Molecular characterization and expression profiling of tandem-repeat galectin-8 from red-spotted grouper (Epinephelus akaara): Potential antibacterial, antiviral, and wound healing activities.

Galectin-8 is a typical ß-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae, Streptococcus parauberis, Lactococcus garvieae, Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper.

Differential gene expression of red-spotted grouper (Epinephelus akaara) in response to lipopolysaccharide, poly I:C, and nervous necrosis virus revealed by RNA-seq data.

Red-spotted grouper (Epinephelus akaara) is a popular aquaculture species with high commercial value in the food industry. However, some infectious diseases may cause mass mortality in cultural practice. Therefore, it is important to understand the immune responses of red-spotted groupers upon pathogenic invasion to develop successful disease prevention mechanisms. Here, we analyzed the transcriptomic profiles of red-spotted grouper head kidney stimulated with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), and nervous necrosis virus (NNV) and identified differentially expressed genes (DEGs) using RNA-sequencing technology. Cluster analysis of the identified DEGs showed DEG distribution in nine separate clusters based on their expression patterns. However, significant upregulation of most DEGs was observed 6 h after poly I:C stimulation. The DEGs were functionally annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, which revealed significant expression of many immune-related signaling pathways, including antiviral, protein translation, cellular protein catabolic process, inflammatory responses, DNA repair, and cell division. Furthermore, selected DEGs were validated by quantitative real-time PCR, confirming the reliability of our findings. Collectively, this study provides insight into the immune responses of red-spotted groupers, thereby expanding the understanding of fish immunity.

Molecular cloning, expression analysis of interleukin 17D (cysteine knot cytokine) from Amphiprion clarkii and their functional characterization and NFκB pathway activation using FHM cells.

Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii. We performed spatial and temporal expression, AcIL-17D-mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin (NKL), Hepcidin antimicrobial peptide-1 (HAMP-1), Defensin-ß (DEFB1)] and some inflammatory genes such as IL-1ß, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits (NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost.

Molecular characterization, antiviral activity, and UV-B damage responses of Caspase-9 from Amphiprion clarkii.

Apoptosis plays a vital role in maintaining cellular homeostasis in multicellular organisms. Caspase-9 (casp-9) is one of the major initiator caspases that induces apoptosis by activating downstream intrinsic apoptosis pathway genes. Here, we isolated the cDNA sequence (1992 bp) of caspase-9 from Amphiprion clarkii (Accasp-9) that consists of a 1305 bp coding region and encodes a 434 aa protein. In silico analysis showed that Accasp-9 has a theoretical isoelectric point of 5.81 and a molecular weight of 48.45 kDa. Multiple sequence alignment revealed that the CARD domain is located at the N-terminus, whereas the large P-20 and small P-10 domains are located at the C-terminus. Moreover, a highly conserved pentapeptide active site (296QACGG301), as well as histidine and cysteine active sites, are also retained at the C-terminus. In phylogenetic analysis, Accasp-9 formed a clade with casp-9 from different species, distinct from other caspases. Accasp-9 was highly expressed in the gill and intestine compared with other tissues analyzed in healthy A. clarkii. Accasp-9 expression was significantly elevated in the blood after stimulation with Vibrio harveyi and polyinosinic:polycytidylic acid (poly I:C; 12-48 h), but not with lipopolysaccharide. The nucleoprotein expression of the viral hemorrhagic septicemia virus was significantly reduced in Accasp-9 overexpressed fathead minnow (FHM) cells compared with that in the control. In addition, other in vitro assays revealed that cell apoptosis was significantly elevated in poly I:C and UV-B-treated Accasp-9 transfected FHM cells. However, H248P or C298S mutated Accasp-9 significantly reduced apoptosis in UV-B irradiated cells. Collectively, our results show that Accasp-9 might play a defensive role against invading pathogens and UV-B radiation and H248 and C298 active residues are significantly involved in apoptosis in teleosts.

Genome-wide association study of VHSV-resistance trait in Paralichthys olivaceus.

In flounder aquaculture, selective breeding plays a vital role in the development of disease-resistant traits and animals with high growth rates. Moreover, superior animals are required to achieve high profits. Unlike growth-related traits, disease-resistant experiments need to be conducted in a controlled environment, as the improper measurement of traits often leads to low genetic correlation and incorrect estimation of breeding values. In this study, viral hemorrhagic septicemia virus (VHSV) resistance was studied using a genome-wide association study (GWAS), and the genetic parameters were estimated. Genotyping was performed using a high-quality 70 K single nucleotide polymorphism (SNP) Affymetrix® Axiom® myDesign™ Genotyping Array of olive flounder. A heritability of ∼0.18 for resistance to VHSV was estimated using genomic information of the fish. According to the GWAS, significant SNPs were detected in chromosomes 21, 24, and contig AGQT02032065.1. Three SNPs showed significance at the genome-wide level (p < 1 × 10-6), while others showed significance above the suggestive cutoff (p < 1 × 10-4). The 3% phenotypic variation was explained by the highest significant SNP, named AX-419319631. Of the important genes for disease resistance, SNPs were associated with plcg1, epha4, clstn2, pik3cb, hes6, meis3, prx6, cep164, siae, and kirrel3b. Most of the genes associated with these SNPs have been previously reported with respect to viral entry, propagation, and immune mechanisms. Therefore, our study provides helpful information regarding VHSV resistance in olive flounder, which can be used for breeding applications.

The possible role of catalase in innate immunity and diminution of cellular oxidative stress: Insights into its molecular characteristics, antioxidant activity, DNA protection, and transcriptional regulation in response to immune stimuli in yellowtail clownfish (Amphiprion clarkii).

Catalase, a key enzyme in the antioxidant defense grid of organisms, scavenges free radicals to curtail their harmful effects on the host, supporting proper immune function. Herein, we report the identification and characterization of a catalase homolog from Amphiprion clarkii (ClCat), followed by its functional characterization. An open reading frame was identified in the cDNA sequence of ClCat at 1581 bp, which encodes a protein of 527 amino acids (aa) with a molecular mass of 60 kDa. In silico analyses of ClCat revealed characteristic features of the catalase family and a lack of a signal peptide. Multiple sequence alignment of ClCat indicated the conservation of functionally important residues among its homologs. According to phylogenetic analysis, ClCat was of vertebrate origin, positioned within the teleost clade. During native conditions, ClCat mRNA was highly expressed in blood, followed by the liver and kidney. Moreover, significant changes in ClCat transcription were observed after stimulation with LPS, poly I:C, and Vibrio harveyi, in a time-dependent manner. Recombinant ClCat (rClCat) was characterized, and its peroxidase activity was determined. Furthermore, the optimum temperature and pH for rClCat were determined to be 30-40 °C and pH 7, respectively. Oxidative stress tolerance and chromatin condensation assays indicated enhanced cell survival and reduced apoptosis, resulting from reactive oxygen species scavenging by rClCat. The DNA-protective function of rClCat was further confirmed via a metal-catalyzed oxidation assay. Taken together, our findings propose that rClCat plays an essential role in maintaining cellular oxidative homeostasis and host immune protection.

Molecular characterization, immune and xenobiotic responses of glutathione S-transferase omega 1 from the big-belly seahorse: Novel insights into antiviral defense.

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification and function by conjugating with the thiol group of glutathione. In this study, we isolated an omega class GST from the big-belly seahorse (Hippocampus abdominalis; HaGSTO1) to study the putative xenobiotic responses and defense ability against viral and bacterial infections in this animal. The isolated HaGSTO1 gene, with a cording sequence of 720 bp, encodes a peptide of 239 amino acids. The predicted molecular mass and theoretical isoelectric point of HaGSTO1 was 27.47 kDa and 8.13, respectively. In-silico analysis of HaGSTO1 revealed a characteristic N-terminal thioredoxin-like domain and a C-terminal domain. Unlike other GSTs, the C-terminal of HaGSTO1 reached up to the N-terminal, and the N-terminal functional group was cysteine rather than tyrosine or serine, as observed in other GSTs. Phylogenetic analysis showed the evolutionary proximity of HaGSTO1 with other identified vertebrate and invertebrate GST orthologs. For the first time, we demonstrated the viral defense capability of HaGSTO1 against viral hemorrhagic septicemia virus (VHSV) infection. All six nucleoproteins of VHSV were significantly downregulated in HaGSTO1-overexpressing FHM cells at 24 h after infection compared with those in the control. Moreover, arsenic toxicity was significantly reduced in HaGSTO1-overexpressing FHM cells, and cell viability increased. Real-time polymerase chain reaction analysis showed that HaGSTO1 transcripts were highly expressed in the pouch and gill when compared with those in other tissues. Blood HaGSTO1 transcripts were significantly upregulated after Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid challenge experiments. Collectively, these findings suggest the involvement of HaGSTO1 in the host defense mechanism of seahorses.

Molecular characterization and immune regulatory, antioxidant, and antiapoptotic activities of thioredoxin domain-containing protein 17 (TXNDC17) in yellowtail clownfish (Amphiprion clarkii).

Thioredoxin domain-containing protein 17 (TXNDC17) is an important, highly conserved oxidoreductase protein, ubiquitously expressed in all living organisms. It is a small (~14 kDa) protein mostly co-expressed with thioredoxin 1 (TRx1). In the present study, we obtained the TXNDC17 gene sequence from a previously constructed yellowtail clownfish (Amphiprion clarkii) (AcTXNDC17) database and studied its phylogeny as well as the protein's molecular characteristics, antioxidant, and antiapoptotic effects. The full length of the AcTXNDC17 cDNA sequence was 862 bp with a 372 bp region encoding a 123 amino acid (aa) protein. The predicted molecular mass and isoelectric point of AcTXNDC17 were 14.2 kDa and 5.75, respectively. AcTXNDC17 contained a TRX-related protein 14 domain and a highly conserved N-terminal Cys43-Pro44-Asp45-Cys46 motif. qPCR analysis revealed that AcTXNDC17 transcripts were ubiquitously and differently expressed in all the examined tissues. AcTXNDC17 expression in the spleen tissue was significantly upregulated in a time-dependent manner upon stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), and Vibrio harveyi. Besides, LPS-induced intrinsic apoptotic pathway (TNF-α, caspase-8, Bid, cytochrome C, caspase-9, and caspase-3) gene expression was significantly lower in AcTXNDC17-overexpressing RAW264.7 cells, as were NF-κB activation and nitric oxide (NO) production. Furthermore, the viability of H2O2-stimulated macrophages was significantly improved under AcTXNDC17 overexpression. Collectively, our findings indicate that AcTXNDC17 is involved in the innate immune response of the yellowtail clownfish.

Transcription profile, NF-ĸB promoter activation, and antiviral activity of Amphiprion clarkii Akirin-2.

Animal defense system constitutes a series of distinct mechanisms that specifically defend against microbial invasion. Understanding these complex biological mechanisms is of paramount importance for implementing disease prevention strategies. In this study, the transcription factor, Akirin-2 was identified from ornamental fish Amphiprion clarkii and its involvement in immune response was characterized. A. clarkii Akirin-2 (AcAkirin-2) was identified as a highly conserved protein with two nuclear localization signals. In-vitro localization analysis in fathead minnow cells revealed that AcAkirin-2 is strictly localized to the nucleus. With regard to tissue-specific expression without immune challenge, AcAkirin-2 expression was highest in the brain and lowest in the liver. Immune challenge experiments revealed that AcAkirin-2 expression was the strongest in response to poly I:C. Overexpression of AcAkirin-2 alone did not enhanced NF-ĸB activity significantly in HEK293T cells; however, it significantly enhanced NF-ĸB activity in the presence of poly I:C. AcAkirin-2-mediated expression of antiviral genes was analyzed using qPCR in mullet kidney cells and plaque assay was performed to decipher the involvement of AcAkirin-2 in antiviral immunity. AcAkirin-2 overexpression significantly enhanced the expression of Viperin but not of Mx. Plaque assays revealed the ability of AcAkirin-2 to enervate VHSV titers. Taken together, this study unveiled the involvement of AcAkirin-2 in NF-ĸB-mediated transcription of antiviral genes.

Expression, subcellular localization, and potential antiviral function of three interferon regulatory factors in the big-belly seahorse (Hippocampus abdominalis).

Interferon regulatory factors (IRFs) are among the most important transcription mediators and have multiple biological functions, such as antiviral and antimicrobial defense, cell differentiation, immune modulation, and apoptosis. Three IRF family members (HaIRF4-like, HaIRF6, and HaIRF8) of the big belly seahorse (Hippocampus abdominalis) were molecularly and functionally characterized at the sequence and transcriptional level. The coding sequences of HaIRF4-like, HaIRF6, and HaIRF8 were 1214, 1485, and 1266 bp in length, encoding proteins of size 46.21, 55.32, and 47.56 kDa, respectively. Potential viral transcription and replication was detected against VHSV infection using qPCR in HaIRFs-transfected FHM cells. IRFs significantly reduced viral gene expression at 24 h and 48 h post infection and the expression of interferon-stimulated genes (ISGs) was modulated at transcriptional level upon HaIRF overexpression in FHM cells. Subcellular HaIRF localization was observed using GFP-tagged expression vectors in FHM cells. HaIRF4-like and HaIRF8 were localized to the nucleus, whereas HaIRF6 was observed in the cytoplasm. All three IRFs were ubiquitously expressed in all analyzed tissues of the big belly seahorse. The mRNA expression of IRF4-like, IRF6, and IRF8 increased significantly post injection in the blood and gills following LPS, poly (I:C), and Streptococcus iniae challenge. These findings demonstrate that seahorse IRFs are involved in host defense mechanisms against immune stimulants and HaIRFs induce interferon and ISGs which trigger antiviral activity against viral infections in the host.

Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly seahorse (Hippocampus abdominalis) and expression upon immune provocation.

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse.

Molecular and functional insights into a novel teleost malectin from big-belly seahorse Hippocampus abdominalis.

Malectin is a carbohydrate-binding lectin protein found in the endoplasmic reticulum (ER). It selectivity binds to Glc2-N-glycan and is involved in a glycoprotein quality control mechanism. Even though malectin may play a role in immunity, its role in innate immunity is not fully known. In the present study, we identified and characterized the malectin gene from Hippocampus abdominalis (HaMLEC). We analyzed sequence features, spatial expression levels, temporal expression profiles upon immune responses, bacterial and carbohydrate binding abilities and anti-viral properties to investigate the potential role of HaMLEC in innate immunity. The molecular weight and isoelectric point (pI) were estimated to be 31.99 kDa and 5.17, respectively. The N-terminal signal peptide, malectin superfamily domain and C-terminal transmembrane region were identified from the amino acid sequence of HaMLEC. The close evolutionary relationship of HaMLEC with other teleosts was identified by phylogenetic analysis. According to quantitative PCR (qPCR) results, HaMLEC expression was observed in all the examined tissues and high expression was observed in the ovary and brain, compared to other tested tissues. Temporal expression of HaMLEC in liver and blood tissues were significant modulated upon exposure to immunogens Edwardasiella tarda, Streptococcus iniae, polyinosinic:polycytidylic and lipopolysaccharide. The presence of carbohydrate binding modules (CBMs) of bacterial glycosyl hydrolases were functionally confirmed by a bacterial binding assay. Anti-viral activity significantly reduced viral hemorrhagic septicemia virus (VHSV) replication in cells overexpressing HaMLEC. The observed results suggested that HaMLEC may have a significant role in innate immunity in Hippocampus abdominalis.

Structural and functional analysis of three Iκb kinases (IKK) in disk abalone (Haliotis discus discus): Investigating their role in the innate immune responses.

The IκB kinases (IKK) are large multiprotein complexes that regulate the activation of the transcription factor NF-κB and are involved in a diverse range of biological processes, including innate immunity, inflammation, and development. To explore the potential roles of invertebrate IKKs on immunity, three IKK encoding genes have been identified from molluscan species disk abalone and designed as AbIKK1, AbIKK2 and AbIKK3 at the transcriptional level. Coding sequences of AbIKK1, AbIKK2 and AbIKK3 encode the peptides of 746, 751 and 713 amino acids with the predicted molecular mass of 86.16, 86.12 and 81.88 kDa respectively. All three AbIKKs were found to share conserved IKK family features including the kinase superfamily domain (KD), ubiquitin-like domain (ULD), and α-helical scaffold/dimerization domain (SDD), similar to their mammalian counterparts. Under normal physiological conditions, AbIKKs were ubiquitously detected in six different tissues, with the highest abundance in the digestive tract and gills. Temporal transcriptional profiles in abalone hemocytes revealed the induction of AbIKK1, AbIKK2, and AbIKK3 expression following exposure to Gram-negative (Vibrio parahemolyticus) and Gram-positive (Listeria monocytogenes) bacteria, viruses (viral hemorrhagic septicemia virus, VHSV), LPS, or poly I:C. The overexpression of AbIKKs in HEK293T or RAW264.7 murine macrophage cells induced NF-κB promoter activation independent of stimulation by TNF-α or LPS. Moreover, iNOS and COX2 expression was induced in AbIKK transfected RAW264.7 murine macrophage cells and the induced state was maintained post-LPS treatment. Furthermore, mRNA levels of three selected cytokine-encoding genes (IL-1ß, IL-6, and TNF-α) were found to be elevated in abalone IKK overexpressed RAW264.7 murine macrophage cells, both with and without LPS exposure. Overall, our findings demonstrated that AbIKKs identified in this study were positively involved in eliciting innate immune responses in abalone. In addition, the data revealed the presence of an evolutionarily conserved signaling mechanism for IKK mediated NF-κB activation in mollusks.

Characterization and expression analysis of rockfish (Sebastes schlegelii) myeloid differentiation factor-88 (SsMyD88) and evaluation of its ability to induce inflammatory cytokines through NF-ĸB.

Innate immunity is characterized by nonspecific, prompt reactions toward armada of antigens. Animals funnel down a repertoire of immune stimulants to activate non-selective defense mechanisms rapidly. This study was conducted to characterize the rockfish (Sebastes schlegelii) adaptor protein MyD88 (SsMyD88), which interacts with both toll-like receptors and interleukin receptors. The tissue expression of unchallenged SsMyD88 was evaluated by quantitative real time PCR (qPCR). Fish were intraperitoneally injected with immune stimulants including poly I:C, lipopolysaccharides, and Streptococcus iniae. Then, the temporal expression of SsMyD88 was analyzed. Finally, the inflammatory gene expression and downstream promoter activation were analyzed. Strongest expressions were reported in the liver, gills and spleen in unchallenged conditions. All diverse immune stimulants were found to be capable of significantly altering SsMyD88 transcription during the challenge experiment. Evaluation of downstream promoter biases by SsMyD88 found a predominant activation of NF-ĸB transcription factors when compared with the AP-1, revealing significant and substantial upregulation of major inflammatory mediators such as IL-1-ß, IL-6, iNOS, COX-2 and TNF-α. Fluorescent detection confirmed an intense production of NO and the predominant differentiation of macrophages into M1 lineage with the overexpression of SsMyD88 in vitro. These results further corroborate the role of SsMyD88 as a mediatory molecule that bridges distinct immune stimulants to induce drastic immune responses in fish.

Glutaredoxin 2 from big belly seahorse (Hippocampus abdominalis) and its potential involvement in cellular redox homeostasis and host immune responses.

Glutaredoxins are oxidoreductases present in almost all living organisms. They belong to the thioredoxin superfamily and share the thioredoxin structure and catalytic motif. Glutaredoxin 2 has been identified as a mitochondrial protein in vertebrates. In this study, the sequence of Glutaredoxin 2 from Hippocampus abdominalis (HaGrx2) was analyzed by molecular, transcriptional, and functional assays. In-silico analysis revealed that HaGrx2 shows the highest homology with Hippocampus comes, while distinctly cluster with fish Grx2 orthologs. Tissue distribution analysis showed that HaGrx2 is ubiquitously expressed in all tissues tested, and the highest expression was observed in the brain and skin. Significant HaGrx2 transcript modulation was identified in blood and liver upon injecting bacterial and Pathogen Associated Molecular Patterns. The redox activity of HaGrx2 was revealed by Dehydroascorbic reduction and insulin disulfide reduction activity assays. Further, the deglutathionylation activity of 1 nM HaGrx2 was found to be equivalent to that of 0.84 nM HaGrx1. HaGrx2 exhibited antiapoptotic activity against H2O2-induced oxidative stress in FHM cells. Altogether, the results of this study suggest that HaGrx2 plays a role in redox homeostasis and innate immune responses in fish.

Molecular and transcriptional insights into viperin protein from Big-belly seahorse (Hippocampus abdominalis), and its potential antiviral role.

Viperin is recognized as an antiviral protein that is stimulated by interferon, viral exposures, and other pathogenic molecules in vertebrate. In this study, a viperin homolog in the Big-belly seahorse (Hippocampus abdominalis; HaVip) was functionally characterized to determine its subcellular localization, expression pattern, and antiviral activity in vitro. The HaVip coding sequence encodes a 348 amino acid polypeptide with predicted molecular weight of 38.48 kDa. Sequence analysis revealed that HaVip comprises three main domains: the N-terminal amphipathic α-helix, a radical S-adenosyl-l-methionine (SAM) domain, and a conserved C-terminal domain. Transfected GFP-tagged HaVip protein was found to localize to the endoplasmic reticulum (ER). Overexpressed-HaVip in FHM cells was found to significantly reduce viral capsid gene expression in VHSV infection in vitro. Under normal physiological conditions, HaVip expression was ubiquitously detected in all 14 examined tissues of the seahorse, with the highest expression observed in the heart, followed by skin and blood. In vivo studies showed that HaVip was rapidly and predominantly upregulated in blood, kidney, and intestinal tissue upon poly (I:C) stimulus. LPS and Streptococus iniae challenges caused a significant increase in expression of HaVip in all the analyzed tissues. The obtained results suggest that HaVip is involved in the immune system of the seahorse, triggering antiviral and antibacterial responses, upon viral and bacterial pathogenic infections.

Immune and xenobiotic responses of glutathione S-Transferase theta (GST-θ) from marine invertebrate disk abalone (Haliotis discus discus): With molecular characterization and functional analysis.

Representing a multifunctional complex group of proteins, glutathione S- transferases (GSTs) play a major role in the phase II detoxification process in a wide range of organisms. This study focused on the potential detoxification ability of disk abalone (Haliotis discus discus) GST theta (AbGST-θ) under different stress conditions with special reference to post immune challenges. Characterization of AbGST-θ revealed with 226 amino acids, 26.6 kDa of predicted molecular mass and 8.9 of theoretical isoelectric point. As illustrated in the multiple sequence alignment, eight glutathione binding sites (G-sites) and ten substrate binding sites (H-sites) were identified in well-distinct N-terminal and C-terminal domains of AbGST-θ, respectively. AbGST-θ exhibited its highest sequence identity with Mizuhopecten yessoensis (59.1%) and the phylogenetic tree clearly positioned AbGST-θ with pre-defined GST-θ molluscan homologues. The AbGST-θ was highly expressed in the digestive tract of un-challenged abalones. Upon administering the challenge experiment, AbGST-θ showed significant modulations in their transcriptional levels depending on the time and the tissue type. The optimum temperature was 37 °C and optimum pH was 7.5 for AbGST-θ. The determined enzyme kinetic parameters of AbGST-θ showed low affinity towards 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. Nonetheless, with Cibacron blue IC50 (half maximal inhibitory concentration) was calculated to be 0.08 µM while observing 100% inhibition with 100 µM. Furthermore, AbGST-θ resulted in significant protection ability towards H2O2, CdCl2, and ZnCl2 in the disk diffusion assay. Collectively, this study provides evidences for the detoxification ability and the immunological host defensive capability of AbGST-θ in disk abalone.

A manganese superoxide dismutase (MnSOD) from red lip mullet, Liza haematocheila: Evaluation of molecular structure, immune response, and antioxidant function.

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant metalloenzyme. The main function of this enzyme is to dismutase the toxic superoxide anion (O2-) into less toxic hydrogen peroxide (H2O2) and oxygen (O2). 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.

Identification of thioredoxin domain-containing protein 17 from big-belly seahorse Hippocampus abdominalis: Molecular insights, immune responses, and functional characterization.

Thioredoxin domain-containing protein 17 (TXNDC17) is a small protein (∼14 kDa) involved in maintaining cellular redox homeostasis via a thiol-disulfide reductase activity. In this study, TXNDC17 was identified and characterized from Hippocampus abdominalis. The open reading frame (ORF) consisted of 369 bp and 123 amino acids. Similar to the other thioredoxins, TXNDC17 contained a conserved WCXXC functional motif. The highest spatial mRNA expressions of HaTXNDC17 were observed in the muscle, brain, and intestine. Interestingly, the mRNA expression of HaTXNDC17 in blood showed significant upregulation at 48 h against all the pathogen associated molecular patterns (PAMPs) and bacteria. Further, HaTXNDC17 transcripts in the trunk kidney were significantly upregulated at 24-48 h by bacterial endotoxin lipopolysaccharides (LPS), viral mimic polyinosinic: polycytidylic acid (poly I:C), and gram-negative bacteria (Edwardsiella tarda). The DPPH assay showed that the radical scavenging activity varies in a concentration-dependent manner. The insulin reduction assay demonstrated a significant logarithmic relationship with the concentration of rHaTXNDC17. Moreover, FHM cells treated with recombinant HaTXNDC17 significantly enhanced cellular viability under oxidative stress. Together, these results show that HaTXNDC17 function is important for maintaining cellular redox homeostasis and that it is also involved in the immune mechanism in seahorses.

Characterization of a Kazal-type serine protease inhibitor from black rockfish Sebastes schlegelii and its possible role in hepatic immune response.

Kazal-type serine protease inhibitors (KSPIs) play important roles in the regulation of endogenous proteases, cell development, blood coagulation, and immune response. In this study, we identified and characterized a KSPI homologue (SsKSPI) in black rockfish, Sebastes schlegelii. The full-length cDNA sequence of SsKSPI was 532 base pairs (bp), including an open reading frame (ORF) of 330 bp, which encodes a polypeptide of 110 amino acids with a signal peptide of 21 amino acids. The greatest value for identity (42.9%) and similarity (50.9%) was observed with Channa striata KSPI. We purified the recombinant protein of SsKSPI and performed protease inhibitory assays using three common serine proteases. The recombinant SsKSPI exhibited specific inhibitory activity against subtilisin A in a dose-dependent manner. Tissue distribution of SsKSPI mRNA has been examined amongst 10 important tissues in healthy rockfish and the liver was found to be the predominant expression organ of SsKSPI. The modulation of SsKSPI expression under immune challenges was also investigated in the liver. The SsKSPI mRNA expression was significantly up-regulated in response to both bacterial (Streptococcus iniae and lipopolysaccharide) and viral (polyinosinic:polycytidylic acid) challenges. Overall, we propose that SsKSPI is potentially involved in the hepatic immune response against bacterial and viral infections in black rockfish.