[Show abstract][Hide abstract] ABSTRACT: 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.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 04/2015; 187. DOI:10.1016/j.cbpb.2015.04.008 · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carboxypeptidases (CPs) are proteases that hydrolyze C-terminal peptide bonds. They are involved in regulating the complement system of the immune system. Here, we report the molecular characterization and immune response of two carboxypeptidases, named carboxypeptidase A (Rb-CPA) and carboxypeptidase N1 (Rb-CPN1), from rock bream. The genomic sequence of Rb-CPA contains 12 exons interrupted by 11 introns, while the genomic sequence of Rb-CPN1 has 9 exons and 8 introns. The cDNA sequence of Rb-CPA encodes a 421-amino-acid (AA) polypeptide (48 kDa), and the cDNA of Rb-CPN1 encodes a 448-AA polypeptide (51 kDa). The amino acid sequences of Rb-CPA and Rb-CPN1 were found to harbor two characteristic Zn-binding signature domains and a peptidase-M14 Zn carboxypeptidase site. Pairwise analysis revealed that Rb-CPA and Rb-CPN1 had the highest identity with the corresponding proteins from Anoplopoma fimbria (87.6%) and Dicentrarchus labrax (96.9%), respectively. qPCR results indicated that Rb-CPA and Rb-CPN1 were constitutively expressed mainly in the kidney, heart, liver, and head kidney. Both genes were transcriptionally regulated in the liver upon challenge with pathogenic bacteria (Streptococcus iniae, Edwardsiella tarda), rock bream iridovirus (RBIV), and the immune modulators polyinosinic:polycytidylic acid and lipopolysaccharide. Taken together, our findings suggest that Rb-CPA and Rb-CPN1 have immune-related functions in rock bream.
Veterinary Immunology and Immunopathology 10/2014; DOI:10.1016/j.vetimm.2014.10.005 · 1.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homologue of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to Epinephelus coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in the spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.
[Show abstract][Hide abstract] ABSTRACT: Superoxide dismutases (SODs) are dedicated to scavenge and dismutate the superoxide anions in order to protect the cells from oxidative stress by establishing the redox homeostasis. In this study, we describe a cytosolic Cu/ZnSOD, the second SOD member from rock bream Oplegnathus fasciatus (Of-cCu/ZnSOD) at molecular, genomic structural-, transcriptional- and functional-levels. The determination of genomic arrangement of Of-cCu/ZnSOD by means of a BAC library revealed that its primary transcript is represented by five exons and encoded a peptide of 154 amino acids. In silico investigation of Of-cCu/ZnSOD indicated the presence of several family characteristics including two Cu/ZnSOD signatures, seven metal liganding residues and eight b-sheets forming a b-barrel topology. Alignment and modeling studies confirmed the conservation of Cu/ZnSOD at primary and tertiary levels. While invertebrate Cu/ZnSOD members mainly demonstrate a tetraexonic structure, the vertebrate members have acquired an additional intron in the third exon resulting in a quinquepartite arrangement with class-specific exon lengths. Although, teleost Cu/ZnSOD members resembled the mammalian orthologs in their genomic organization; they shared a proximal position with molluscan members in the phylogeny. The antioxidant (AO) activity of Of-cCu/ZnSOD was affirmed by a recombinant protein which was also used to examine the biophysical and biochemical properties. The pronounced activity was detected when the rOf-cCu/ZnSOD was expressed with the Cu2 + and Zn2 + supplementation. The optimum activities were observed at pH 10 and 25 °C, and KCN strongly inhibited the activity of the rOf-cCu/ZnSOD. Furthermore, a constitutive mRNA expression of Of-cCu/ZnSOD with higher levels in blood > liver > heart and brain was observed, which was consistent with the transcriptional profile of Of-mMnSOD, suggesting important physiological role(s). This idea was further strengthened by the temporal assessment of Of-cCu/ZnSOD transcripts in animals under pathological (bacteria- or viral-induced) and physiological (H2O2-induced oxidative) stress conditions using qPCR, in which it exhibited significantly up-regulated levels. Screening of Of-cCu/ZnSOD 5'-flanking region revealed the presence of several important transcription factor binding sites that potentially govern the Cu/ZnSOD expression. These findings conjointly contribute to expand our understanding regarding the piscine Cu/ZnSODs and; in particular, the AO enzyme network of rock bream.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 10/2014; 176(1). DOI:10.1016/j.cbpb.2014.07.004 · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial immune defense is evolutionarily preserved in all organisms. Mammals have developed robust, protein-based antiviral defenses, which are under constant investigation. Studies have provided evidences for the various fish immune factors sharing similarity with those of mammals. In this study, we have identified an ortholog of mitochondrial antiviral signaling protein from rock bream, Oplegnathus fasciatus. RbMAVS cDNA possesses an open reading frame (ORF) of 1758 bp coding for a protein of 586 amino acids with molecular mass of approximately 62 kDa and isoelectric point of 4.6. In silico analysis of RbMAVS protein revealed a caspase recruitment domain (CARD), a proline rich domain and a transmembrane domain. RbMAVS protein also contains a putative TRAF2 binding motif, 319PVQDT323. Primary sequence comparison of RbMAVS with other orthologues revealed heterogeneity towards the C-terminus after the CARD region. RbMAVS transcripts were evident in all the examined tissues. RbMAVS expression was induced in vivo after poly I:C challenge in peripheral blood cells, liver, head kidney and spleen tissues. Over-expression of RbMAVS potently inhibited marine birnavirus (MABV) infection in rock bream heart cells and induced various cytokines and signaling molecules in vitro. Thus, RbMAVS is an antiviral protein and potentially involved in the recognition and signaling of antiviral defense mechanism in rock bream.
[Show abstract][Hide abstract] ABSTRACT: Ferritins are iron binding proteins made out of 24 subunits, involved in iron homeostasis and metabolism in cellular environments. Here, we sought to identify and functionally characterize a one type of subunits of ferritin (ferritin H-like subunit) from rock bream (Oplegnathus fasciatus; RbFerH). The complete coding sequence of RbFerH was 531 bp in length, encoding a 177-amino acid protein with a predicted molecular mass of 20.8 kDa. The deduced protein structure possessed the domain architecture characteristic of known ferritin H subunits, including metal ligands for iron binding, a ferroxidase center, and two iron-binding region signatures. As expected, the 5' untranslated region of the RbFerH cDNA sequence contained a putative iron response element region, a characteristic regulatory element involved in its translation. The RbFerH gene comprised 5 exons and 4 introns spanning a 4195 bp region. Overexpressed recombinant RbFerH protein demonstrated prominent Fe(II) ion depriving activity, bacteriostatic properties, and protective effects against oxidative double-stranded DNA damage. Using quantitative polymerase chain reaction (qPCR), we found that RbFerH was expressed ubiquitously in the majority of physiologically important tissues in rock bream. A greater abundance of the mRNA transcripts were detected in blood and liver tissues. Upon administering different microbial pathogens and pathogen-derived mitogens, RbFerH transcription was markedly elevated in the blood of rock bream. Taken together, our findings suggest that RbFerH acts as a potent iron sequestrator in rock bream and may actively participate in antimicrobial as well as antioxidative defense.
[Show abstract][Hide abstract] ABSTRACT: Immunoproteasomes are primarily induced upon infection and formed by replacing constitutive beta subunits with inducible beta subunits which possess specific cleavage properties that aid in the release of peptides necessary for MHC class I antigen presentation. In this study, we report the molecular characterization and expression analysis of the inducible immunosubunits PSMB8, PSMB9, PSMB9-L, and PSMB10 from rock bream, Oplegnathus fasciatus. The three subunits shared common active site residues and were placed in close proximity to fish homologues in the reconstructed phylogenetic tree, in which the mammalian homologues formed separate clades, indicating a common ancestral origin. The rock bream immunosubunits possessed higher identity and similarity with the fish homologues. RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 were multi-exonic genes with 6, 6, 7 and 8 exons, respectively. These four genes were constitutively expressed in all the examined tissues. Immunostimulants such as lipopolysaccharide and poly I:C induced RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 in liver and head kidney, suggesting their possible involvement in immune defense in rock bream.
[Show abstract][Hide abstract] ABSTRACT: The membrane-attack complex/perforin (MACPF) domain-containing proteins play an important role in the innate immune response against invading microbial pathogens. In the current study, a member of the MACPF domain-containing proteins, macrophage expressed gene-1 (MPEG1) encoding 730 amino acids with the theoretical molecular mass of 79.6 kDa and an isoelectric point (pI) of 6.49 was characterized from disk abalone Haliotis discus discus (AbMPEG1). We found that the characteristic MACPF domain (Val(131)-Tyr(348)) and transmembrane segment (Ala(669)-Ile(691)) of AbMPEG1 are located in the N- and C-terminal ends of the protein, respectively. Ortholog comparison revealed that AbMPEG1 has the highest sequence identity with its pink abalone counterpart, while sequences identities of greater than 90% were observed with MPEG1 members from other abalone species. Likewise, the furin cleavage site KRRRK was highly conserved in all abalone species, but not in other species investigated. We identified an intron-less genomic sequence within disk abalone AbMPEG1, which was similar to other mammalian, avian, and reptilian counterparts. Transcription factor binding sites, which are important for immune responses, were identified in the 5'-flanking region of AbMPEG1. qPCR revealed AbMPEG1 transcripts are present in every tissues examined, with the highest expression level occurring in mantle tissue. Significant up-regulation of AbMPEG1 transcript levels was observed in hemocytes and gill tissues following challenges with pathogens (Vibrio parahaemolyticus, Listeria monocytogenes and viral hemorrhagic septicemia virus) as well as pathogen-associated molecular patterns (PAMPs: lipopolysaccharides and poly I:C immunostimulant). Finally, the antibacterial activity of the MACPF domain was characterized against Gram-negative and -positive bacteria using a recombinant peptide. Taken together, these results indicate that the biological significance of the AbMPEG1 gene includes a role in protecting disk abalone through the ability of AbMPEG1 to initiate an innate immune response upon pathogen invasion.
[Show abstract][Hide abstract] ABSTRACT: The interferon regulatory factor 5 (IRF5) is a key mediator of the Toll-like receptor (TLR)7 and TLR8 signaling pathways. In this study, we describe the identification of IRF5 (Rb-IRF5) from rock bream fish (Oplegnathus fasciatus) and its characteristics features at the genomic and expression levels. The full-length Rb-IRF5 sequence was identified from a cDNA library and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The genomic sequence is comprised of 8 exons interrupted by 7 introns. The complete coding sequence of Rb-IRF5 is 1497 bp in length and encodes for 498 amino acids. The putative Rb-IRF5 protein consists of 3 important conserved domains: a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus. Based on pairwise sequence analysis, the highest sequence similarity/identity for Rb-IRF5 was observed with the IRF5 gene from turbot fish (>87%) and Japanese flounder (83%). Several important putative transcription factor-binding sites shared by the IRF gene family, including the NF-κB, Ap-1, IRF-1, and ICSBP/ISRE sites, were found in the 5′ flanking region of Rb-IRF5. The predicted tertiary structure of the dimerized IAD and VAD of the Rb-IRF5 protein resembled that of its orthologs from humans. In healthy rock bream, the highest constitutive expression of Rb-IRF5 was detected in the liver. After iridovirus and polyinosinic-polycytidylic acid (poly(I:C)) challenge, Rb-IRF5 expression was significantly induced in the head kidney. Furthermore, rock bream recombinant type I interferon (Rb-IFN1) was also found to be an efficient inducer of Rb-IRF5 in a head kidney primary cell culture model. Upon IRF5 transfection, rock bream Mx (Rb-Mx), interferon I (Rb-IFN1) and tumor necrosis factor α (Rb-TNFα) genes get significantly upregulated in rock bream heart cells. The findings of the present study explain the involvement of Rb-IRF5 in the induction of interferons and pro-inflammatory cytokines and thereby provide a model for how IRF5 modulates immune responses against viral infections in rock bream.
[Show abstract][Hide abstract] ABSTRACT: Complement component 1q (C1q) is a subcomponent of the C1 complex and the key protein that recognizes and binds to a broad range of immune and non-immune ligands to initiate the classical complement pathway. In the present study, we identified and characterized three novel C1q family members from rock bream, Oplegnathus fasciatus. The full-length cDNAs of C1q A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like (RbC1qCL) consist of 780, 720 and 726 bp of nucleotide sequence encoding polypeptides of 260, 240 and 242 amino acids, respectively. All three RbC1qs possess a leading signal peptide and collagen-like region(s) (CLRs) in the N-terminus, and a C1q domain at the C-terminus. The C1q characteristic Gly-X-Y repeats are present in all three RbC1qs, while the CLR-associated sequence that enhances phagocytic activity is present in RbC1qAL ((49)GEKGEP(54)) and RbC1qCL ((70)GEKGEP(75)). Moreover, the coding region was distributed across six exons in RbCqAL and RbC1qCL, but only five exons in RbC1qBL. Phylogenetic analysis revealed that the three RbC1qs tightly cluster with the fish clade. All three RbC1qs are most highly expressed in the spleen and liver, as indicated by qPCR tissue profiling. In addition, all three are transcriptionally responsive to immune challenge, with liver expression being significantly up-regulated in the early phase of infection with intact, live bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus) and in the late phase of exposure to purified endotoxin (lipopolysaccharide). These data collectively suggest that the RbC1qs may play defense roles as an innate immune response to protect the rock bream from bacterial and viral infections.
[Show abstract][Hide abstract] ABSTRACT: Lysozymes are ubiquitously distributed enzymes with hydrolytic activity against bacterial peptidoglycan and function to protect organisms from microbial pathogens. In this study, an invertebrate goose-type lysozyme, designated as abLysG, was identified in the disk abalone, Haliotis discus discus. The full-length cDNA of abLysG was 894 bp in length with an open reading frame of 789 bp encoding a polypeptide of 263 amino acids containing a signal peptide and a characteristic soluble lytic transglycosylase domain. Six cysteine residues and two catalytic residues (Glu(142) and Asp(168)) conserved among molluscs were also identified. The 3D homology structural models of abLysG and hen egg white lysozyme had similar conformations of the active sites involved in the binding of substrate. BAC sequence data revealed that the genomic structure of disk abalone g-type lysozyme comprises 7 exons with 6 intervening introns. The deduced amino acid sequence of abLysG shared 45.2-61.6% similarity with those of other molluscs and vertebrates. The TFSEARCH server predicted a variety of transcription factor-binding sites in the 5'-flanking region of the abLysG gene, some of which are involved in transcriptional regulation of the lysozyme gene. abLysG expression was detected in multiple tissues with the highest expression in mantle. Moreover, qPCR analysis of abLysG mRNA expression demonstrated significant up-regulation in gill in response to infection by live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia) and bacterial mimics (LPS and PGN). Expression of the recombinant disk abalone g-type lysozyme in Escherichia coli BL21, demonstrated its bacteriolytic activity against several Gram-negative and Gram-positive bacterial species. Collectively these data suggest that abLysG is an antimicrobial enzyme with a potential role in the disk abalone innate immune system to protect it from bacterial and viral infections.
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rockbream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, Edwardsiella tarda, and Streptococcus iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.
[Show abstract][Hide abstract] ABSTRACT: Catalases are known to be antioxidant enzymes that can mainly dismutate hydrogen peroxide into water and oxygen in order to prevent oxidative stress. The complete genomic DNA (gDNA) sequence of the catalase gene from rock bream (Oplegnathus fasciatus) was identified from our custom-constructed BAC genomic DNA library and designated as RbCat. RbCat consists of 13 exons, separated by 12 introns, within a 13,722-bp gDNA sequence. The complete cDNA sequence (3,303 bp) of RbCat is comprised of a 1,581-bp coding region, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 60 kDa and a theoretical isoelectric point of 8.34. The anticipated promoter region of RbCat contains several transcription factor binding sites, including sites that bind with immune- and antioxidant-responsive signaling molecules, suggesting its substantial transcriptional regulation. RbCat resembles the typical catalase family signature, i.e., it is composed of the catalase proximal active site motif along with a catalase proximal heme-ligand signature motif and shares great homology with its fish counterparts. According to multiple sequence alignment, functionally important aa present in RbCat were thoroughly conserved among its vertebrate counterparts. Phylogenetic analysis revealed that RbCat evolved from a vertebrate origin, and further positioned it in the fish clade. Recombinant RbCat had noticeable peroxidase activity against its substrate, hydrogen peroxide, in a dose-dependent manner. However, it demonstrated substantial peroxidase activity within a broad range of temperatures and pH values. Constitutive RbCat mRNA expression of different magnitudes was detected in a tissue-specific manner, suggesting its diverse role in physiology with respect to the tissue type. Moreover, immune challenge experiments using Edwardsiella tarda and rock bream iridovirus (RBIV) as live pathogens and polyinosinic:polycytidylic acid and lipopolysaccharide as mitogens revealed that the transcription of RbCat can be modulated by immune stimulation. Collectively, the results obtained in this study suggest that RbCat can function as a potent antioxidant enzyme in rock bream and may play a role in post-immune responses with respect to its peroxidase activity.
[Show abstract][Hide abstract] ABSTRACT: Lysozyme is an important enzyme in the innate immune system that plays a vital role in fighting microbial infections. In the current study, we identified, cloned, and characterized a gene that encodes an invertebrate-type lysozyme from the disk abalone, Haliotis discus discus (abLysI). The full-length cDNA of abLysI consisted of 545 bp with an open reading frame of 393 bp that encodes 131 amino acids. The theoretical molecular mass of mature abLysI was 12.3 kDa with an isoelectric point of 8.03. Conserved features in other homologs, such as catalytic sites for lytic activity (Glu(30) and Asp(41)), isopeptidase activity (His(107)), and ten cysteine residues were identified in abLysI. Genomic sequence analysis with respect to its cDNA showed that abLysI was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative promoter region. Homology and phylogeny analysis of abLysI depicted high identity and closer proximity, respectively, with an annelid i-type lysozyme from Hirudo medicinalis, and indicated that abLysI is a novel molluscan i-type lysozyme. Tissue-specific expressional studies revealed that abLysI is mainly transcribed in hepatopancreas followed by mantle. In addition, abLysI mRNA expression was induced following bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus) challenges. Recombinantly expressed abLysI [(r)abLysI] demonstrated strong lytic activity against Micrococcus lysodeikticus, isopeptidase activity, and antibacterial activity against several Gram-positive and Gram-negative bacteria. Moreover, (r)abLysI showed optimum lytic activity at pH 4.0 and 60°C, while exhibiting optimum isopeptidase activity at pH 7.0. Taken together, these results indicate that abLysI is potentially involved in immune responses of the disk abalone to protect it from invaders.
[Show abstract][Hide abstract] ABSTRACT: Akirins are conserved nuclear resident NF-κB signaling pathway molecules. Isoforms of akirins found in various organisms are known to play diverse roles. In this study, we have characterized two akirin2 homologues from rock bream, OfAk2(1) and OfAk2(2). The proteins derived from OfAk2(1) and OfAk2(2) revealed the presence of nuclear localization signal. Multiple sequence alignment and pairwise alignment of OfAk2(1) and OfAk2(2) with the akirin homologues, revealed high conservation and identity. Phylogenetic tree analysis revealed that the distinct position of OfAk2(1) and OfAk2(2) was close to the fish homologues and separated from the mammals and invertebrates. Genomic structure characterization revealed two distinct structures. OfAk2(1) possessed 6 exons interrupted by 5 introns whereas OfAk2(2) possessed 5 exons interrupted by 4 introns. The promoter analysis revealed the presence of significant transcription factors, which suggests its regulation by diverse stimuli. In addition, transcript expression analysis using real time quantitative reverse-transcriptase polymerase chain reaction post immune challenges with lipopolysaccharide, Edwardsiella tarda and poly I:C revealed upregulation of both OfAk2(1) and OfAk2(2) in liver, spleen and head kidney.
[Show abstract][Hide abstract] ABSTRACT: Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule playing vital roles in various signaling cascades, including cell proliferation, activation of immune responses against infections. It is well known as a pivotal regulator of innate immunity. In this study, we have rescued and characterized two members of the MIF family, macrophage migration inhibitory factor (OfMIF) and D-Dopachrome tautomerase (OfDDT) from rock bream, Oplegnathus fasciatus. The deduced OfMIF and OfDDT protein sequences revealed the presence of the catalytic oxidoreductase (CXXC), motif. They also possessed highly conserved proline (P(2)) and lysine residues (K(33)), responsible for their isomerase and tautomerase functions. Rock bream MIF and DDT homologues shared higher identity with fish homologues and also with mammals and occupied a distinct position in the phylogenetic tree, depicting their evolutionary conservation. The spatial expression analysis revealed the highest expression of both OfMIF and OfDDT in liver, while portraying constitutive expression in other tissues. The recombinant proteins purified using the E. coli system revealed potent oxidoreductase activity against insulin with both dithiothreitol and glutathione as reducing agents. Stimulation of rock bream head kidney cells with recombinant OfMIF and OfDDT proteins induced the expression of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and interleukin-1β (IL-1β). These results together suggest their involvement in rock bream immune defense and this study on the novel MIF family member DDT from rock bream will pave the way for further studies of this homologue in other teleosts and delineate its multiple functions.