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>

Molecular, structural, and functional comparison of N lobe and C lobe of the transferrin from rock bream, <i>Oplegnathus fasciatus,</i> with respect to its immune response

Perera, N.C.N.,Godahewa, G.I.,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>The iron-withholding strategy of innate immunity is an effective antimicrobial defense mechanism that combats microbial infection by depriving microorganisms of Fe<SUP>3+</SUP>, which is important for their growth and propagation. Transferrins (Tfs) are a group of iron-binding proteins that exert their antimicrobial function through Fe<SUP>3+</SUP> sequestration. The current study describes both structural and functional characteristics of a transferrin ortholog from rock bream <I>Oplegnathus fasciatus</I> (RbTf). The RbTf cDNA possesses an open reading frame (ORF) of 2079 bp encoding 693 amino acids. It has a molecular mass of approximately 74 kDa and an isoelectric point of 5.4. <I>In silico</I> analysis revealed that RbTf has two conserved domains: N-terminal domain and C-terminal domain. Pairwise homology analysis and phylogenetic analysis revealed that RbTf shared the highest identity (82.6%) with <I>Dicentrarchus labrax</I> Tf. According to the genomic analysis, RbTf possesses 17 exons and 16 introns, similar to the other orthologs. Here, we cloned the N terminal and C terminal domains of RbTf to evaluate their distinct functional features. Results obtained through the CAS (chrome azurol S) assay confirmed the iron-binding ability of the RbTf, and it was further determined that the iron-binding ability of rRbTfN was higher than that of rRbTfC. The antimicrobial functions of the rRbTfN and the rRbTfC were confirmed via the iron-dependent bacterial growth inhibition assay. Tissue distribution profiling revealed a ubiquitous expression with intense expression in the liver. Temporal assessment revealed that <I>RbTf</I> increased after stimulation of LPS, <I>Edwardsiella tarda,</I> and <I>Streptococcus iniae</I> post injection (p.i.). These findings demonstrated that RbTf is an important antimicrobial protein that can combat bacterial pathogens.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Transferrin was identified from rock bream (<I>RbTf</I>). </LI> <LI> N terminal and C terminal domains of RbTf was separately cloned and evaluated their distinct functional features. </LI> <LI> CAS (chrome azurol S) assay confirmed the iron-binding ability of the RbTf. </LI> <LI> Transcriptional level of <I>RbTf</I> was modulated by pathological stress. </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>

Immune responses upon bacterial and viral stimulants of carboxypeptidase N1 from mullet Chelon haematocheilus

N.C.N. Perera,G. I. Godahewa,Jehee Lee 한국수산해양기술학회(구 한국어업기술학회) 2017 한국수산해양기술학회 학술발표대회 Vol.2017 No.11

Copper-zinc-superoxide dismutase (CuZnSOD), an antioxidant gene from seahorse (Hippocampus abdominalis); molecular cloning, sequence characterization, antioxidant activity and potential peroxidation function of its recombinant protein

Perera, N.C.N.,Godahewa, G.I.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.57 No.-

Copper-zinc-superoxide dismutase (CuZnSOD) from Hippocampus abdominalis (HaCuZnSOD) is a metalloenzyme which belongs to the ubiquitous family of SODs. Here, we determined the characteristic structural features of HaCuZnSOD, analyzed its evolutionary relationships, and identified its potential immune responses and biological functions in relation to antioxidant defense mechanisms in the seahorse. The gene had a 5' untranslated region (UTR) of 67 bp, a coding sequence of 465 bp and a 3' UTR of 313 bp. The putative peptide consists of 154 amino acids. HaCuZnSOD had a predicted molecular mass of 15.94 kDa and a theoretical pI value of 5.73, which is favorable for copper binding activity. In silico analysis revealed that HaCuZnSOD had a prominent Cu-Zn_superoxide_dismutase domain, two Cu/Zn signature sequences, a putative N-glycosylation site, and several active sites including Cu<SUP>2+</SUP> and Zn<SUP>2+</SUP> binding sites. The three dimensional structure indicated a β-sheet barrel with 8 β-sheets and two short α-helical regions. Multiple alignment analyses revealed many conserved regions and active sites among its orthologs. The highest amino acid identity to HaCuZnSOD was found in Siniperca chuatsi (87.4%), while Maylandia zebra shared a close relationship in the phylogenetic analysis. Functional assays were performed to assess the antioxidant, biophysical and biochemical properties of overexpressed recombinant (r) HaCuZnSOD. A xanthine/XOD assay gave optimum results at pH 9 and 25 <SUP>o</SUP>C indicating these may be the best conditions for its antioxidant action in the seahorse. An MTT assay and flow cytometry confirmed that rHaCuZnSOD showed peroxidase activity in the presence of HCO<SUB>3</SUB><SUP>-</SUP>. In all the functional assays, the level of antioxidant activity of rHaCuZnSOD was concentration dependent; metal ion supplementation also increased its activity. The highest mRNA expressional level of HaCuZnSOD was found in blood. Temporal assessment under pathological stress showed a delay response by HaCuZnSOD. Our findings demonstrated that HaCuZnSOD is an important antioxidant, which might be involved in the host antioxidant defense mechanism against oxidative stress.

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>

Analysis of complete genome and pathogenicity studies of the spring viremia of carp virus isolated from common carp (<i>Cyprinus carpio carpio</i>) and largemouth bass (<i>Micropterus salmoides</i>): An indication of SVC disease threat in Korea

Godahewa, G.I.,Lee, Seongdo,Kim, Jeongeun,Perera, N.C.N.,Kim, Myoung-Jin,Kwon, Mun Gyeong,Jee, Bo Young,Hwang, Seong Don,Lee, Jehee ELSEVIER 2018 VIRUS RESEARCH Vol.255 No.-

<P><B>Abstract</B></P> <P>A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea.</P> <P><B>Highlights</B></P> <P> <UL> <LI> SVCV isolation from common carp and largemouth bass. </LI> <LI> Complete genome analysis revealed 11,034 bp nucleotides in length. </LI> <LI> Phylogenetic analysis revealed the Korean SVCV were clustered within the Asian clade. </LI> <LI> Virus transmission studies showed that the highest virulence is at 15 °C. </LI> <LI> Pathogenicity of SVCV was high in injection method than the other methods. </LI> </UL> </P>

An Inflammatory cytokine, Macrophage Migration Inhibitory Factor; insights in to molecular, structural and functional aspects from Korean black rockfish (Sebastes Schligelli)

N. C. N. Perera,G. I. Godahewa,Jehee Lee 한국수산과학회 양식분과 2018 한국수산과학회 양식분과 학술대회 Vol.2018 No.5

Transcriptional analysis with its molecular characterization of beta catenin gene identified from rock bream (Oplegnathus fasciatus)

N. C. N. Perera,G. I. Godahewa,Jehee Lee 한국수산과학회 양식분과 2015 한국수산과학회 양식분과 학술대회 Vol.2015 No.10

Isolation and involvement of peroxiredoxin 2 from Sebastes schlegelii in both antioxidant and immune defense systems

N. C. N. Perera,G. I. Godahewa,Jehee Lee 제주대학교 해양과학연구소 2018 해양과환경연구소 연구논문집 Vol.42 No.-