Molecular characterization, cytoprotective, DNA protective, and immunological assessment of peroxiredoxin-1 (Prdx1) from yellowtail clownfish (Amphiprion clarkii).

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.

Molecular features, antioxidant potential, and immunological expression assessment of thioredoxin-like protein 1 (TXNL1) in yellowtail clownfish (Amphiprion clarkii).

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H2O2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii.

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 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.

PDI family thioredoxin from disk abalone (Haliotis discus discus): Responses to stimulants (PAMPs, bacteria, and viral) and functional characterization.

Thioredoxin, a highly conserved class of proteins involved in redox signaling, is found in a range of organisms from bacteria to higher-level eukaryotes. Thioredoxin acts as an active regulatory enzyme to eliminate excessive reactive oxygen species, thereby preventing cellular damage. In this study, the cDNA sequence of thioredoxin domain-containing 5 (AbTXNDC5) from the disk abalone transcriptomic database was characterized. An in silico analysis of AbTXNDC5 was performed, and its spatial and temporal expression patterns in hemocytes and gills in response to bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), viral hemorrhagic septicemia virus, and pathogen-associated molecular pattern molecules were observed. Furthermore, AbTXNDC5 expression was examined in different developmental stages. Functional assays to explore insulin disulfide reduction, anti-apoptotic activity, and protection against hypoxic cell death of AbTXNDC5 were conducted through recombinant proteins or overexpression in cells. AbTXNDC5 contains a 1179-bp open reading frame coding for 392 amino acids. Conserved thiol-disulfide cysteine residues within two Cys-X-X-Cys motifs were found in AbTXNDC5. Quantitative real-time polymerase chain reaction indicated that healthy digestive tract and hemocyte tissues expressed high levels of AbTXNDC5 mRNA, which may protect the host from invading pathogens. Immune-challenged abalone hemocytes and gills exhibited upregulated expression of AbTXNDC5 at different time points. rAbTXNDC5 also exhibited a functional insulin disulfide reductase activity. AbTXNDC5 conferred protection to cultured cells from apoptosis and hypoxia-induced stress, compared to the pcDNA3.1(+) transfected control cells. Therefore, AbTXNDC5 can be considered an important gene in abalones in relation to the primary immune system and regulation of redox homeostasis and confers protection from stress.

Molecular characterization, immune responses, and functional activities of manganese superoxide dismutase in disk abalone (Haliotis discus discus).

Superoxide dismutases (SODs) are metalloenzymes that convert superoxide radicals to H2O2 and O2. Although SODs have been extensively studied in mammals and other species, comparative studies in invertebrates, such as abalones, are lacking. Here, we aimed to characterize manganese superoxide dismutase in disk abalone (Haliotis discus discus) (AbMnSOD) by assessing its transcriptional levels at different embryonic developmental stages. Additionally, the temporal expression of AbMnSOD in different abalone tissues in response to bacterial, viral, and pathogen-associated molecular pattern (PAMP) stimuli was investigated. SOD activity was measured at various recombinant protein concentrations via the xanthine oxidase/WST-1 system. Cell viability upon exposure to H2O2, wound healing ability, and subcellular localization were determined in AbMnSOD-transfected cells. AbMnSOD was 681 bp long and contained the SOD-A domain. AbMnSOD expression was higher at the trochophore stage than at the other stages. When challenged with immune stimulants, AbMnSOD showed the highest expression at 6 h post-injection (p.i.) for all stimulants except lipopolysaccharides. In the gills, the highest AbMnSOD expression was observed at 6 h p.i., except for the Vibrio parahaemolyticus challenge. Recombinant AbMnSOD showed concentration-dependent xanthine oxidase activity. Furthermore, AbMnSOD-transfected cells survived H2O2-induced apoptosis and exhibited significant wound gap closure. As expected, AbMnSOD was localized in the mitochondria of the cells. Our findings suggest that AbMnSOD is an essential antioxidant enzyme that participates in regulating developmental processes and defense mechanisms against oxidative stress in hosts.

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.

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

Peroxiredoxins (Prxs) are ubiquitously expressed antioxidant proteins that can protect aerobic organisms from oxidative stress. Here, we characterized the HaPrx3 homolog at the molecular level from big-belly seahorse (Hippocampus abdominalis) and analyzed its functional activities. The coding sequence of HaPrx3 consists of 726 bp, which encodes 241 amino acids. The predicted molecular weight and theoretical isoelectric point (pI) of HaPrx3 was 26.20 kDa and 7.04, respectively. Multiple sequence alignments revealed that the arrangements of domains, catalytic triads, dimers, and decamer interfaces of HaPrx3 were conserved among Prx sequences of other organisms. According to the phylogenetic analysis, HaPrx3 is clustered with the teleost Prx3 subclade. The highest transcript level of HaPrx3 was detected in the ovary tissue among fourteen healthy fish tissues. The mRNA levels of HaPrx3 in blood and liver tissues were significantly (P < 0.05) upregulated in response to lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), Edwardsiella tarda, and Streptococcus iniae, suggesting its involvement in immune responses. Under functional properties, insulin disulfide reduction assay confirmed the oxidoreductase activity of recombinant HaPrx3. A cell viability assay and Hoechst staining indicated cell survival ability and reduction of apoptotic activity, respectively. Moreover, a peroxidase activity assay verified peroxidase activity, while a metal-catalyzed oxidation (MCO) assay indicated the DNA protection ability of HaPrx3. Collectively, it is concluded that HaPrx3 may play a significant role in oxidative stress and immune responses against pathogenic infections in big-belly seahorses.

Glutathione-S-transferase alpha-4 in Hippocampus abdominalis (big-belly seahorse): Molecular characterization, antioxidant properties, and its potent immune response.

Glutathione-S-transferase (GST) is a key enzyme in the phase-II detoxification process and is a biomarker of oxidative stress. In this study, we analyzed the molecular, biochemical, and antioxidant properties of GST alpha-4 from Hippocampus abdominalis (HaGSTA-4). Also, the spatial and temporal expression of HaGSTA-4 upon immune challenge with abiotic and biotic stimulants were evaluated. The HaGSTA-4 ORF encodes 223 amino acids with a molecular weight of 25.7 kDa, and an estimated isoelectric point (pI) of 8.47. It consists of the GST_C superfamily and thioredoxin-like superfamily domain. The phylogenetic tree revealed that HaGSTA-4 is evolutionarily conserved with its GST alpha class counterparts. From pairwise alignment, the highest values of identity (78.5%) and similarity (85.7%) were with Parambassis ranga GSTA-4. Protein rHaGSTA-4 exhibited the highest conjugation activity towards 1-chloro-2,4-dinitrobenzene (CDNB) at pH 7 and 20 °C. A disk diffusion assay showed that rHaGSTA-4 significantly protects cells from the stress of exposure to ROS inducers such as CuSO4, CdCl2, and ZnCl2. Furthermore, overexpressed HaGSTA-4 defended cells against oxidative stress caused by H2O2; evidence of selenium-independent peroxidase activity. From qPCR, the tissue-specific expression profile demonstrates that HaGSTA-4 is most highly expressed in the kidney, followed by the intestine and stomach, among fourteen different tissues extracted from healthy seahorses. The mRNA expression profile of HaGSTA-4 upon immune challenge varied depending on the tissue and the time after challenge. Altogether, this study suggests that HaGSTA-4 may be involved in protection against oxidative stress, in immune defense regulation, and xenobiotic metabolism.

Identification, molecular characterization, expression analysis and wound-healing ability of multifunctional calreticulin from big-belly seahorse Hippocampus abdominalis.

Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)-specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5'-KHEQSIDCGGGYVKVF-3' and 5'-LMFGPDICG-3'), triplicate repeats (5'-IKDPEAKKPEDWD-3', 5'-IPDPDDTKPEDWD-3', 5'-IPDPDAKKPDDWD-3'), signal peptide and an ER-targeting 5'-KDEL-3' sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patterns-binding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and interleukin-1ß (IL-1ß). Furthermore, rHaCRT exhibited wound-healing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.

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.

Molecular insights and immune responses of big belly seahorse syndecan-2 (CD362): Involvement of ectodomain in regulating cell survival, proliferation, and wound healing.

Syndecan-2, also known as CD362, is a transmembrane heparan sulfate proteoglycan which regulates cell growth, proliferation, cell adhesion, wound healing, and recruits immune cells. In the present study, we performed bioinformatics, spatial and temporal expression analyses of Hippocampus abdominalis syndecan-2 (HaSDC-2). Additionally, functional assays were conducted. HaSDC-2 has five major domains; an extracellular heparan sulfate attachment domain, a co-receptor binding domain, a transmembrane domain, two conserved domains (C1 domain, C2 domain), and a variable (V) domain. The ectodomain contained a signal peptide and GAG attachment sites. In-silico analysis revealed that HaSDC-2 contained a 798 bp long ORF and protein sequence of 265 amino acid residues. Further analysis of the amino acid sequence predicted a 28.9 kDa molecular weight and a 4.13 theoretical isoelectric point. The spatial expression of HaSDC-2 was ubiquitous in all tested tissues. HaSDC-2 expression in the liver was upregulated 24 h post-injection in response to all stimuli. Further, HaSDC-2 expression in blood cells was upregulated at 12 and 72 h post-injection in response to all the stimuli. HaSDC-2 + pcDNA™3.1(+) transfected cells exhibited significant survival in response to cell stressors such as H2O2 and HED. The ectodomain of recombinant HaSDC-2 treated cells showed significant cell proliferation in a concentration-dependent manner. The scratch wound healing assay showed significant Δ gap closures with increasing concentrations of HaSDC-2. Collectively, these results indicated that syndecan-2 was involved in regulating immune responses and cell stress conditions.

Molecular characterization and functional analysis of glutathione S-transferase kappa 1 (GSTκ1) from the big belly seahorse (Hippocampus abdominalis): Elucidation of its involvement in innate immune responses.

Glutathione S-transferases (GSTs) are essential enzymes for the bioactivation of xenobiotics through the conjugation of the thiol group of glutathione (GSH). In this study, a kappa class of GST was identified from the big belly seahorse (Hippocampus abdominalis) (HaGSTκ1) and its biochemical and functional properties were analyzed. HaGSTκ1 has 231 amino acids encoded by a 696 bp open reading frame (ORF). The protein has a predicted molecular mass of 26.04 kDa and theoretical isoelectric point (pI) of 8.28. It comprised a thioredoxin domain, disulfide bond formation protein A (DsbA) general fold, and Ser15 catalytic site as well as GSH-binding and polypeptide-binding sites. Phylogenetic analysis revealed that HaGSTκ1 is closely clustered with the kappa class of GSTs from teleost fishes. The recombinant (rHaGSTκ1) protein exhibited activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrobenzyl (4-NBC), and 4-nitrophenethyl bromide (4-NPB) but not 1,2-dichloro-4-nitrobenzene (DCNB). The optimum pH and temperature were 8 and 30 °C, respectively, for the catalysis of CDNB and the universal substrate of GSTs. The rHaGSTκ1 activity was efficiently inhibited in the presence of Cibacron blue (CB) as compared with hematin. Most prominent expression of HaGSTκ1 was observed in the liver and kidney among the fourteen different tissues of normal seahorse. After challenge with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), gram-negative Edwardsiella tarda, and gram-positive Streptococcus iniae, HaGSTκ1 expression was significantly modulated in the liver and blood tissues. Altogether, our study proposes the plausible important role of HaGSTκ1 in innate immunity and detoxification of harmful xenobiotics.

Glutaredoxin 1 from big-belly seahorse (Hippocampus abdominalis): Molecular, transcriptional, and functional evidence in teleost immune responses.

Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. In this study, we characterized glutaredoxin 1 (HaGrx1) from big-belly seahorse, Hippocampus abdominalis. In-silico analysis showed that HaGrx1 contained the classical glutaredoxin 1 structure with a CSYC thioredoxin active site motif. According to multiple sequence alignment and phylogenetic reconstruction, HaGrx1 presented the highest homology to the Grx1 ortholog from Hippocampus comes. Transcriptional studies demonstrated the ubiquitous distribution of HaGrx1 transcripts in all the seahorse tissues tested. Significant modulation (p < 0.05) of HaGrx1 transcripts were observed in blood upon stimulation with pathogen-associated molecular patterns and live pathogens. The ß-hydroxyethyl disulfide reduction assay confirmed the antioxidant activity of recombinant HaGrx1. Further, dehydroascorbate reduction and insulin disulfide reduction assays revealed the oxidoreductase activity of HaGrx1. HaGrx1 utilized 1,4-dithiothreitol, l-cysteine, 2-mercaptoethanol, and reduced l-glutathione as reducing agent with different dehydroascorbate reduction activity levels. Altogether, our results suggested a vital role of HaGrx1 in redox homeostasis as well as the host innate immune defense system.

Molecular characterization of kappa class glutathione S-transferase from the disk abalone (Haliotis discus discus) and changes in expression following immune and stress challenges.

Glutathione S-transferase (GST; EC 2.5.1.18) isoenzymes represent a complex group of proteins that are involved in phase II detoxification in several organisms. In this study, GST kappa (GSTκ) from the disk abalone (Haliotis discus discus; AbGSTκ) was characterized at both the transcriptional and functional levels to determine its potential capacity to perform as a detoxification agent under conditions of different stress. The predicted AbGSTκ protein consists of 227 amino acids, with a predicted molecular weight of 25.6 kDa and a theoretical isoelectric point (pI) of 7.78. In silico analysis reveals that AbGSTκ is a disulfide bond formation protein A (DsbA), consisting of a thioredoxin domain, GSH binding sites (G-sites), and a catalytic residue. In contrast, no hydrophobic ligand binding site (H-site), or signal peptides, were detected. AbGSTκ showed the highest sequence identity with the orthologue from pufferfish (Takifugu obscurus) (60.0%). In a phylogenetic tree, AbGSTκ clustered closely together with other fish GSTκs, and was evolutionarily distanced from other cytosolic GSTs. The predicted three-dimensional structure clearly demonstrates that the dimer adopts a butterfly-like shape. A tissue distribution analysis revealed that GSTκ was highly expressed in the digestive tract, suggesting it has detoxification ability. Depending on the tissue and time, AbGSTκ showed different expression patterns, and levels of expression, following challenge of the abalone with immune stimulants. Enzyme kinetics of the purified recombinant proteins demonstrated its conjugating ability using 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates, and suggested it has a low affinity for both substrates. The optimum temperature and pH for the rAbGSTκ GSH: CDNB conjugating activity were found to be 35 °C and pH 8, respectively indicating that the abalone is well adapted to a wide range of environmental conditions. Cibacron blue (100 µM) was capable of completely inhibiting rAbGSTκ (100%) with an IC50 (half maximal inhibitory concentration) of 0.05 µM. A disk diffusion assay revealed that rAbGSTκ could significantly protect cells from H2O2, CdCl2, and ZnCl2. Altogether, this current study suggests that AbGSTκ is involved in detoxification and immunological host defense mechanisms and allows abalones to overcome stresses in order for them to have an increased chance of survival.

Identification and characterization of molluscan caveolin-1 ortholog from Haliotis discus discus: Possible involvement in embryogenesis and host defense mechanism against pathogenic stress.

Caveolins are principal membrane proteins of caveolae that play a central role in signal transduction, substrate transport, and membrane trafficking in various cell types. Numerous studies have reported the crucial role of caveolin-1 (CAV1) in response to invading microbes; yet, very little is known about molluscan CAV1. In this study, we identified and characterized CAV1 ortholog from the disk abalone, Haliotis discus discus (HdCAV1). The cDNA sequence of HdCAV1 is 826 bp long and encodes a 127-amino acid polypeptide. Characteristic caveolin superfamily domain (Glu3 - Lys126) and two possible transmembrane domains (Cys48 - Tyr67 and Ile103 - Phe120) were identified in the HdCAV1 protein. Homology analysis revealed that HdCAV1 shared higher identity (>47%) with molluscans, but lower identity with other species. Phylogenetic tree constructed by the neighbor-joining (NJ) method revealed a distinct evolutionary pathway for molluscans. Transcriptional analysis by SYBR Green qPCR showed the highest expression of HdCAV1 mRNA in late veliger stage, as compared to that in other embryonic developmental stages of disk abalone. In adult animals, gill tissue showed highest HdCAV1 transcript levels under normal physiological condition. Stimulations with two bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), viral hemorrhagic septicemia virus, and two pathogen-associated molecular patterns (LPS and poly I:C) significantly modulated the expression of HdCAV1 transcripts. Collectively, these data suggest that CAV1 plays an important role in embryogenesis and host immune defense in disk abalone.

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system.

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS)]. Collectively, these findings suggest that AbCD63 and AbTSPAN33 are involved in innate immune responses in disk abalone during pathogenic stress.

A cysteine protease (cathepsin Z) from disk abalone, Haliotis discus discus: Genomic characterization and transcriptional profiling during bacterial infections.

Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense via phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. AbCTSZ encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, N-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from Crassostrea gigas. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of AbCTSZ genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of AbCTSZ in early developmental stages and in eight different tissues. Higher levels of AbCTSZ transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (Vibrio parahaemolyticus and Listeria monocytogenes) and bacterial lipopolysaccharides significantly modulated AbCTSZ transcription. Collectively, these lines of evidences suggest that AbCTSZ plays an indispensable role in the innate immunity of disk abalone.

Molecular characterization of a bactericidal permeability-increasing protein/lipopolysaccharide-binding protein from black rockfish (Sebastes schlegelii): Deciphering its putative antibacterial role.

Bactericidal permeability-increasing protein (BPI)/lipopolysaccharide (LPS) binding proteins (LBPs) are well-known proteins that play an indispensable role in host antimicrobial defense. Herein, we report a homolog of BPI/LBP from black rockfish (Sebastes schlegelii) (designated as RfBPI/LBP) and characterize its structural and functional features at the molecular level. We identified the putative complete open reading frame (1422 bp) of RfLBP that encodes a 474 amino acid protein with a predicted molecular mass of ∼51.5 kDa. The primary protein sequence of RfBPI/LBP contains domain features of BPI/LBP family proteins and shares significant sequence consistency with its homologs. Our phylogenetic analysis clearly demonstrated the vertebrate ancestral origin of RfBPI/LBP, further reinforcing its evolutionary relationship with teleostean homologs. Recombinant RfBPI/LBP demonstrated in vitro LPS-binding activity and antibacterial activity against Escherichia coli, but not against Streptococcus iniae. Moreover, RfBPI/LBP exhibited temporal transcriptional activation against pathogens and pathogen-associated molecular patterns. Collectively, our findings suggest that RfBPI/LBP plays an essential role in host antimicrobial defense, plausibly through selective eradication of invading bacteria.