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