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.

A newly discovered teleost disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), from big-belly seahorse (Hippocampus abdominalis): Insights into its molecular and functional properties and immune regulatory functions.

The thioredoxin domain containing 5 (TXNDC5) is a recently discovered member of the protein disulfide isomerase family (PDI), which is mainly involved in the proper folding of and the correct formation of disulfide bonds in newly synthesized proteins via its disulfide isomerase and chaperone activities. Although the structural and functional features of mammalian TXNDC5 have been explored in previous studies, no studies have reported the functional characteristics of TXNDC5 in teleost fish. In this study, we report the identification and characterization of TXNDC5 from big-belly seahorse (Hippocampus abdominalis) (ShTXNDC5) accompanied by functional studies. The in-silico analysis revealed that the gene encodes a 433 amino acid (aa) long polypeptide chain with a predicted molecular weight of 49.3 kDa. According to homology analysis, ShTXNDC5 shares more than 55% sequence similarity with other teleost TXNDC5 proteins, and the alignment of the gene sequence convincingly reflects the accepted phylogeny of teleost. Analysis of the spatial distribution of ShTXNDC5 expression showed that its highest expression was observed in the ovary, gill, and pouch of seahorses. Moreover, significant upregulation of ShTXNDC5 transcription was noted in seahorse blood and kidney tissues in a time-dependent manner upon viral and bacterial immune challenges. Furthermore, considerable NADPH turnover, insulin reduction ability and significant cell survival effects of ShTXNDC5 were determined by the functional assay, revealing its capability to overcome cellular oxidative stress. Altogether, these findings expand our understanding of TXNDC5 at the molecular and functional levels, and its putative role in seahorse immunity.

ß-hydroxybutyrate (ß-HB) exerts anti-inflammatory and antioxidant effects in lipopolysaccharide (LPS)-stimulated macrophages in Liza haematocheila.

Poly-ß-hydroxybutyrate (PHB) can be hydrolyzed to ß-hydroxybutyrate (ß-HB) in the intestinal tract of animals, and dietary PHB supplementation could enhance the immunity and disease resistance of aquatic animals. Antioxidant system is responsive to PHB stimuli via MAPK/PI3K-Akt/TNF/NF-κB/TCR/TLR signaling pathways. However, the precise immunopotentiation mechanism needs further study. In this study, macrophages from spleen in Liza haematocheila was used to study the effect of ß-HB on cell viability and antioxidant function to illustrate the immunopotentiation mechanism of PHB. The results showed that ß-HB (100 µg/mL) promoted the viability of macrophages and balanced the production of reactive oxygen species, but inhibited the excessive production of intracellular nitric oxide. In order to further explore the immunopotentiation mechanism of ß-HB, LPS (100 µg/mL) was used to induce the inflammation and investigated the inhibitory effect of ß-HB on inflammation. The results showed that LPS could induce inflammation successfully, and ß-HB exerted anti-inflammatory and antioxidant effects in LPS-stimulated macrophages. Compared with LPS stimuli alone, the expression of anti-inflammatory genes NF-κBIA, MAP3K8 and TLR5 in ß-HB pretreatment group was up-regulated, and the expression of pro-inflammatory genes TNFSF6, TNF-α, PI3K, NF-κB and TLR1 down-regulated. It suggested that ß-HB inhibited the inflammatory response by up-regulation of anti-inflammatory genes such as NF-κBIA, thereby enhancing the immunity of the body.

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.

TLR13, TLR22, TRAF6, and TAK1 in the soiny mullet (Liza haematocheila): Molecular characterization and expression profiling analysis.

Toll-like receptors (TLRs) and their associated signaling pathways play pivotal roles in the immune response to invading pathogens. Here, TLR13, TLR22, tumor necrosis factor receptor-associated factor 6 (TRAF6), and transforming growth factor-ß-activated kinase1 (TAK1) were characterized in the soiny mullet (Liza haematocheila), representative mugilid species that is widely cultured in Asia. The four mullet genes, which shared characteristic features with their counterparts in other teleosts, were ubiquitously expressed in all of the examined tissues, albeit with different expression patterns. Following Streptococcus dysgalactiae infection, the four genes were upregulated to different degrees in various mullet tissues. These results indicated that the four genes were involved in the mullet immune response to bacterial infection. To the best of our knowledge, this is the first characterization of these four genes in mullet. Our results provide a basis for future studies of TLR signaling pathways in mullet, as well as for similar studies in other mugilids.

Immune defense parameters of wild fish as sensitive biomarkers for ecological risk assessment in shallow sea ecosystems: A case study with wild mullet (Liza haematocheila) in Liaodong Bay.

Environmental monitoring is important to the health management of an ecosystem. Biomarkers are particularly relevant because they are direct indicators of any toxic effects on organisms and are cheaper to use compared with chemical indicators, especially for extremely low-level organic contaminants. Fish can be significantly affected by pollutants, given their high trophic levels in aquatic food chains. Their immune function is closely related to their survival. The present study compared immune function-related parameters of wild mullet (Liza haematocheila) samples from low (Jinzhou) and high (Yingkou) polluted sites during the pre-winter (PW) and pre-breeding (PB) periods in Liaodong Bay, to evaluate the effect of water pollution on fish health and to explore potential biomarkers of coast water pollution. Compared with Jinzhou mullet, there was a significantly higher level of hematocrit in Yingkou mullet, but a significantly lower serum lysozyme level (P < 0.001), indicating that these fish were immunosuppressed. Significant differences occurred in the spleen between the two site populations. The abnormal: normal fish ratio in Yingkou L. haematochila was significantly higher than that of Jinzhou L. haematochila (2.5 times of that of Jinzhou during PB and nine times during PW). The splenic index of male Yingkou L. haematochila was 47.2% higher than that of Jinzhou L. haematochila in PW (P = 0.001). Moreover, histological observations showed that the spleen of the former was more congestive, with increased numbers (39.6% more) of melanomacrophage centers (MMCs) and changes in pigments (hemosiderin 8.3% higher and melanin 29.4% higher), compared with the latter. The splenic MMC area of Yingkou L. haematochila was significantly smaller than that of Jinzhou L. haematochila (P < 0.05) in PB, but showed no clear difference in PW (P > 0.05). Splenic MMC number was significantly higher in individual Yingkou L. haematochila with abnormal livers compared with normal Yingkou L. haematochila during both sampling periods. The splenic MMC area in abnormal livers was approximately four times those of normal individuals during PB in Yingkou L. haematochila. The number of splenic melanomacrophages (MM) in abnormal livers was approximately nine times those of the normal livers during PW. There were also differences in pigments in normal Yingkou individuals compared with normal Jinzhou samples during PW (melanin 29.4% higher and hemosiderin 8.3% higher). Based on these results, we suggest that serum lysozyme activity, splenic MM number and MMC (both number and area), and melanin of local fish have potential as sensitive biomarkers for the assessment of coastal water pollution.

Identification, expression profiling and functional characterization of interleukin 11a ortholog from redlip mullet Liza haematocheila: Insight into its roles in the inflammation and apoptosis regulation.

Interleukin 11 (IL-11) is a secretory cytokine with pleotropic properties, including anti-inflammatory and anti-apoptotic functions. This study aimed to functionally characterize a teleostean IL-11a ortholog from redlip mullet (LhIL-11a) through bioinformatic analysis, transcriptional expression profiling and protein function assays. The deduced LhIL-11a protein sequence is 200 amino acids long, with a predicted molecular weight of 23.168 kDa. Multiple sequence alignment indicates that LhIL-11a has a typical four-bundle architecture of α-helixes as observed in other IL-11s. The identity-similarity matrix show a higher identity between LhIL-11a and other fish IL-11a sequences. Phylogenetic analysis demonstrated that LhIL-11a falls within a clade including other fish counterparts. In the tissue distribution analysis, the highest constitutive expression of LhIL-11a mRNA was observed in the mullet gastrointestinal tract and brain tissues. Following the challenges with LPS, poly I:C and Lactococcus garvie, the transcription levels of LhIL-11a were significantly upregulated in both PBCs and liver. In the biological functional assay, recombinant LhIL-11a protein showed strong activities of suppressing pro-inflammatory cytokines and apoptotic gene expression in mullet kidney cells and reducing LPS stimulated NO production in murine macrophage cells. Overall, the findings in this study provide the experimental clues to understanding the functional roles of fish IL-11a in inflammation and apoptosis regulation during host defense against invading microbial pathogens.

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.

Antioxidant system of soiny mullet (Liza haematocheila) is responsive to dietary poly-ß-hydroxybutyrate (PHB) supplementation based on immune-related enzyme activity and de novo transcriptome analysis.

As a dietary supplement, poly-ß-hydroxybutyrate (PHB) has been reported to positively influence growth, boost the immune system and enhance disease resistance in fish and shellfish. However, the protective mechanism is little known. Thus, the present study was conducted to evaluate the effect of PHB supplementation on immune-related enzyme activity and transcriptome-based gene expression in soiny mullet (Liza haematocheila). Results showed that dietary PHB supplementation could increase antioxidant enzyme activity, including total antioxidant capacity, catalase and superoxide dismutase. A total of 7,082,094,175 and 7,650,341,357 raw reads with mean length of 757 bp were obtained from control and PHB (dietary PHB supplementation at 2%) groups, respectively. There were 46,106 differentially expressed genes (DEGs) between control and PHB groups, including 21,828 upregulated and 24,278 downregulated DEGs. All the DEGs were classified into three gene ontology categories, and 312 DEGs related with immune system process and 760 with the response to a stimulus. Additionally, all DEGs were allocated to 261 Kyoto Encyclopedia of Gene and Genome pathways, and major immune-related pathways were detected, including MAPK/PI3K-Akt/TNF/NF-κB/TCR/TLR signaling pathways. Moreover, the regulation of several observed immune-related genes was confirmed by qRT-PCR. Altogether, this study suggests that antioxidant system is more effective for dietary PHB supplementation and lays the foundation for further study on the precise immunostimulatory mechanism of PHB. Hopefully, it provides insights into exploring biomarker for assessment of immunostimulants in fish culture.

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.

Molecular cloning, characterization, and expression level analysis of a marine teleost homolog of catalase from big belly seahorse (Hippocampus abdominalis).

Organisms possess a cellular antioxidant defense system inclusive of ROS scavengers to maintain the homeostasis of antioxidant levels. Catalase is a major ROS scavenger enzyme that plays a significant role in the antioxidant defense mechanism of organisms by reducing toxic hydrogen peroxide molecules into a nontoxic form of oxygen and water with a high turnover rate. In the present study, we performed molecular and functional characterization of the catalase homolog from Hippocampus abdominalis (HaCat). The HaCat cDNA sequence was identified as a 1578 bp ORF (open reading frame) that encodes a polypeptide of 526 amino acids with 59.33 kDa molecular weight. Its estimated pI value is 7.7, and it does not have any signal sequences. HaCat shared a conserved domain arrangement including the catalase proximal active site signature and heme ligand signature domain with the previously identified catalase counterparts. Phylogenetic analysis displayed close evolutionary relationships between HaCat and catalases from other teleost fish. According to our qPCR results, ubiquitous expression of HaCat transcripts were observed in all the tested tissues with high expression in the kidney followed by liver. Significant modulations of HaCat transcription were observed in blood, liver, and kidney tissues post-challenge with Streptococcus iniae, Edwardsiella tarda, poly I:C, and LPS. Peroxidase activity of recombinant HaCat (rHaCat) was evaluated using an ABTS assay and the ROS removal effect was further confirmed by oxidative DNA damage protection and cell viability assays. The rHaCat showed more than 97% activity over a temperature and pH range of 10 °C-40 °C and 5 to 6, respectively. The above results suggest that HaCat plays an indispensable role in the oxidative homeostasis of the seahorse during pathogenic attack.

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.

Sequence characterization and expression pattern analysis of six kinds of IL-17 family genes in the Asian swamp eel (Monopterus albus).

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens.

Sex-biased regulation of respiratory burst, phagocytic activity and plasma immune factors in lined seahorse (Hippocampus erectus) after subchronic benzo[a]pyrene exposure.

Both wild and aquacultured seahorses are currently under great threat from marine pollution, notably from the potent contaminant and carcinogen benzo[a]pyrene (BaP). However, very little data are available regarding the immunomodulating effects of BaP in seahorses. Therefore, in this study, we exposed lined seahorses (Hippocampus erectus) for 7 d to BaP at three dosages (0.5, 5, and 50 µg/L) to evaluate sexual dimorphism in immune response. We measured eight immune parameters in the blood, including respiratory burst (RB), phagocytic activity (PA), monocytes/leucocytes, immunoglobulin M, complement 3, complement, interferon-a, and interleukin-2. Male seahorses had significantly higher parameters than females, except in terms of monocytes/leucocytes (P < 0.05). Although flow cytometry showed that RB and PA variation per BaP dose were roughly similar across sexes, RB and PA exhibited distinct patterns. Additionally, fluorescence intensity and leucocyte percentage were positively correlated in PA but not RB for all treatment and sex combinations. Through ELISA, we showed that the other six parameters had complex responses that nevertheless varied in a BaP-dosage and sex-dependent manner. Overall, adult male seahorses had higher immunocompetence than females before BaP exposure, and sexual dimorphism continued to be apparent during BaP exposure. Furthermore, all eight parameters were sensitive to BaP. Based on these results, we highly recommend H. erectus as a sentinel species for crude contamination, whereas PA and RB are valuable bioindicators of marine contaminants such as BaP.

Characterization of four C1q/TNF-related proteins (CTRPs) from red-lip mullet (Liza haematocheila) and their transcriptional modulation in response to bacterial and pathogen-associated molecular pattern stimuli.

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses.

Two phospholipid scramblase 1-related proteins (PLSCR1like-a & -b) from Liza haematocheila: Molecular and transcriptional features and expression analysis after immune stimulation.

Phospholipid scramblases (PLSCRs) are a family of transmembrane proteins known to be responsible for Ca2+-mediated bidirectional phospholipid translocation in the plasma membrane. Apart from the scrambling activity of PLSCRs, recent studies revealed their diverse other roles, including antiviral defense, tumorigenesis, protein-DNA interactions, apoptosis regulation, and cell activation. Nonetheless, the biological and transcriptional functions of PLSCRs in fish have not been discovered to date. Therefore, in this study, two new members related to the PLSCR1 family were identified in the red lip mullet (Liza haematocheila) as MuPLSCR1like-a and MuPLSCR1like-b, and their characteristics were studied at molecular and transcriptional levels. Sequence analysis revealed that MuPLSCR1like-a and MuPLSCR1like-b are composed of 245 and 228 amino acid residues (aa) with the predicted molecular weights of 27.82 and 25.74 kDa, respectively. A constructed phylogenetic tree showed that MuPLSCR1like-a and MuPLSCR1like-b are clustered together with other known PLSCR1 and -2 orthologues, thus pointing to the relatedness to both PLSCR1 and PLSCR2 families. Two-dimensional (2D) and 3D graphical representations illustrated the well-known 12-stranded ß-barrel structure of MuPLSCR1like-a and MuPLSCR1like-b with transmembrane orientation toward the phospholipid bilayer. In analysis of tissue-specific expression, the highest expression of MuPLSCR1like-a was observed in the intestine, whereas MuPLSCR1like-b was highly expressed in the brain, indicating isoform specificity. Of note, we found that the transcription of MuPLSCR1like-a and MuPLSCR1like-b was significantly upregulated when the fish were stimulated with poly(I:C), suggesting that such immune responses target viral infections. Overall, this study provides the first experimental insight into the characteristics and immune-system relevance of PLSCR1-related genes in red lip mullets.

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.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge.

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1-4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1-3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

Molecular characterization of thioredoxin-like protein 1 (TXNL1) from big-belly seahorse Hippocampus abdominalis in response to immune stimulation.

Thioredoxin is a highly conserved protein found in both prokaryotes and eukaryotes. Reactive oxygen species (ROS) are produced in response to metabolic processes, radiation, metal oxidation, and pathological infections. High levels of ROS lead to cell death via autophagy. However, thioredoxin acts as an active regulatory enzyme in response to excessive ROS. Here, we performed in-silico analysis, immune challenge experiments, and functional assays of seahorse thioredoxin-like protein 1 (ShTXNL1). Evolutionary identification showed that ShTXNL1 protein belongs to the thioredoxin superfamily comprising 289 amino acids. It possesses an N-terminal active thioredoxin domain and C-terminal proteasome-interacting thioredoxin domain (PITH) of ShTXNL1 which is a component of 26S proteasome and binds to the matrix or cell. Pairwise alignment results showed 99.0% identity and 99.7% similarity with the sequence of Hippocampus species. Conserved thiol-disulfide cysteine residue containing Cys-X-X-Cys motif may be found in the first few amino acids in the second beta sheet starting from the N-terminus. This motif can be discovered in ShTXNL1 as 14CRPC17 and comprised two N-linked glycosylation sites at 72NISA75 and 139NESD142. According to the quantitative real-time polymerase chain reaction analysis from healthy seahorses, highest ShTXNL1 mRNA expression was observed in muscle, followed by ovary, brain, gill, and blood tissues. Moreover, significant temporal expression of ShTXNL1 was observed in gill and blood tissues after bacterial stimuli. Thus, the ShTXNL1 gene may be identified as an immunologically important gene in seahorse.

A comparative study of three akirin genes from big belly seahorse Hippocampus abdominalis: Molecular, transcriptional and functional characterization.

Akirins, members of the NF-κB signaling pathway, are highly conserved nuclear proteins, which regulate gene expression in many physiological processes, including immunity, myogenesis, carcinogenesis, and embryogenesis. The akirin family in teleost fish consists of two to three genes. In the present study, three akirin genes from Hippocampus abdominalis were identified from a transcriptome database and designated as HaAkirin1, HaAkirin2(1), and HaAkirin2(2). The nuclear localization of HaAkirin1 and HaAkirin2(1) was confirmed by subcellular localization analysis. In contrast, diffused localization of HaAkirin2(2) was identified in the nucleus and cytoplasm that confirmed the aberrant nature of the nuclear localization signal. Phylogenetic analysis revealed a closer relationship of HaAkirins with other known teleost akirins. All three HaAkirin transcripts were ubiquitously expressed in all examined tissues with higher expression in ovary tissue. Immune challenge with LPS, poly I:C, and Streptococcus iniae exhibited a significant increase in the expression of all three HaAkirins in kidney and liver tissues. NF-κB luciferase assays revealed that relative luciferase activity was significantly higher for all three HaAkirin genes than mock controls. These results suggest that HaAkirin genes might play a role in regulating NF-κB dependent immune gene expression and their expression could be induced by bacterial and viral pathogen recognition molecular patterns.