Identification and characterization of a carboxypeptidase N1 from red lip mullet (<i>Liza haematocheila</i>); revealing its immune relevance

Perera, N.C.N.,Godahewa, G.I.,Jung, Sumi,Kim, Myoung-Jin,Nam, Bo-Hye,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.84 No.-

<P><B>Abstract</B></P> <P>Complement system orchestrates the innate and adaptive immunity <I>via</I> the activation, recruitment, and regulation of immune molecules to destroy pathogens. However, regulation of the complement is essential to avoid injuries to the autologous tissues. The present study unveils the characteristic features of an important complement component, anaphylatoxin inactivator from red lip mullet at its molecular and functional level. Mullet carboxypeptidase N1 (MuCPN1) cDNA sequence possessed an open reading frame of 1347 bp, which encoded a protein of 449 amino acids with a predicted molecular weight of 51 kDa. <I>In silico</I> analysis discovered two domains of PM14-Zn carboxypeptidase and a C-terminal domain of M14 N/E carboxypeptidase, two zinc-binding signature motifs, and an N-glycosylation site in the MuCPN1 sequence. Homology analysis revealed that most of the residues in the sequence are conserved among the other selected homologs. Phylogeny analysis showed that MuCPN1 closely cladded with the <I>Maylandia zebra</I> CPN1 and clustered together with the teleostean counterparts. A challenge experiment showed modulated expression of MuCPN1 upon polyinosinic:polycytidylic acid and <I>Lactococcus garviae</I> in head kidney, spleen, gill, and liver tissues. The highest upregulation of MuCPN1 was observed 24 h post infection against poly I:C in each tissue. Moreover, the highest relative expressions upon <I>L. garviae</I> challenge were observed at 24 h post infection in head kidney tissue and 48 h post infection in spleen, gill, and liver tissues. MuCPN1 transfected cells triggered a 2.2-fold increase of nitric oxide (NO) production upon LPS stimulation compared to the un-transfected controls suggesting that MuCPN1 is an active protease which releases arginine from complement C3a, C4a, and C5a. These results have driven certain way towards enhancing the understanding of immune role of MuCPN1 in the complement defense mechanism of red lip mullet.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Carboxypeptidase N1 complement component was identified from the red lip mullet. </LI> <LI> Ubiquitous expression of MuCPN1 was observed in healthy mullet tissues. </LI> <LI> Modulated transcriptions of MuCPN1 revealed the importance in the immune responses. </LI> <LI> MuCPN1 was enhanced the nitric oxide production at an inflammatory condition. </LI> </UL> </P>

Manganese-superoxide dismutase (MnSOD), a role player in seahorse (<i>Hippocampus abdominalis</i>) antioxidant defense system and adaptive immune system

Perera, N.C.N.,Godahewa, G.I.,Lee, Seongdo,Kim, Myoung-Jin,Hwang, Jee Youn,Kwon, Mun Gyeong,Hwang, Seong Don,Lee, Jehee Elsevier 2017 Fish & shellfish immunology Vol.68 No.-

<P><B>Abstract</B></P> <P>Manganese superoxide dismutase (MnSOD) is a metaloenzyme that catalyzes dismutation of the hazardous superoxide radicals into less hazardous H<SUB>2</SUB>O<SUB>2</SUB> and H<SUB>2</SUB>O. Here, we identified a homolog of MnSOD from big belly seahorse (<I>Hippocampus abdominalis</I>; <I>HaMnSOD</I>) and characterized its structural and functional features. HaMnSOD transcript possessed an open reading frame (ORF) of 672 bp which codes for a peptide of 223 amino acids. Pairwise alignment showed that HaMnSOD shared highest identity with rock bream MnSOD. Results of the phylogenetic analysis of HaMnSOD revealed a close proximity with rock bream MnSOD which was consistent with the result of homology alignment. The intense expression of <I>HaMnSOD</I> was observed in the ovary, followed by the heart and the brain. Further, immune related responses of <I>HaMnSOD</I> towards pathogenic stimulation were observed through bacterial and viral challenges. Highest <I>HaMnSOD</I> expression in response to stimulants <I>Edwardsiella tarda</I>, <I>Streptococcus iniae</I>, lipopolysaccharide (LPS), and polyinosinic-polycytidylic acid (Poly I:C) was observed in the late stage in the blood tissue. Xanthine/xanthine oxidase assay (XOD assay) indicated the ROS-scavenging ability of purified recombinant HaMnSOD (rHaMnSOD). The optimum conditions for the SOD activity of rHaMnSOD were pH 9 and the 25 °C. Collectively, the results obtained through the expressional analysis profiles and the functional assays provide insights into potential immune related and antioxidant roles of <I>HaMnSOD</I> in the big belly seahorse.</P> <P><B>Highlights</B></P> <P> <UL> <LI> MnSOD was identified from big belly seahorse (HaMnSOD). </LI> <LI> HaMnSOD was cloned and expressed to evaluate its distinct functional features. </LI> <LI> XOD (Xanthine oxidase) assay confirmed the superoxide scavenging ability of HaMnSOD. </LI> <LI> Transcriptional level of <I>HaMnSOD</I> was modulated by pathological stress. </LI> </UL> </P>

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>

Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis

Godahewa, G.I.,Perera, N.C.N.,Bathige, S.D.N.K.,Nam, B.H.,Noh, J.K.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.55 No.-

<P>The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277 amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CID. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451 bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12 h p.i.), Streptococcus iniae (12 h p.i.), rock bream iridovirus (RBIV) (6-12 h p.i.), and poly I:C (6 h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Molecular characterization and expression analysis of B cell activating factor from rock bream (Oplegnathus fasciatus)

Godahewa, G.I.,Perera, N.C.N.,Umasuthan, N.,Wan, Q.,Whang, I.,Lee, J. Pergamon Press ; Elsevier Science 2016 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.55 No.-

<P>B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) ligand family. BAFF has been shown to induce survival and proliferation of lymphocytes. We characterized the gene encoding BAFF (RbBAFF) in rock bream (Oplegnathus fasciatus), and attempted to determine its biological functions upon immune responses. In silk analysis of RbBAFF demonstrated the presence of common TNF ligand family features, including a TNF domain, a D-E loop, and three cysteine residues that are crucial for trimer formation. Amino acid sequence alignment confirmed that RbBAFF and its homologs were conserved at secondary and tertiary levels. Transcriptional analysis indicated that RbBAFF mRNAs were ubiquitously expressed in wide array of tissues. The higher levels of constitutive expression were observed in the kidney, head kidney and spleen, suggesting an important physiological relationship with lymphocytes. Under pathological conditions, RbBAFF mRNA levels were significantly elevated. The role of RbBAFF in lymphocyte survival and proliferation was confirmed by MIT assays and flow cytometry. Recombinant RbBAFF protein (10 mu g/mL) was able to prolong the survival and/or enhance the proliferation of rock bream lymphocytes by approximately 30%. Transcription of IL-10 and NF kappa B-1 was significantly stimulated by RbBAFF. Our findings provide further information regarding fish BAFF gene and its role in adaptive immunity. (C) 2015 Elsevier Ltd. All rights reserved.</P>

Molecular structure and immune-stimulated transcriptional modulation of the first teleostean IFP35 counterpart from rockfish (Sebastes schlegelii)

Perera, N.C.N.,Godahewa, G.I.,Nam, B.H.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.56 No.-

<P>Interferons (IFNs) and IFN-inducible proteins play numerous physiological roles, particularly in antiviral defense mechanisms of the innate immune response with the presence of pathogens. IFN-induced protein-35 kDa (IFP35) is induced by Type II IFN (IFN-gamma); it is a cytoplasmic protein that can be trans located to the nucleus via the stimulation of IFN. In this study, we report the complete molecular characterization of the IFP35 cDNA sequence from the black rockfish in an effort to understand its role in the immune response. The coding sequence of RfIFP35 encoded a putative peptide of 371 amino acids containing two characteristic Nmi/IFP 35 domains (NIDs), which are highly conserved among its counterparts. The protein showed a molecular mass of 42.2 kDa with a theoretical pI of 5.05 and was predicted to be unstable because of its high instability index (4937). Therefore, the protein-protein interaction is essential for its stability, which may be facilitated by the intrinsically disordered regions in this protein. According to cellular location prediction, the RflFP35 protein is cytosolic. Phylogenetic analysis showed that RfIFP35 was cladded within the fish counterparts. Tissue distribution profiling revealed a ubiquitous presence of the protein in all examined tissues, with highest expression in the blood followed by the spleen tissues. The expression of RfIFP35 during immune challenge with poly I:C and lipopolysaccharide treatments affirms its putative importance in the first-line host defense system. RfIFN-gamma mRNA was significantly expressed at 6 h p.i. in blood and 3 h p.i. in the spleen following treatment with different immune stimulants, and its expression was higher compared to that of RfIFP35 mRNA. Therefore, the modulation patterns of both RfIFP35 and RfIFN-gamma suggest that RfIFP35 may be induced by RfEN-gamma. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Two metalloenzymes from rockfish (<i>Sebastes schligellii</i>): Deciphering their potential involvement in redox homeostasis against oxidative stress

Perera, N.C.N.,Godahewa, G.I.,Nam, Bo-Hye,Park, Jung Youn,Lee, Jehee Elsevier 2018 FISH AND SHELLFISH IMMUNOLOGY Vol.80 No.-

<P>Disturbance in the balance between pro-oxidants and anti-oxidants result oxidative stress in aerobic organisms. However, oxidative stress can be inhibited by enzymatic and non-enzymatic defense mechanisms. Superoxide dismutases (SODs) are well-known scavengers of superoxide radicals, and they protect cells by detoxifying hazardous reactive oxygen species. Here, we have identified and characterized two different SODs, CuZnSOD and MnSOD, from black rockfish (RfCuZnSOD and RfMnSOD, respectively). In silica analysis revealed the well conserved molecular structures comprising all essential properties of CuZnSOD and MnSOD. Phylogenetic analysis revealed that both RfCuZnSOD and RfMnSOD cladded with their fish counterparts. The recombinant RfSOD proteins demonstrated their potential superoxide scavenging abilities through a xanthine oxidase assay. The optimum temperature and pH conditions for both rRfSODs were 25 degrees C and pH 8, respectively. Moreover, the potential peroxidation function of rRfCuZnSOD was observed in the presence of HCO3-. The highest peroxidation activity was observed at 100 mu g/mL of rRfCuZnSOD using the MTT cell viability assay and flow cytometry. The analogous tissue-specific expression profile indicated ubiquitous expression of both RfCuZnSOD and RfMnSOD in selected tissues of healthy juvenile rockfish. An immune challenge experiment illustrated the altered expression profiles of both RfCuZnSOD and RfMnSOD against lipopolysaccharide, Streptococcus iniae, and polyinosinic-polycytidylic acid (poly I:C). Collectively, these results strengthen the general understanding of the structural and functional characteristics of SODs within the host defense system.</P>

A homolog of Kunitz-type serine protease inhibitor from rock bream, Oplegnathus fasciatus: Molecular insights and transcriptional modulation in response to microbial and PAMP stimulation, and tissue injury

Bathige, S.D.N.K.,Umasuthan, N.,Godahewa, G.I.,Jayasinghe, J.D.H.E.,Whang, I.,Noh, J.K.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.46 No.2

Serine proteases and their inhibitors play vital roles in diverse biological processes. In this study, we identified and characterized cDNA coding for a Kunitz-type serine protease inhibitor (SPI), which we designated as RbKSPI, in a commercially important species, rock bream. The full-length cDNA sequence of RbKSPI consisted of 2452 bp with an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acid (aa) residues. In the RbKSPI protein, MANEC, PKD, LDLa, and two Kunitz domains responsible for various functions were identified as characteristic features. Homology analysis revealed that RbKSPI shared the highest identity with the Kunitz homolog in Takifugu rubripes (77.6%). Phylogenetic analysis indicated that RbKSPI clusters with other teleostean KSPIs. In tissue-specific expression analysis, RbKSPI transcripts were detected in all the tested tissues, with the highest expression in gill tissue, followed by kidney and intestine. The mRNA expression of RbKSPI significantly increased in blood cells upon stimulation with two strains of bacteria (Edwardsiella tarda and Streptococcus iniae) and two pathogen-associated molecular patterns (PAMPs; LPS and poly I:C). Meanwhile, down-regulated expression of RbKSPI was observed in response to tissue injury. Collectively, these results suggest that the RbKSPI may be involved in essential immune defense against microbial pathogens and in the wound-healing process.

Molecular insights of two STAT1 variants from rock bream (Oplegnathus fasciatus) and their transcriptional regulation in response to pathogenic stress, interleukin-10, and tissue injury

Bathige, S.D.N.K.,Umasuthan, N.,Godahewa, G.I.,Thulasitha, W.S.,Jayasinghe, J.D.H.E.,Wan, Q.,Lee, J. Academic Press 2017 Fish & shellfish immunology Vol.69 No.-

Signal transducers and activators of transcription 1 (STAT1) is critically involved in mediating cytokine-driven signaling, and triggers the transcription of target genes to activate cellular functions. Although the structural and functional aspects of STAT members have been well described in mammals, only limited information is available for the STAT genes in teleost fishes. In the present study, two variants of STAT1 genes (RbSTAT1 and RbSTAT1L) were identified from rock bream and characterized at the cDNA and genomic sequence levels. RbSTAT1 and RbSTAT1L were found to share a common domain architecture with mammalian STAT1. Phylogenetic analysis revealed that RbSTAT1 shows a common evolutionary trajectory with other STAT1 counterparts, whereas RbSTAT1L showed a separate path, implying that it could be a novel member of the STAT family. The genomic organizations of RbSTAT1 and RbSTAT1L illustrated a similar exon-intron pattern with 23 exons in the coding sequence. Transcription factor-binding sites, which are mostly involved in the regulation of immune responses, were predicted at the putative promoter regions of the RbSTAT1 and RbSTAT1L genes. SYBR Green qPCR analysis revealed the ubiquitous expression of RbSTAT1 and RbSTAT1L transcripts in different fish tissues with the highest level observed in peripheral blood cells. Significantly modulated transcripts were noted upon viral (rock bream iridovirus [RBIV]), bacterial (Edwardsiella tarda and Streptococcus iniae), and pathogen-associated molecular pattern (lipopolysaccharide and poly I:C) stimulations. The WST-1 cell viability assay affirmed the potential antiviral capacity of RbSTAT1 and RbSTAT1L against RBIV. A possible role of RbSTAT1 and RbSTAT1L in the wound healing process was revealed according to their modulated expression in injured fish. In addition, the transcriptional regulation of RbSTAT1 and RbSTAT1L was analyzed by qPCR following stimulation with rock bream interleukin-10. Taken together, these findings suggest that the STAT1-mediated Janus kinase/STAT pathway might at least in part be involved in the regulatory mechanisms underlying the immune defensive roles against microbial pathogens and the wound healing process.

Molecular characterization and comparative expression analysis of two teleostean pro-inflammatory cytokines, IL-1β and IL-8, from Sebastes schlegeli

Herath, H.M.L.P.B.,Elvitigala, D.A.S.,Godahewa, G.I.,Umasuthan, N.,Whang, I.,Noh, J.K.,Lee, J. Elsevier/North-Holland 2016 Gene Vol.575 No.2

Interleukin 1β (IL-1β) and interleukin 8 (IL-8) are two major pro-inflammatory cytokines which play a central role in initiation of inflammatory responses against bacterial- and viral-infections. IL-1β is a member of the interleukin 1 family proteins and IL-8 is classified as a CXC-chemokine. In the current study, putative IL-1β and IL-8 counterparts were identified from a black rockfish transcriptomic database and designated as RfIL-1β and RfIL-8. The RfIL-1β cDNA sequence consists of 1140 nucleotides with a 759bp open reading frame (ORF) which encodes a 252 amino acid (aa) protein, whereas the RfIL-8 cDNA sequence (898bp) harbors a 300bp ORF encoding a 99 aa protein. Furthermore, the RfIL-1β aa sequence contains an IL-1 super family-like domain and an N-terminal IL-1 super family propeptide, while the amino acid sequence of RfIL-8 consists of a typical chemokine-CXC domain. Analysis of sequenced BAC clones containing RfIL-1β and RfIL-8 showed each gene to contain 4 exons interrupted by 3 introns. Pairwise comparison and phylogeny analysis of these cytokine sequences clearly revealed their closer relationship with other corresponding members of teleosts compared to birds and mammals. Constitutive differences in RfIL-1β and RfIL-8 mRNA expression were detected in a tissue-specific manner with the highest expression of each mRNA in spleen tissue. Two immune challenge experiments were conducted with Streptococcus iniae and polyinosinic:polycytidylic acid (poly I:C; a viral double stranded RNA mimic), and transcripts were quantified in spleen and peripheral blood cells. Significantly increased RfIL-1β and RfIL8 transcript levels were detected with almost similar profile patterns, further suggesting a putative involvement of these pro-inflammatory cytokines in the rockfish immunity.