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.

Galectin-8-like isoform X1 mediates antibacterial, antiviral, and antioxidant responses in red-lip mullet (Planiliza haematocheilus) through positive modulation of pro-inflammatory cytokine, chemokine, and enzymatic antioxidant activity.

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to ß-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 µg/mL or 100 µg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H2O2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet.

Galectin 9 restricts viral replication in teleost via autophagy-antiviral pathway and polarizes M2 macrophages for anti-inflammatory response: New insights into functional properties of fish Galectin-9 from Planiliza haematocheilus.

Galectin 9 (Gal9) is a tandem repeat type ß-galactoside-binding galectin that mediates various cellular biochemical and immunological functions. Many studies have investigated the functional properties of Gal9 in mammals; however, knowledge of fish Gal9 is limited to antibacterial studies. In this context, our aim was to clone Gal9 from Planiliza haematocheilus (PhGal9) and investigate its structural and functional characteristics. We discovered the PhGal9 open reading frame, which was 969 base pairs long and encoded a 322 amino acid protein. PhGal9 had a projected molecular weight of 35.385 kDa but no signal peptide sequence. PhGal9 mRNA was ubiquitously produced in all investigated tissues but was predominant in the intestine, spleen, and brain. Its mRNA expression was increased in response to stimulation by Poly(I:C), LPS, and L. garvieae. The rPhGal9 exhibited a dose-dependent agglutination potential toward gram-positive and gram-negative bacteria at a minimum concentration of 50 µg/mL. Overexpression of PhGal9 promoted M2-like phenotype changes in mouse macrophages, and RT-qPCR analysis of M1 and M2 marker genes confirmed M2 polarization with upregulation of M2 marker genes. In the antiviral assay, the expression levels of Viral Hemorrhagic Septicemia Virus (VHSV) glycoproteins, phosphoproteins, nucleoproteins, non-virion proteins, matrix proteins, and RNA polymerase were significantly reduced in PhGal9-overexpressed cells. Furthermore, the mRNA expression of autophagic genes (sqstm1, tax1bp1b, rnf13, lc3, and atg5) and antiviral genes (viperin) were upregulated in PhGal9 overexpressed cells. For the first time in teleosts, our study demonstrated that PhGal9 promotes M2 macrophage polarization by upregulating M2-associated genes (egr2 and cmyc) and suppressing M1-associated genes (iNOS and IL-6). Furthermore, our results show that exogenous and endogenous PhGal9 prevented VHSV attachment and replication by neutralizing virion and autophagy, respectively. Gal9 may be a potent modulator of the antimicrobial immune response in teleost fish.

TRAF family member-associated NF-κB activator (TANK) regulates the antiviral function and NF-κB activation in red-spotted grouper (Epinephelus akaara).

The TRAF family member-associated nuclear factor kappa B (NF-κB) activator (TANK) regulates the NF-κB activation through the TRAF-mediated signaling pathway and is involved in the antiviral pathway by inducing the interferon (IFN) production. In the present study, we identified a TANK ortholog from the red-spotted grouper (Epinephelus akaara) and analyzed its immunological functions. The coding sequence of EaTANK consists of 1047 base pairs and encodes a 348 amino acids protein. The predicted molecular weight and theoretical isoelectric point (pI) were 38.92 kDa and 5.39, respectively. According to the phylogenetic analysis, EaTANK was closely clustered with fish TANK orthologs, exhibiting the highest identity (97.1 %) and similarity (97.1 %) to that of Epinephelus lanceolatus. A highly conserved TBK1/IKKi binding domain (TBD) was identified between 110 and 164 residues. Our tissue distribution analysis showed that EaTANK mRNA was ubiquitously expressed in 12 tested tissues, with the highest expression in the spleen and peripheral blood cells (PBCs). According to the immune challenge experiments, EaTANK mRNA expression in PBCs was significantly elevated following stimulation with polyinosinic:polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS), or nervous necrosis virus (NNV). We also observed a significant elevation in the mRNA expression of downstream antiviral pathway-related genes (ISG15, IRF3, and IRF7) in EaTANK-overexpressing fathead minnow (FHM) cells against poly (I:C) stimulation. Moreover, the replication of 6 genes in the VHSV genome was inhibited by the overexpression of EaTANK. Finally, we confirmed that the expression of NFKB1 mRNA and promoter binding activity of NF-κB was significantly increased in poly (I:C)-stimulated EaTANK-overexpressing FHM cells. In conclusion, the results of this study suggest that TANK significantly contributes to the antiviral response and regulation of NF-κB activity in red-spotted grouper.

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.

Expression profile and molecular function of beclin-1 in Epinephelus akaara in response to immune stimuli and oxidative stress.

Beclin-1, the mammalian ortholog of the yeast autophagy-related gene 6 (Atg 6), is a key regulator of autophagy. A variety of health and disease conditions in mammals are intricately related to the broad spectrum of beclin-1 functions. Nevertheless, few studies have investigated the role of beclin-1 in fish. In this study, we identified and cloned the beclin-1 cDNA (EaBECN-1) of Epinephelus akaara (red-spotted grouper) and carried out in silico analysis, tissue-specific expression analysis, immune challenge experiment, and in vitro analysis of its roles against viral infection and oxidative stress. The open reading frame was 1344 bp long and encoded 447 amino acids with a molecular weight of 51.2 kDa. Beclin-1 consisted of a conserved N-terminal BH3 and APG6 domains, and shared more than 88% identity with other vertebrates, according to a pairwise sequence alignment. EaBECN-1 expression profile analysis in E. akaara revealed that it is mostly expressed in the blood. Moreover, transcriptional modulation of EaBECN-1 was observed following stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly (I:C)), and nervous necrosis virus. During the viral hemorrhagic septicemia virus challenge, increased viral gene expression was observed at 12 h post-infection in FHM cells ectopically expressing EaBECN-1, and decreased thereafter at 24 h post-infection compared to control cells. However, increased antiviral gene expression at 12 and 24 h confirmed the antiviral function of EaBECN-1. Furthermore, EaBECN-1 overexpression protected the cells against H2O2-mediated apoptosis, as evidenced by the MTT assay, analysis of mRNA expression levels of apoptotic genes, and AO-EtBr staining. Overall, our study demonstrated the protective role of EaBECN-1 against viral pathogenesis and oxidative stress through autophagy, increasing our understanding of the role of beclin-1 in fish.

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.

Identification of reactive oxygen species modulator 1 (Romo 1) from black rockfish (Sebastes schlegelii) and deciphering its molecular characteristics, immune responses, oxidative stress modulation, and wound healing properties.

Reactive oxygen species modulator 1 (Romo1) is a mitochondrial inner membrane protein that induces mitochondrial reactive oxygen species (ROS) generation. In this study, we identified the Romo1 homolog from the black rockfish (Sebastes schlegelii), named it as SsRomo1, and characterized it at the molecular as well as functional levels. An open reading frame consisting of 240 bp was identified in the SsRomo1 complementary DNA (cDNA) sequence that encodes a 79 amino acid-long polypeptide with a molecular weight of 8,293 Da and a theoretical isoelectric point (pI) of 9.89. The in silico analysis revealed the characteristic features of SsRomo1, namely the presence of a transmembrane domain and the lack of a signal peptide. Homology analysis revealed that SsRomo1 exhibits the highest sequence identity with its fish counterparts (>93%) and shares a similar percentage of sequence identity with mammals (>92%). Additionally, it is closely clustered together with the fish clade in the constructed phylogenetic tree. The subcellular localization analysis confirmed its mitochondrial localization within the fathead minnow (FHM) cells. Under normal physiological conditions, the SsRomo1 mRNA is highly expressed in the rockfish ovary, followed by the blood and testis, indicating the abundance of mitochondria in these tissues. Furthermore, the significant upregulation of SsRomo1 in cells treated with lipopolysachharide (LPS), polyinosinic:polycytidylic acid, and Streptococcus iniae suggest that the increased ROS production is induced by SsRomo1 to eliminate pathogens during infections. Incidentally, we believe that this study is the first to determine the involvement of SsRomo1 in LPS-mediated nitric oxide (NO) production in RAW267.4 cells, based on their higher NO production as compared to that in the control. Moreover, overexpression of SsRomo1 enhanced the wound healing ability of FHM cells, indicating its high invasion and migration properties. We also determined the hydrogen peroxide-mediated cell viability of SsRomo1-overexpressed FHM cells and observed a significant reduction in viability, which is possibly due to increased ROS production. Collectively, our observations suggest that SsRomo1 plays an important role in oxidative stress modulation upon immune stimulation and in maintenance of tissue homeostasis in black rockfish.

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.

Molecular delineation, expression profiling, immune response, and anti-apoptotic function of a novel clusterin homolog from big-belly seahorse (Hippocampus abdominalis).

Clusterin (CLU) is a glycoprotein that contains α- and ß-chains. CLU exerts multifunctional activities and plays a role in different cell signaling pathways that are associated with various diseases such as proteotoxic and oxidative stress, as well as cell death and survival. However, its role in marine teleost fish remains unclear. Therefore, the present study was carried out to characterize and investigate the immune responses and anti-apoptotic effects of CLU of the big-belly seahorse (Hippocampus abdominalis) (HaCLU) on oxidative stress-induced cell death. The HaCLU open reading frame was 1389 bp long and encoded a protein with 462 amino acids, a molecular weight of 51.28 kDa and an isoelectric point of 5.41. In-silico results demonstrated that HaCLU has a signal peptide in the 1-29 amino acid region, while the α- and ß-chains were in the 34-227 and 228-455 amino acid regions, respectively. Multiple sequence alignment clarified the low homology of the α-chain with other orthologs. The highest HaCLU mRNA expression level was observed in the liver, followed by the heart, spleen, and brain tissues of healthy big-belly seahorses. Further, HaCLU mRNA expression level was elevated in the liver in response to different stimuli, including lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae. HaCLU potentiates cell viability and weakens chromatin condensation in the nucleus of FHM cells following H2O2-induced oxidative stress and subsequent cell death. HaCLU overexpression resulted in a reduced Bax/Bcl-2 mRNA expression ratio. This study revealed the role of HaCLU in immune regulation against pathogenic infections and its anti-apoptotic effects on oxidative stress-induced cell death.

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 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 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.

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.

A molluscan calreticulin ortholog from Haliotis discus discus: Molecular characterization and transcriptional evidence for its role in host immunity.

Calreticulin (CALR), a Ca(2+) binding chaperone of the endoplasmic reticulum (ER) and mainly involved in Ca(2+) storage and signaling. In this study, we report the molecular characterization and immune responses of CALR homolog from disk abalone (AbCALR). The full length AbCALR cDNA (1837 bp) had an ORF of 1224 bp. According to the multiple alignments analysis, N- and P-domains were highly conserved in all the selected members of CALRs. In contrast, the C-domain which terminated with the characteristic ER retrieval signal (HDEL) was relatively less conserved. The phylogenetic analysis showed that all the selected molluscan homologs clustered together. Genomic sequence of AbCALR revealed that cDNA sequence was dispersed into ten exons interconnected with nine introns. AbCALR mRNA expression shows the significant (P < 0.05) up-regulation of AbCALR transcripts in hemocytes upon bacterial (Listeria monocytogenes and Vibrio parahaemolyticus), viral (Viral haemorrhagic septicaemia virus; VHSV) and immune stimulants (LPS and poly I:C) challenges at middle and/or late phases. These results collectively implied that AbCALR is able to be stimulated by pathogenic signals and might play a potential role in host immunity.

Identification and molecular characterization of peroxiredoxin 6 from Japanese eel (Anguilla japonica) revealing its potent antioxidant properties and putative immune relevancy.

Peroxiredoxins (Prdx) are thiol specific antioxidant enzymes that play a pivotal role in cellular oxidative stress by reducing toxic peroxide compounds into nontoxic products. In this study, we identified and characterized a peroxiredoxin 6 counterpart from Japanese eel (Anguilla japonica) (AjPrdx6) at molecular, transcriptional and protein level. The identified full-length coding sequence of AjPrdx6 (669 bp) coded for a polypeptide of 223 aa residues (24.9 kDa). Deduced protein of AjPrdx6 showed analogy to characteristic structural features of 1-cysteine peroxiredoxin sub-family. According to the topology of the generated phylogenetic reconstruction AjPrdx6 showed closest evolutionary relationship with Salmo salar. As detected by Quantitative real time PCR (qPCR), AjPrdx6 mRNA was constitutively expressed in all the tissues examined. Upon the immune challenges with Edwardsiella tarda, lipopolysaccharides and polyinosinic:polycytidylic acid, expression of AjPrdx6 mRNA transcripts were significantly induced. The general functional properties of Prdx6 were confirmed using purified recombinant AjPrdx6 protein by deciphering its potent protective effects on cultured vero cells (kidney epithelial cell from an African green monkey) against H2O2-induced oxidative stress and protection against oxidative DNA damage elicited by mixed function oxidative (MFO) system. Altogether, our findings suggest that AjPrdx6 is a potent antioxidant protein in Japanese eels and its putative immune relevancy in pathogen stress mounted by live-bacteria or pathogen associated molecular patterns (PAMPs).