Molecular and functional insights into a novel teleost malectin from bigbelly seahorse Hippocampus abdominalis

Sarithaa Sellaththurai,K.A.S.N. Shanaka,D.S. Liyanage,Hyerim Yang,Thanthrige Thiunuwan Priyathilaka,Jehee Lee 제주대학교 해양과학연구소 2020 해양과환경연구소 연구논문집 Vol.44 No.-

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

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

Omeka, W.K.M.,Liyanage, D.S.,Priyathilaka, Thanthrige Thiunuwan,Godahewa, G.I.,Lee, Seongdo,Lee, Sukkyoung,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.90 No.-

<P><B>Abstract</B></P> <P>Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. In this study, we characterized glutaredoxin 1 (HaGrx1) from big-belly seahorse, <I>Hippocampus abdominalis. In-silico</I> 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 <I>Hippocampus comes</I>. Transcriptional studies demonstrated the ubiquitous distribution of <I>HaGrx1</I> transcripts in all the seahorse tissues tested. Significant modulation (<I>p</I> < 0.05) of <I>HaGrx1</I> transcripts were observed in blood upon stimulation with pathogen-associated molecular patterns and live pathogens. The β-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, <SMALL>L</SMALL>-cysteine, 2-mercaptoethanol, and reduced <SMALL>L</SMALL>-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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Grx1 from <I>Hippocampus abdominalis</I> were characterized. </LI> <LI> CSYC thioredoxin active motif and classical Grx1 structure was found. </LI> <LI> Spatial and temporal expression patterns of <I>HaGrx1</I> were observed. </LI> <LI> Antioxidant activity of Grx1 assessed by different functional assays<I>.</I> </LI> </UL> </P>

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

Samaraweera, Anushka Vidurangi,Sandamalika, W.M Gayashani,Liyanage, D.S.,Lee, Sukkyoung,Priyathilaka, Thanthrige Thiunuwan,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.92 No.-

<P><B>Abstract</B></P> <P>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 (<I>Hippocampus abdominalis</I>) (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 <I>HaGSTκ1</I> 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 <I>Edwardsiella tarda</I>, and gram-positive <I>Streptococcus iniae</I>, <I>HaGSTκ1</I> 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> HaGSTκ1 was identified and characterized from big-belly seahorse. </LI> <LI> HaGSTκ1 was highly expressed in Kidney and liver tissues. </LI> <LI> The elevation of transcript levels of HaGSTκ1 was observed after pathogenic attack. </LI> <LI> Enzyme kinetics, Optimum pH, temperature and inhibition were measured. </LI> </UL> </P>

A Study on Salt-fermented Seahorse added with Proteolytic Enzyme (Protamex)

LEE, In-Sook,LEE, Min-Ho,JANG, Kyung-Tae Korea FoodHealth Convergence Association 2020 식품보건융합연구 (KJFHC) Vol.6 No.6

We compared the fermentation of 0 to 4 weeks by manufacturing a rapid low salt-fermented seahorse with a commercial Protamex added to the functional food, Hippocampus abdominalis. We studied amino acid composition, content and major amino acids related to flavor during the fermentation process of salt-fermented seahorse. In the enzyme-free group, it showed little change in the content of non-protein nitrogenous compounds, the content of amino acids and degree of hydrolysis. The Protamex enzyme treatment group was rapidly hydrolyzed in one week of ripening, resulting in increased non-protein nitrogenous compounds content, amino acid content and degree of hydrolysis, and minimal changes in the four weeks. The total amino acid contents ratio showed the highest content of glutamic acid in the enzyme additive group, glycine, alanine, which indicates sweet taste, and serine, the content of glycine, alanine, serine, and lysine, indicating sweet taste, has increased significantly over the enzyme-free group. Twenty species of free amino acid in the four-week of salt-fermented seahorse were detected. It detected 43.0% (6 species) in the enzyme-free group and 63.96% (7 species) in the enzyme additive group.

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

Liyanage, D.S.,Omeka, W.K.M.,Yang, Hyerim,Godahewa, G.I.,Kwon, Hyukjae,Nam, Bo-Hye,Lee, Jehee Elsevier 2019 Fish & shellfish immunology Vol.86 No.-

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

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

Tharuka, M.D. Neranjan,Priyathilaka, Thanthrige Thiunuwan,Yang, Hyerim,Pavithiran, Amirthalingam,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.86 No.-

<P><B>Abstract</B></P> <P>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 (<I>Hippocampus abdominalis</I>; HaVip) was functionally characterized to determine its subcellular localization, expression pattern, and antiviral activity <I>in vitro</I>. The <I>HaVip</I> 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-<SMALL>L</SMALL>-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 <I>in vitro</I>. Under normal physiological conditions, <I>HaVip</I> 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. <I>In vivo</I> studies showed that <I>HaV</I>ip was rapidly and predominantly upregulated in blood, kidney, and intestinal tissue upon poly (I:C) stimulus. LPS and <I>Streptococus iniae</I> challenges caused a significant increase in expression of <I>HaVip</I> 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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Identified and characterized viperin from big-belly seahorse. </LI> <LI> The HaVip gene was ubiquitously expressed in unchallenged-fish tissues. </LI> <LI> Modulated HaVip transcription pattern revealed its contribution in the immune response. </LI> <LI> Overexpression of HaVip in FHM cells showed the antiviral activity. </LI> </UL> </P>

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

Liyanage, D.S.,Omeka, W.K.M.,Godahewa, G.I.,Lee, Jehee Elsevier 2018 Fish & shellfish immunology Vol.75 No.-

<P><B>Abstract</B></P> <P>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 <I>in-silico</I> 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 <I>Hippocampus</I> 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 <SUP>14</SUP>CRPC<SUP>17</SUP> and comprised two N-linked glycosylation sites at <SUP>72</SUP>NISA<SUP>75</SUP> and <SUP>139</SUP>NESD<SUP>142</SUP>. According to the quantitative real-time polymerase chain reaction analysis from healthy seahorses, highest <I>ShTXNL1</I> mRNA expression was observed in muscle, followed by ovary, brain, gill, and blood tissues. Moreover, significant temporal expression of <I>ShTXNL1</I> was observed in gill and blood tissues after bacterial stimuli. Thus, the <I>ShTXNL1</I> gene may be identified as an immunologically important gene in seahorse.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Big-belly seahorse thioredoxin-like protein 1 maintain redox homeostasis. </LI> <LI> TXNL1 is a component of 26S proteasome. </LI> <LI> Thiol active CXXC conserved motif consists in ShTXNL1. </LI> <LI> Spatial and temporal mRNA expression was evaluated. </LI> <LI> Radical scavenging ability, antioxidant activity, ferric-reducing activity measured. </LI> </UL> </P>

Effects of enzymatic hydrolysate from seahorse Hippocampus abdominalis on testosterone secretion from TM3 Leydig cells and in male mice

Kim, Young Mee,Jeon, You Jin,Huh, Jung Sik,Kim, Sung Dae,Park, Kyung Kgi,Cho, Moonjae The Korean Society for Applied Biological Chemistr 2016 Applied Biological Chemistry (Appl Biol Chem) Vol.59 No.6

Protein hydrolysates, the so-called bioactive peptides, are specific protein fragments that have positive effects on several body functions and may improve human health. Marine organism-derived protein hydrolysates and bioactive peptides have shown to possess many physiological functions. Seahorses, used in traditional medicine, are generally ground to powder form either for direct application or for application following dissolution in warm water. In this present study, we purified, hydrolyzed, and characterized two bioactive peptides (ALC and PEP) obtained from seahorse (Hippocampus abdominalis). The hydrolysates derived from seahorse significantly upregulated the expression of cyclin D and cyclin E and increased cell proliferation and testosterone level in the TM3 mouse Leydig cell line. These findings suggest that the hydrolysates stimulate the proliferation of TM3 cells via the AKT, ERK, and JNK pathways. The decline in the circulating testosterone levels in older men is associated with various adverse health effects. Following daily intake of hydrolysates for 12 weeks, the circulating level of testosterone and the sperm count in mice were measured. We found increased sperm motility (sperm count) as well as an increase in the testosterone level in male mice following a 12-week intake of hydrolysates derived from H. abdominalis. Hence, it can be suggested that seahorse-derived hydrolysates play an important role in improving male health by improving the serum testosterone level.

Effects of enzymatic hydrolysate from seahorse Hippocampus abdominalis on testosterone secretion from TM3 Leydig cells and in male mice

김영미,전유진,허정식,김성대,박경기,조문제 한국응용생명화학회 2016 Applied Biological Chemistry (Appl Biol Chem) Vol.59 No.6

Protein hydrolysates, the so-called bioactive peptides, are specific protein fragments that have positive effects on several body functions and may improve human health. Marine organism-derived protein hydrolysates and bioactive peptides have shown to possess many physiological functions. Seahorses, used in traditional medicine, are generally ground to powder form either for direct application or for application following dissolution in warm water. In this present study, we purified, hydrolyzed, and characterized two bioactive peptides (ALC and PEP) obtained from seahorse (Hippocampus abdominalis). The hydrolysates derived from seahorse significantly upregulated the expression of cyclin D and cyclin E and increased cell proliferation and testosterone level in the TM3 mouse Leydig cell line. These findings suggest that the hydrolysates stimulate the proliferation of TM3 cells via the AKT, ERK, and JNK pathways. The decline in the circulating testosterone levels in older men is associated with various adverse health effects. Following daily intake of hydrolysates for 12 weeks, the circulating level of testosterone and the sperm count in mice were measured. We found increased sperm motility (sperm count) as well as an increase in the testosterone level in male mice following a 12-week intake of hydrolysates derived from H. abdominalis. Hence, it can be suggested that seahorse-derived hydrolysates play an important role in improving male health by improving the serum testosterone level.

Anti-fatigue activity of a mixture of seahorse (Hippocampus abdominalis) hydrolysate and red ginseng

강나래,전유진,김서영,고주영,노섬 한국수산과학회 2017 Fisheries and Aquatic Sciences Vol.20 No.1

Seahorse, a syngnathidae fish, is one of the important organisms used in Chinese traditional medicine. Hippocampus abdominalis, a seahorse species successfully cultured in Korea, was validated for use in food by the Ministry of Food and Drug Safety in February 2016; however. the validation was restricted to 50% of the entire composition. Therefore, to use H. abdominalis as a food ingredient, H. abdominalis has to be prepared as a mixture by adding other materials. In this study, the effect of H. abdominalis on muscles was investigated to scientifically verify its potential bioactivity. In addition, the anti-fatigue activity of a mixture comprising H. abdominalis and red ginseng (RG) was evaluated to commercially utilize H. abdominalis in food industry. H. abdominalis was hydrolyzed using Alcalase, a protease, and the effect of H. abdominalis hydrolysate (HH) on the muscles was assessed in C2C12 myoblasts by measuring cell proliferation and glycogen content. In addition, the mixtures comprising HH and RG were prepared at different percentages of RG to HH (20, 30, 40, 50, 60, 70, and 80% RG), and the anti-fatigue activity of these mixtures against oxidative stress was assessed in C2C12 myoblasts. In C2C12 myoblasts, H2O2-induced oxidative stress caused a decrease in viability and physical fatigue-related biomarkers such as glycogen and ATP contents. However, treatment with RG and HH mixtures increased cell viability and the content of fatigue-related biomarkers. In particular, the 80% RG mixture showed an optimum effect on cell viability and ATP synthesis activity. In this study, all results indicated that HH had anti-fatigue activity at concentrations approved for use in food by the law in Korea. Especially, an 80% RG to HH mixture can be used in food for ameliorating fatigue.