search tool

    J Mol Biol

    (1990)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

    Methods

    (2001)
  • M.M. Bradford

    A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal Biochem

    (1976)
  • C. Nikapitiya et al.

    Molecular cloning, characterization and expression analysis of peroxiredoxin 6 from disk abalone Haliotis discus discus and the antioxidant activity of its recombinant protein

    Fish Shellfish Immun

    (2009)
  • P. Nagy et al.

    Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: a kinetic and computational study

    J Biol Chem

    (2011)
  • L. Li et al.

    Increased susceptibility of MER5 (peroxiredoxin III) knockout mice to LPS-induced oxidative stress

    Biochem Biophys Res Commun

    (2007)
  • J. Van Horn et al.

    Molecular cloning and expression of a 2-Cys peroxiredoxin gene in the crustacean Eurypanopeus depressus induced by acute hypo-osmotic stress

    Com Biochem Phys B

    (2010)
  • J. Chen et al.

    Increased liver protein and mRNA expression of natural killer cell-enhancing factor B (NKEF-B) in ayu (Plecoglossus altivelis) after Aeromonas hydrophila infection

    Fish Shellfish Immun

    (2009)
  • Q. Zhang et al.

    Molecular cloning, expression of a peroxiredoxin gene in Chinese shrimp Fenneropenaeus chinensis and the antioxidant activity of its recombinant protein

    Mol Immunol

    (2007)
  • Cited by (22)

    • Molecular profiling and functional delineation of peroxiredoxin 3 (HaPrx3) from the big-belly seahorses (Hippocampus abdominalis) and understanding their immunological responses

      2021, Gene
      Citation Excerpt :

      The regulation of apoptotic signaling in mitochondria with human Prx3 mRNA was examined using HeLa cells (Joly et al., 1997), and the bactericidal activity of Prx3 was investigated in Salmonella enterica by Prx3 knockdown in THP-1 cells (Pérez-Sánchez et al., 2011). Furthermore, previous studies on Prx3 have been reported for other fish species, including Oplegnathus fasciatus (Godahewa et al., 2015), Oryzias latipes (Joly et al., 1997), and Sparus aurata (Pérez-Sánchez et al., 2011). The immune response of Prx3 was investigated against bacteria [Edwardsiella tarda (E. tarda), Streptococcus iniae (S. iniae)], and virus (RBIV) infections in Oplegnathus fasciatus (Godahewa et al., 2015).

    View all citing articles on Scopus
    View full text