A C1 inhibitor ortholog from rock bream (Oplegnathus fasciatus): Molecular perspectives of a central regulator in terms of its genomic arrangement, transcriptional profiles and anti-protease activities of recombinant peptide

Umasuthan, N.,Bathige, S.D.N.K.,Revathy, K.S.,Wickramaarachchi, W.D.N.,Wan, Q.,Whang, I.,Kim, E.,Park, M.A.,Park, H.C.,Lee, J. Pergamon Press ; Elsevier Science 2014 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.42 No.2

C1 inhibitor (C1Inh), a member of serpin superfamily, is a crucial regulator of the activation of various plasmatic cascades associated with immunity and inflammation. This study describes the identification and characterization of a C1Inh gene from rock bream Oplegnathus fasciatus (OfC1Inh) at structural, expressional and functional levels. The cDNA-(2245bp) and corresponding gDNA-sequences (5.2kbp) of OfC1Inh were isolated from rock bream transcriptome- and BAC-libraries, respectively. Predicted amino acid sequence of OfC1Inh revealed a two-domain architecture composed of an N-terminal region with two Ig-like domains and a C-terminal region with a serpin domain. Tertiary model of OfC1Inh disclosed its active site topology. In the multi-exonic genomic arrangement of OfC1Inh, it consisted of eleven exons disjoined by ten introns as observed in few other fish homologs. Our comparative analysis indicated that the teleostean C1Inhs were distinct from their non-teleostean vertebrate counterparts in terms of their (1) extended N-terminal domains, (2) evolutionary divergence and (3) exon-intron distribution. The OfC1Inh had a TATA-deficient promoter with a putative initiator element, and two tandemly arranged downstream promoter elements. Several components associated with the immune and inflammatory transcriptional activation were also predicted to exist in 5' flanking region of OfC1Inh. The exclusive mRNA levels in liver and moderate levels in extra-hepatic tissues intimated the diversified importance of OfC1Inh in rock bream physiology. We also provide an evidence for the involvement of OfC1Inh in immune balance, based on its modulated transcription upon different PAMP (lipopolysaccharide and poly I:C)- or pathogen (Streptococcus iniae and rock bream irido virus)-challenges. A recombinantly expressed fusion protein [(r)OfC1Inh] was employed in demonstrating the anti-protease function of OfC1Inh. The (r)OfC1Inh exhibited detectable inhibitory activity against C1 esterase and thrombin, where the anti-C1 esterase role was shown to be potentiated by heparin. Taken together, the results of this study provide the first line of evidence for the possible involvement of a teleostean C1Inh in fish immunity, based on its expressional response(s) and inhibitory properties against two enzymes involved in biological cascades.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus)

Umasuthan, N.,Bathige, S.D.N.K.,Noh, J.K.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.47 No.1

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38β (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (~15.6 kb) and OfMAPK14 (~13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

Heparin cofactor II (RbHCII) from rock bream (Oplegnathus fasciatus): Molecular characterization, cloning and expression analysis

Umasuthan, N.,Whang, I.,Lee, Y.,Lee, S.,Kim, Y.,Kim, H.,Jung, S.J.,Oh, M.J.,Choi, C.Y.,Yeo, S.Y.,Lee, S.J.,Lee, J. Academic Press 2011 Fish & shellfish immunology Vol.30 No.1

Heparin cofactor (HCII) is a serine protease inhibitor (SPI), and plays important physiological roles in various biological events including hemostasis. The gene encoding the HCII was isolated from GS-FLX(TM) genomic data of rock bream (Oplegnathus fasciatus), designated as RbHCII. The RbHCII (1950 bp) consists of a 1512 bp open reading frame (ORF) encoding 504 amino acids (aa), with a signal peptide of 19 aa residues. The predicted molecular mass and the estimated isoelectric point of RbHCII were 58 kDa and 5.9, respectively. The deduced aa sequence of RbHCII displayed a characteristic serpin domain and a serpin signature motif (FTVDQPFLFLI). RbHCII demonstrated homology with vertebrate HCIIs and the greatest degree of similarity (90.1%) was observed with Gasterosteus aculeatus HCII. Various functional domains including the reactive center loop (RCL), glycosaminoglycan (GAG) and thrombin binding sites and acidic repeats of human and RbHCII were found to be orthologs through the molecular modeling studies. Phylogenetic analysis revealed that RbHCII belongs to the clade D serpins, and is closely related to the clade A members. Constitutive expression of RbHCII mRNA was detected at different levels in various tissues in a tissue-specific manner. Interestingly, RbHCII transcription was significantly downregulated (p < 0.05) in liver after challenge with lipopolysaccharide (LPS), Edwardsiella tarda and rock bream iridovirus (RBIV). However, after the immune challenges, RbHCII showed a significant downregulation in blood tissue only at the late-phase of investigation. The recombinant RbHCII (rRbHCII) was overexpressed in Rosetta-gami (DE3) cells and purified using the pMAL(TM) system. The rRbHCII inhibited thrombin and chymotrypsin in a dose-dependent manner. Remarkably, heparin was found to be an enhancer of RbHCII's thrombin-inhibitory activity. Correlating the heparin-dependent thrombin-inhibition activity of RbHCII with its temporal downregulation against immune stimulants, it could be suggested that it is not only involved in the blood coagulation cascade, but also plays an incognito role in immune modulation.

Mitochondrial thioredoxin-2 from Manila clam (Ruditapes philippinarum) is a potent antioxidant enzyme involved in antibacterial response

Umasuthan, N.,Saranya Revathy, K.,Lee, Y.,Whang, I.,Lee, J. Academic Press 2012 Fish & shellfish immunology Vol.32 No.4

Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561bp) consists of a 498bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic <SUP>89</SUP>WCGPC<SUP>93</SUP> catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams.

A teleostean angiotensinogen from <i>Oplegnathus fasciatus</i> responses to immune and injury challenges

Umasuthan, Navaneethaiyer,Whang, Ilson,Saranya Revathy, Kasthuri,Oh, Myung-Joo,Jung, Sung-Ju,Choi, Cheol Young,Lee, Jeong-Ho,Noh, Jae Koo,Lee, Jehee Elsevier 2012 FISH AND SHELLFISH IMMUNOLOGY Vol.32 No.5

<P><B>Abstract</B></P><P>Angiotensinogen (AGT) is the precursor of the renin-angiotensin system and contributes to osmoregulation, acute-phase and immune responses. A full-length cDNA of the <I>AGT</I> (2004 bp with a 1389 bp coding region) was isolated from rock bream (Rb), <I>Oplegnathus fasciatus</I>. The encoded polypeptide of 463 amino acids had a predicted molecular mass of 51.6 kDa. <I>RbAGT</I> possessed a deduced signal peptide of 22 residues upstream of a putative angiotensin I sequence (<SUP>23</SUP>NRVYVHPFHL<SUP>32</SUP>). <I>RbAGT</I> possessed a specific domain profile and a signature motif which are characteristics of the serpin family. Sequence homology and phylogenetic analysis indicated that <I>RbAGT</I> was evolutionarily closest to AGT of <I>Rhabdosargus sarba</I>. The mRNA expression profile of <I>RbAGT</I> was determined by quantitative RT-PCR and it demonstrated a constitutive and tissue-specific expression with the highest transcript level in the liver. Significantly up-regulated <I>RbAGT</I> expression was elicited by systemic injection of a lipopolysaccharide, rock bream iridovirus (RBIV) and bacteria (<I>Edwardsiella tarda</I> and <I>Streptococcus iniae</I>), revealing its pathogen inducibility. <I>RbAGT</I> manifested a down-regulated response to systemic injury, contemporaneously with two other serpins, protease nexin-1 (<I>PN-</I>1), and heparin cofactor II (<I>HCII</I>). In addition, a synchronized expression pattern was elicited by <I>RbAGT</I> and <I>RbTNF-α</I> in response to injury, suggesting that TNF-α might be a potential modulator of <I>AGT</I> transcription.</P> <P><B>Graphical abstract</B></P><P><ce:figure id='dfig1'></ce:figure></P><P><B>Highlights</B></P><P>► Molecular characterization of angiotensinogen from rock bream (<I>RbAGT</I>). ► Tissue-specific transcriptional profile of <I>RbAGT.</I> ► Response of hepatic <I>RbAGT</I> against LPS, bacteria and iridovirus. ► Temporal expression of hematic <I>RbAGT</I> upon injury. ► Expressional relationship between <I>RbAGT</I>, <I>HCII</I>, <I>PN-1</I> and <I>TNF-α</I>.</P>

Insights into molecular profiles and genomic evolution of an <i>IRAK4</i> homolog from rock bream (<i>Oplegnathus fasciatus</i>): Immunogen- and pathogen-induced transcriptional expression

Umasuthan, Navaneethaiyer,Bathige, S.D.N.K.,Whang, Ilson,Lim, Bong-Soo,Choi, Cheol Young,Lee, Jehee Elsevier 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.43 No.2

<P><B>Abstract</B></P> <P>As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This study investigates the molecular and expressional profiles of an <I>IRAK4-like</I> homolog from <I>Oplegnathus fasciatus</I> (<I>OfIRAK4</I>). The <I>OfIRAK4</I> gene (8.2?kb) was structured with eleven exons and ten introns. A putative coding sequence (1395bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4 protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain (PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initiation site revealed the presence of putative regulatory elements, including NF-κB-binding sites, which are possibly involved in transcriptional control of <I>OfIRAK4</I>. Quantitative real-time PCR (qPCR) was employed to assess the transcriptional expression of <I>OfIRAK4</I> in different juvenile tissues and post-injection of different immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected with highest level in liver. <I>In?vivo</I> flagellin (FLA) challenge significantly intensified its mRNA levels in intestine, liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expression was also determined in liver and head kidney of animals challenged with potent immunogens (LPS and poly I:C) and pathogens (<I>Edwardsiella tarda</I> and <I>Streptococcus iniae</I> and rock bream iridovirus (RBIV)). Taken together, these data implicate that <I>OfIRAK4</I> might be engaged in antibacterial and antiviral immunity in rock bream.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Rock bream <I>IRAK4</I> (<I>OfIRAK4</I>) had 11 exons encoding its protein with a bipartite domain structure. </LI> <LI> Teleost IRAK4s are distinct from tetrapod IRAK4s in terms of gene structure and phylogeny. </LI> <LI> Liver was the primary site of <I>OfIRAK4</I>'s transcription. </LI> <LI> <I>OfIRAK4</I>'s putative role in flagellin sensing was evidenced from its transcriptional response. </LI> <LI> Immunogenic PAMPs/pathogens induced <I>OfIRAK4</I>'s expression implied its association with immunity. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Molecular genomic- and transcriptional-aspects of a teleost TRAF6 homolog: Possible involvement in immune responses of Oplegnathus fasciatus against pathogens

Umasuthan, N.,Bathige, S.D.N.K.,Revathy, K.S.,Nam, B.H.,Choi, C.Y.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.42 No.1

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial docking molecule for TNFR superfamily and Interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. As an adaptor protein in pathogen-induced signaling cascades, TRAF6 modulates both adaptive- and innate-immunity. In order to understand the immune responses of teleost TRAF6, Oplegnathus fasciatus TRAF6-like gene (OfTRAF6) was identified and characterized. Genomic length of OfTRAF6 (4 kb), obtained by means of a genomic BAC library, spanned seven exons which represented a putative coding sequence of 1716 bp and encoded 571 amino acids (aa) with an estimated molecular weight of 64 kDa. This putative protein demonstrated the classical tetra-domain architecture composed of a zinc finger RING-type profile, two zinc finger TRAF-type profiles, a coiled-coil region and a MATH domain. While the sequence similarity with human TRAF6 was 66.5%, OfTRAF6 shared a higher overall similarity with teleost homologs (~75-92%). Phylogeny of TRAF-family was examined and TRAF6-subfamily appeared to be the precursor of other subfamilies. In addition, the clustering pattern confirmed that OfTRAF6 is a novel member of TRAF6subfamily. Based on comparative genomic analysis, we found that vertebrate TRAF6 exhibits two distinct structures in teleost and tetrapod lineages. An intron-loss event has probably occurred in TRAF6 gene during the evolution of tetrapods from teleosts. Inspection of putative OfTRAF6 promoter revealed the presence of several immune responsive transcription factor binding sites. Real-time qPCR assay detected OfTRAF6 transcripts in eleven juvenile fish tissues with higher levels in peripheral blood cells followed by liver. Putative role of OfTRAF6 in response to flagellin, LPS, poly I:C, pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and rock bream iridovirus (RBIV) was profiled in different tissues and OfTRAF6 revealed up-regulated transcript levels. Altogether, these findings implicate that OfTRAF6 is not only involved in flagellin-induced signaling cascade, but also contributes to the antibacterial- and antiviral-responses.

Molecular aspects, genomic arrangement and immune responsive mRNA expression profiles of two CXC chemokine receptor homologs (CXCR1 and CXCR2) from rock bream, Oplegnathus fasciatus

Umasuthan, N.,Wan, Q.,Revathy, K.S.,Whang, I.,Noh, J.K.,Kim, S.,Park, M.A.,Lee, J. Academic Press 2014 FISH AND SHELLFISH IMMUNOLOGY Vol.40 No.1

The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5'-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 and CXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that of OfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream.

Identification of a gene encoding a membrane-anchored toll-like receptor 5 (TLR5M) in <i>Oplegnathus fasciatus</i> that responds to flagellin challenge and activates NF-κB

Umasuthan, Navaneethaiyer,Bathige, S.D.N.K.,Thulasitha, William Shanthakumar,Jayasooriya, R.G.P.T.,Shin, Younhee,Lee, Jehee Elsevier 2017 Fish & shellfish immunology Vol.62 No.-

<P><B>Abstract</B></P> <P>Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, <I>Oplegnathus fasciatus.</I> Both <I>Oftlr5m</I> (6.7 kb) and <I>Ofmyd88</I> (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the <I>tlr5m</I> gene has evolved with extensive rearrangement; whereas, the <I>myd88</I> gene has maintained a stable structure throughout the evolution. Inspection of 5′ flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected <I>Oftlr5m</I> mRNA in eleven tissues with the highest abundance in liver. <I>In vivo</I> flagellin administration strongly induced the transcripts of both <I>Oftlr5m</I> and <I>Ofmyd88</I> in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with <I>Oftlr5m</I> and <I>Ofmyd88</I> demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> genes in rock bream display quinquepartite structure. </LI> <LI> While gene structure of <I>tlr5m</I> is evolved with rearrangements, <I>myd88</I> is preserved. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> showed similar tissue mRNA profile with highest level in liver. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> were induced by ultrapure flagellin in gill and head kidney. </LI> <LI> They individually and synergetically activated NF-κB upon flagellin-stimulation. </LI> </UL> </P>

Rock bream (Oplegnathus fasciatus) serpin, protease nexin-1: Transcriptional analysis and characterization of its antiprotease and anticoagulant activities

Umasuthan, N.,Whang, I.,Kim, J.O.,Oh, M.J.,Jung, S.J.,Choi, C.Y.,Yeo, S.Y.,Lee, J.H.,Noh, J.K.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.7

Protease nexin-1 (PN-1) is a serine protease inhibitor (SERPIN) protein with functional roles in growth, development, patho-physiology and injury. Here, we report our work to clone, analyze the expression profile and characterize the properties of the PN-1 gene in rock bream (Rb), Oplegnathus fasciatus. RbPN-1 encodes a peptide of 397 amino acids (AA) with a predicted molecular mass of 44kDa and a 23 AA signal peptide. RbPN-1 protein was found to harbor a characteristic SERPIN domain comprised of a SERPIN signature and having sequence homology to vertebrate PN-1s. The greatest identity (85%) was observed with PN-1 from the three-spined stickleback fish, Gasterosteus aculeatus. The functional domains, including a heparin binding site and reactive centre loop were conserved between RbPN-1 and other fish PN-1s; in particular, they were found to correspond to components of the human plasminogen activator inhibitor 1, PAI-1. Phylogenetic analysis indicated that RbPN-1 was closer to homologues of green spotted pufferfish and Japanese pufferfish. Recombinant RbPN-1 demonstrated antiprotease activity against trypsin (48%) and thrombin (89%) in a dose-dependent manner, and its antithrombotic activity was potentiated by heparin. The anticoagulant function prolonged clotting time by 3.7-fold, as compared to the control in an activated partial thromboplastin time assay. Quantitative real-time PCR results indicated that RbPN-1 is transcribed in many endogenous tissues at different levels. Lipopolysaccharide (LPS) stimulated a prolonged transcriptional response in hematic cells, and Rb iridovirus up-regulated the RbPN-1 mRNA level in hematic cells to a maximum of 3.4-fold at 12h post-infection. Interestingly, LPS and Edwardsiella tarda significantly induced the RbPN-1 transcription at the late phase of infection. In vivo studies indicated that injury response caused a temporal suppression in RbPN-1 transcription, in conjunction with that of another SERPIN, rock bream heparin cofactor II, RbHCII. Taken together, our findings suggest that PN-1 functions as an antiprotease and anticoagulant and that SERPINs (PN-1 and HCII) are likely to contribute to immunity and post-injury responses.