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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 ( 112 T, 115 C, 191 R) were identified in the SsPrx4 protein sequence. A peroxidatic cysteine ( 115 C P ) and resolving cysteines ( 236 C R ) 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 H 2 O 2 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., (Copyright © 2019. Published by Elsevier Ltd.)

Complement system orchestrates the innate and adaptive immunity via the activation, recruitment, and regulation of immune molecules to destroy pathogens. However, regulation of the complement is essential to avoid injuries to the autologous tissues. The present study unveils the characteristic features of an important complement component, anaphylatoxin inactivator from red lip mullet at its molecular and functional level. Mullet carboxypeptidase N1 (MuCPN1) cDNA sequence possessed an open reading frame of 1347 bp, which encoded a protein of 449 amino acids with a predicted molecular weight of 51 kDa. In silico analysis discovered two domains of PM14-Zn carboxypeptidase and a C-terminal domain of M14 N/E carboxypeptidase, two zinc-binding signature motifs, and an N-glycosylation site in the MuCPN1 sequence. Homology analysis revealed that most of the residues in the sequence are conserved among the other selected homologs. Phylogeny analysis showed that MuCPN1 closely cladded with the Maylandia zebra CPN1 and clustered together with the teleostean counterparts. A challenge experiment showed modulated expression of MuCPN1 upon polyinosinic:polycytidylic acid and Lactococcus garviae in head kidney, spleen, gill, and liver tissues. The highest upregulation of MuCPN1 was observed 24 h post infection against poly I:C in each tissue. Moreover, the highest relative expressions upon L. garviae challenge were observed at 24 h post infection in head kidney tissue and 48 h post infection in spleen, gill, and liver tissues. MuCPN1 transfected cells triggered a 2.2-fold increase of nitric oxide (NO) production upon LPS stimulation compared to the un-transfected controls suggesting that MuCPN1 is an active protease which releases arginine from complement C3a, C4a, and C5a. These results have driven certain way towards enhancing the understanding of immune role of MuCPN1 in the complement defense mechanism of red lip mullet., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant metalloenzyme. The main function of this enzyme is to dismutase the toxic superoxide anion (O 2 -) into less toxic hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ). Structural analysis of mullet MnSOD (MuMnSOD) was performed using different bioinformatics tools. Pairwise alignment revealed that the protein sequence matched to that derived from Larimichthys crocea with a 95.2% sequence identity. Phylogenetic tree analysis showed that the MuMnSOD was included in the category of teleosts. Multiple sequence alignment showed that a SOD Fe-N domain, SOD Fe-C domain, and Mn/Fe SOD signature were highly conserved among the other examined MnSOD orthologs. Quantitative real-time PCR showed that the highest MuMnSOD mRNA expression level was in blood cells. The highest expression level of MuMnSOD was observed in response to treatment with both Lactococcus garvieae and lipopolysaccharide (LPS) at 6 h post treatment in the head kidney and blood. Potential ROS-scavenging ability of the purified recombinant protein (rMuMnSOD) was examined by the xanthine oxidase assay (XOD assay). The optimum temperature and pH for XOD activity were found to be 25 °C and pH 7, respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. Activity of rMuMnSOD was inhibited by potassium cyanide (KCN) and N-N'-diethyl-dithiocarbamate (DDC). Moreover, expression of MuMnSOD resulted in considerable growth retardation of both gram-positive and gram-negative bacteria. Results of the current study suggest that MuMnSOD acts as an antioxidant enzyme and participates in the immune response in mullet., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Disturbance in the balance between pro-oxidants and anti-oxidants result oxidative stress in aerobic organisms. However, oxidative stress can be inhibited by enzymatic and non-enzymatic defense mechanisms. Superoxide dismutases (SODs) are well-known scavengers of superoxide radicals, and they protect cells by detoxifying hazardous reactive oxygen species. Here, we have identified and characterized two different SODs, CuZnSOD and MnSOD, from black rockfish (RfCuZnSOD and RfMnSOD, respectively). In silico analysis revealed the well-conserved molecular structures comprising all essential properties of CuZnSOD and MnSOD. Phylogenetic analysis revealed that both RfCuZnSOD and RfMnSOD cladded with their fish counterparts. The recombinant RfSOD proteins demonstrated their potential superoxide scavenging abilities through a xanthine oxidase assay. The optimum temperature and pH conditions for both rRfSODs were 25 °C and pH 8, respectively. Moreover, the potential peroxidation function of rRfCuZnSOD was observed in the presence of HCO 3 - . The highest peroxidation activity was observed at 100 μg/mL of rRfCuZnSOD using the MTT cell viability assay and flow cytometry. The analogous tissue-specific expression profile indicated ubiquitous expression of both RfCuZnSOD and RfMnSOD in selected tissues of healthy juvenile rockfish. An immune challenge experiment illustrated the altered expression profiles of both RfCuZnSOD and RfMnSOD against lipopolysaccharide, Streptococcus iniae, and polyinosinic-polycytidylic acid (poly I:C). Collectively, these results strengthen the general understanding of the structural and functional characteristics of SODs within the host defense system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Manganese superoxide dismutase (MnSOD) is a metaloenzyme that catalyzes dismutation of the hazardous superoxide radicals into less hazardous H 2 O 2 and H 2 O. Here, we identified a homolog of MnSOD from big belly seahorse (Hippocampus abdominalis; HaMnSOD) and characterized its structural and functional features. HaMnSOD transcript possessed an open reading frame (ORF) of 672 bp which codes for a peptide of 223 amino acids. Pairwise alignment showed that HaMnSOD shared highest identity with rock bream MnSOD. Results of the phylogenetic analysis of HaMnSOD revealed a close proximity with rock bream MnSOD which was consistent with the result of homology alignment. The intense expression of HaMnSOD was observed in the ovary, followed by the heart and the brain. Further, immune related responses of HaMnSOD towards pathogenic stimulation were observed through bacterial and viral challenges. Highest HaMnSOD expression in response to stimulants Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide (LPS), and polyinosinic-polycytidylic acid (Poly I:C) was observed in the late stage in the blood tissue. Xanthine/xanthine oxidase assay (XOD assay) indicated the ROS-scavenging ability of purified recombinant HaMnSOD (rHaMnSOD). The optimum conditions for the SOD activity of rHaMnSOD were pH 9 and the 25 °C. Collectively, the results obtained through the expressional analysis profiles and the functional assays provide insights into potential immune related and antioxidant roles of HaMnSOD in the big belly seahorse., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The iron-withholding strategy of innate immunity is an effective antimicrobial defense mechanism that combats microbial infection by depriving microorganisms of Fe 3+ , which is important for their growth and propagation. Transferrins (Tfs) are a group of iron-binding proteins that exert their antimicrobial function through Fe 3+ sequestration. The current study describes both structural and functional characteristics of a transferrin ortholog from rock bream Oplegnathus fasciatus (RbTf). The RbTf cDNA possesses an open reading frame (ORF) of 2079 bp encoding 693 amino acids. It has a molecular mass of approximately 74 kDa and an isoelectric point of 5.4. In silico analysis revealed that RbTf has two conserved domains: N-terminal domain and C-terminal domain. Pairwise homology analysis and phylogenetic analysis revealed that RbTf shared the highest identity (82.6%) with Dicentrarchus labrax Tf. According to the genomic analysis, RbTf possesses 17 exons and 16 introns, similar to the other orthologs. Here, we cloned the N terminal and C terminal domains of RbTf to evaluate their distinct functional features. Results obtained through the CAS (chrome azurol S) assay confirmed the iron-binding ability of the RbTf, and it was further determined that the iron-binding ability of rRbTfN was higher than that of rRbTfC. The antimicrobial functions of the rRbTfN and the rRbTfC were confirmed via the iron-dependent bacterial growth inhibition assay. Tissue distribution profiling revealed a ubiquitous expression with intense expression in the liver. Temporal assessment revealed that RbTf increased after stimulation of LPS, Edwardsiella tarda, and Streptococcus iniae post injection (p.i.). These findings demonstrated that RbTf is an important antimicrobial protein that can combat bacterial pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants. AbMAPK1 harbors a 5' untranslated region (UTR) of 23 bps, a coding sequence of 1104 bps, and a 3' UTR of 448 bp. The putative peptide comprises a predicted molecular mass of 42.2 kDa, with a theoretical pI of 6.28. Based on the in silico analysis, AbMAPK1 possesses two N-glycosylation sites, one S_TK catalytic domain, and a conserved His-Arg-Asp domain (HRD). In addition, a conservative glycine rich ATP-phosphate-binding loop and a threonine-x-tyrosine motif (TEY) important for the autophosphorylation were also identified in the protein. Homology assessment of AbMAPK1 showed several conserved regions, and ark clam (Aplysia californica) showed the highest sequence identity (87.9%). The phylogenetic analysis supported close evolutionary kinship with molluscan orthologs. Constitutive expression of AbMAPK1 was observed in six different tissues of disk abalone, with the highest expression in the digestive tract, followed by the gills and hemocytes. Highest AbMAPK1 mRNA expression level was detected at the trochophore developmental stage, suggesting its role in abalone cell differentiation and proliferation. Significant modulation of AbMAPK1 expression under pathogenic stress suggested its putative involvement in the immune defense mechanism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Copper-zinc-superoxide dismutase (CuZnSOD) from Hippocampus abdominalis (HaCuZnSOD) is a metalloenzyme which belongs to the ubiquitous family of SODs. Here, we determined the characteristic structural features of HaCuZnSOD, analyzed its evolutionary relationships, and identified its potential immune responses and biological functions in relation to antioxidant defense mechanisms in the seahorse. The gene had a 5' untranslated region (UTR) of 67 bp, a coding sequence of 465 bp and a 3' UTR of 313 bp. The putative peptide consists of 154 amino acids. HaCuZnSOD had a predicted molecular mass of 15.94 kDa and a theoretical pI value of 5.73, which is favorable for copper binding activity. In silico analysis revealed that HaCuZnSOD had a prominent Cu-Zn_superoxide_dismutase domain, two Cu/Zn signature sequences, a putative N-glycosylation site, and several active sites including Cu(2+) and Zn(2+) binding sites. The three dimensional structure indicated a β-sheet barrel with 8 β-sheets and two short α-helical regions. Multiple alignment analyses revealed many conserved regions and active sites among its orthologs. The highest amino acid identity to HaCuZnSOD was found in Siniperca chuatsi (87.4%), while Maylandia zebra shared a close relationship in the phylogenetic analysis. Functional assays were performed to assess the antioxidant, biophysical and biochemical properties of overexpressed recombinant (r) HaCuZnSOD. A xanthine/XOD assay gave optimum results at pH 9 and 25 °C indicating these may be the best conditions for its antioxidant action in the seahorse. An MTT assay and flow cytometry confirmed that rHaCuZnSOD showed peroxidase activity in the presence of HCO3(-). In all the functional assays, the level of antioxidant activity of rHaCuZnSOD was concentration dependent; metal ion supplementation also increased its activity. The highest mRNA expressional level of HaCuZnSOD was found in blood. Temporal assessment under pathological stress showed a delay response by HaCuZnSOD. Our findings demonstrated that HaCuZnSOD is an important antioxidant, which might be involved in the host antioxidant defense mechanism against oxidative stress., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

1-cysteine peroxiredoxin (Prx6) is an antioxidant enzyme that protects cells by detoxifying multiple peroxide species. This study aimed to describe molecular features, functional assessments and potential immune responses of Prx6 identified from the big-belly seahorse, Hippocampus abdominalis (HaPrx6). The complete ORF (666 bp) of HaPrx6 encodes a polypeptide (24 kDa) of 222 amino acids, and harbors a prominent peroxiredoxin super-family domain, a peroxidatic catalytic center, and a peroxidatic cysteine. The deduced amino acid sequence of HaPrx6 shares a relatively high amino acid sequence similarity and close evolutionary relationship with Oplegnathus fasciatus Prx6. The purified recombinant HaPrx6 protein (rHaPrx6) was shown to protect plasmid DNA in the Metal Catalyzed Oxidation (MCO) assay and, together with 1,4-Dithiothreitol (DTT), protected human leukemia THP-1 cells from extracellular H2O2-mediated cell death. In addition, quantitative real-time PCR revealed that HaPrx6 mRNA was constitutively expressed in 14 different tissues, with the highest expression observed in liver tissue. Inductive transcriptional responses were observed in liver and kidney tissues of fish after treating them with bacterial stimuli, including LPS, Edwardsiella tarda, and Streptococcus iniae. These results suggest that HaPrx6 may play an important role in the immune response of the big-belly seahorse against microbial infection. Collectively, these findings provide structural and functional insights into HaPrx6., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Interferons (IFNs) and IFN-inducible proteins play numerous physiological roles, particularly in antiviral defense mechanisms of the innate immune response with the presence of pathogens. IFN-induced protein-35 kDa (IFP35) is induced by Type II IFN (IFN-γ); it is a cytoplasmic protein that can be translocated to the nucleus via the stimulation of IFN. In this study, we report the complete molecular characterization of the IFP35 cDNA sequence from the black rockfish in an effort to understand its role in the immune response. The coding sequence of RfIFP35 encoded a putative peptide of 371 amino acids containing two characteristic Nmi/IFP 35 domains (NIDs), which are highly conserved among its counterparts. The protein showed a molecular mass of 42.2 kDa with a theoretical pI of 5.05 and was predicted to be unstable because of its high instability index (49.37). Therefore, the protein-protein interaction is essential for its stability, which may be facilitated by the intrinsically disordered regions in this protein. According to cellular location prediction, the RfIFP35 protein is cytosolic. Phylogenetic analysis showed that RfIFP35 was cladded within the fish counterparts. Tissue distribution profiling revealed a ubiquitous presence of the protein in all examined tissues, with highest expression in the blood followed by the spleen tissues. The expression of RfIFP35 during immune challenge with poly I:C and lipopolysaccharide treatments affirms its putative importance in the first-line host defense system. RfIFN-γ mRNA was significantly expressed at 6 h p.i. in blood and 3 h p.i. in the spleen following treatment with different immune stimulants, and its expression was higher compared to that of RfIFP35 mRNA. Therefore, the modulation patterns of both RfIFP35 and RfIFN-γ suggest that RfIFP35 may be induced by RfIFN-γ., (Copyright © 2016 Elsevier Ltd. All rights reserved.)