National Fisheries and Research and Developmental Institute

Busan, South Korea

National Fisheries and Research and Developmental Institute

Busan, South Korea

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Premachandra H.K.A.,Jeju National University | Elvitigala D.A.S.,Jeju National University | Whang I.,Jeju National University | Kim E.,Korea University | And 7 more authors.
Fish and Shellfish Immunology | Year: 2013

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (. Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon invivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response. © 2013 Elsevier Ltd.


Wickramaarachchi W.D.N.,Jeju National University | Whang I.,Jeju National University | Wan Q.,Jeju National University | Bathige S.D.N.K.,Jeju National University | And 5 more authors.
Developmental and Comparative Immunology | Year: 2013

The complement component 8α and 8β are glycoproteins that mediate formation of the membrane attack complex (MAC) on the surface of target cells. Full-length complement C8α (Rb-C8α) and C8β (Rb-C8β) sequences were identified from a cDNA library of rock bream (Oplegnathus fasciatus), and their genomic sequences were obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The Rb-C8α gene contains 64. bp of 5'-UTR, open reading frame (ORF) of 1794. bp, which encodes a polypeptide of 598 amino acids, 212. bp of 3'-UTR. The Rb-C8β gene contains 5'-UTR of 27. bp, open reading frame (ORF) of 1761. bp, which encodes a polypeptide of 587 amino acids, 3'-UTR of 164. bp. Rb-C8α consists of 11 exons interrupted by 10 introns and Rb-C8β consists of 12 exons interrupted by 11 introns. Sequence analysis revealed that both Rb-C8α and Rb-C8β contain thrombospondin type-1, a low-density lipoprotein receptor domain class A, membrane attack complex/perforin (MACPF) domain and epidermal growth factor like domain. The promoter regions of both genes contain important putative transcription factor binding sites including those for NF-κB, SP-1, C/EBP, AP-1, and OCT-1. Rb-C8α and Rb-C8β showed the highest amino acid identity of 62% and 83% to rainbow trout C8α and Japanese flounder C8β respectively. Quantitative real-time PCR analysis confirmed that Rb-C8α and Rb-C8β were constitutively expressed in all examined tissues, isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus led to up regulation of Rb-C8α and Rb-C8β in liver. Positive regulations upon bacterial and viral challenges, and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C8α and Rb-C8β important immune genes, likely involved in the complement system lytic pathway of rock bream. © 2012 Elsevier Ltd.


Wickramaarachchi W.D.N.,Jeju National University | Wan Q.,Jeju National University | Lim B.-S.,Jeju National University | Jung H.-B.,Jeju National University | And 4 more authors.
Fish and Shellfish Immunology | Year: 2014

The interferon regulatory factor 5 (IRF5) is a key mediator of the Toll-like receptor (TLR)7 and TLR8 signaling pathways. In this study, we describe the identification of IRF5 (Rb-IRF5) from rock bream fish (Oplegnathus fasciatus) and its characteristics features at the genomic and expression levels. The full-length Rb-IRF5 sequence was identified from a cDNA library and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The genomic sequence is comprised of 8 exons interrupted by 7 introns. The complete coding sequence of Rb-IRF5 is 1497bp in length and encodes for 498 amino acids. The putative Rb-IRF5 protein consists of 3 important conserved domains: a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus. Based on pairwise sequence analysis, the highest sequence similarity/identity for Rb-IRF5 was observed with the IRF5 gene from turbot fish (>87%) and Japanese flounder (83%). Several important putative transcription factor-binding sites shared by the IRF gene family, including the NF-κB, Ap-1, IRF-1, and ICSBP/ISRE sites, were found in the 5' flanking region of Rb-IRF5. The predicted tertiary structure of the dimerized IAD and VAD of the Rb-IRF5 protein resembled that of its orthologs from humans. In healthy rock bream, the highest constitutive expression of Rb-IRF5 was detected in the liver. After iridovirus and polyinosinic-polycytidylic acid (poly(I:C)) challenge, Rb-IRF5 expression was significantly induced in the head kidney. Furthermore, rock bream recombinant type I interferon (Rb-IFN1) was also found to be an efficient inducer of Rb-IRF5 in a head kidney primary cell culture model. Upon IRF5 transfection, rock bream Mx (Rb-Mx), interferon I (Rb-IFN1) and tumor-necrosis factor α (Rb-TNFα) genes get significantly upregulated in rock bream heart cells. The findings of the present study explain the involvement of Rb-IRF5 in the induction of interferons and pro-inflammatory cytokines and thereby provide a model for how IRF5 modulates immune responses against viral infections in rock bream. © 2014 Elsevier Ltd.


Bathige S.D.N.K.,Jeju National University | Whang I.,Jeju National University | Umasuthan N.,Jeju National University | Lim B.-S.,Jeju National University | And 4 more authors.
Fish and Shellfish Immunology | Year: 2012

The interferon regulatory factor (IRF) members IRF4 and IRF8 contribute to B-lymphocyte development and can act as regulators of immunoglobulin (Ig) light chain gene transcription. These two IRFs are closely interrelated and are expressed at high levels in the lymphoid and myeloid cells of the immune system. In this study, the complete cDNA and genomic sequences of rock bream IRF4 (RbIRF4) and IRF8 (RbIRF8) were identified by homology screening of a multi-tissue normalized cDNA library and a BAC library, respectively, which had been established using Roche 454 GS-FLX™ technology. The full-length RbIRF4 cDNA is composed of 3442 bp and encodes a polypeptide of 462 amino acids; the genomic DNA is 9262 bp in length, consisting of eight exons and seven introns. The full-length RbIRF8 cDNA is composed of 2186 bp and encodes a 422 amino acid polypeptide; the genomic DNA is 4120 bp in length, consisting of nine exons and eight introns. The deduced amino acid sequences of RbIRF4 and RbIRF8 include a conserved DNA-binding domain (DBD) encompassing a tryptophan pentad-repeat and an IRF-association domain (IAD). Several putative transcription factor binding sites were also identified in 5' flanking region of both RbIRF4 and RbIRF8, and include those of immune-related factors. Quantitative real time PCR analysis of healthy rock bream detected the highest expression levels of RbIRF4 and RbIRF8 in lymphomyeloid-rich tissues. In addition, viral (rock bream iridovirus) and bacterial (Edwardsiella tarda and Streptococcus iniae) infection stimulated RbIRF4 and RbIRF8 expressions in head kidney and spleen. These results suggest not only that RbIRF4 and RbIRF8 may have a protective function against virus and bacteria pathogen invasion in rock bream, but also that IRFs may be immunomodulatory factors of teleost fish. © 2012 Elsevier Ltd.


Bathige S.D.N.K.,Jeju National University | Whang I.,Jeju National University | Umasuthan N.,Jeju National University | Wickramaarachchi W.D.N.,Jeju National University | And 4 more authors.
Fish and Shellfish Immunology | Year: 2013

Complement component 1q (C1q) is a subcomponent of the C1 complex and the key protein that recognizes and binds to a broad range of immune and non-immune ligands to initiate the classical complement pathway. In the present study, we identified and characterized three novel C1q family members from rock bream, Oplegnathus fasciatus. The full-length cDNAs of C1q A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like (RbC1qCL) consist of 780, 720 and 726bp of nucleotide sequence encoding polypeptides of 260, 240 and 242 amino acids, respectively. All three RbC1qs possess a leading signal peptide and collagen-like region(s) (CLRs) in the N-terminus, and a C1q domain at the C-terminus. The C1q characteristic Gly-X-Y repeats are present in all three RbC1qs, while the CLR-associated sequence that enhances phagocytic activity is present in RbC1qAL (49GEKGEP54) and RbC1qCL (70GEKGEP75). Moreover, the coding region was distributed across six exons in RbCqAL and RbC1qCL, but only five exons in RbC1qBL. Phylogenetic analysis revealed that the three RbC1qs tightly cluster with the fish clade. All three RbC1qs are most highly expressed in the spleen and liver, as indicated by qPCR tissue profiling. In addition, all three are transcriptionally responsive to immune challenge, with liver expression being significantly up-regulated in the early phase of infection with intact, live bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus) and in the late phase of exposure to purified endotoxin (lipopolysaccharide). These data collectively suggest that the RbC1qs may play defense roles as an innate immune response to protect the rock bream from bacterial and viral infections. © 2013 Elsevier Ltd.


Bathige S.D.N.K.,Jeju National University | Umasuthan N.,Jeju National University | Saranya Revathy K.,Jeju National University | Lee Y.,Jeju National University | And 5 more authors.
Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology | Year: 2014

Glutathione S-transferases (GSTs) are enzymes that catalyze xenobiotic metabolism in the phase II detoxification process. GSTs have a potential for use as indicators or biomarkers to assess the presence of organic and inorganic contaminants in aquatic environments. In this study, a full-length cDNA of a mu (μ) class GST (RpGSTμ) was identified from Manila clam (Ruditapes philippinarum) and biochemically characterized. The 1356 bp of the cDNA included an open reading frame of 651 bp encoding a polypeptide of 217 amino acid residues with a molecular mass of 25.04 kDa and an estimated pI of 6.34. Sequence analysis revealed that the RpGSTμ possessed several characteristic features of μ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione (GSH), a C-terminal domain containing substrate binding sites, and a μ loop. The recombinant RpGSTμ (rRpGSTμ) protein exhibited GSH-conjugating catalytic activity towards several substrates, and significantly strong activity was detected against 4-nitrophenethyl bromide (5.77 ± 0.55) and 1-chloro-2,4-dinitrobenzene (CDNB, 3.19 ± 0.05). Kinetic analysis as a function of GSH and CDNB concentrations revealed relatively low Km values of 1.03 ± 0.46 mM and 0.56 ± 0.20 mM, respectively, thereby indicating a GSH-conjugation attributed with high rates. The optimum pH and temperature for the catalytic activity of the rRpGSTμ protein were 7.7 and 37 °C, respectively. The effect of two inhibitors, Cibacron blue and hematin, on the activity of rRpGSTμ was evaluated and the IC50 values of 0.65 μM and 9 μM, respectively, were obtained. While RpGSTμ transcripts were highly expressed in gills and hemocytes, a significant elevation in mRNA levels was detected in these tissues after lipopolysaccharide (LPS), polyinosinic- polycytidylic acid (poly I:C) and live bacterial (Vibrio tapetis) challenges. These findings collectively suggest that RpGSTμ functions as a potent detoxifier of xenobiotic toxicants present in the aquatic environment, and that its mRNA expression could be modulated by pathogenic stress signal(s). © 2014 Elsevier Inc.


Umasuthan N.,Jeju National University | Elvitigala D.A.S.,Jeju National University | Saranya Revathy K.,Jeju National University | Lee Y.,Jeju National University | And 4 more authors.
Gene | Year: 2013

Troponin C (TnC) is one of the subunits composing the troponin complex, which is primarily expressed in muscle tissue and plays a major role in regulating contractility. We have identified a novel TnC-like gene (RpTnC) from the Ruditapes philippinarum Manila clam. Sequence analysis indicated that RpTnC has a 450bp coding sequence, encoding a 150 amino acid protein with a molecular mass of 17.4kDa. The RpTnC protein consisted of four EF-hand motifs (I-IV), each with a Ca2+-binding site. In silico comparative analysis of protein sequence showed that only site IV, demonstrating a conserved stretch (DxDxSx6E), is functionally active for Ca2+-coordination. Moreover, RpTnC was homologically (61.3% identity) and phylogenetically closest to Japanese flying squid TnC. The mRNA expression analysis using quantitative real-time PCR revealed a differential basal-expression of RpTnC transcripts in six different clam tissues, with higher levels in adductor muscle and mantle. Intramuscular administration of CaCl2 caused a prominent upregulation of RpTnC transcripts in adductor muscle (~5 fold). Collectively, our findings suggest that the TnC homolog of Manila clam identified in this study may be involved in important role(s) in clam physiology, mainly in its muscle tissues, and its transcription could be significantly influenced by increased Ca2+ levels. © 2012 Elsevier B.V.


Umasuthan N.,Jeju National University | Wan Q.,Jeju National University | Revathy K.S.,Jeju National University | Whang I.,Jeju National University | And 4 more authors.
Fish and Shellfish Immunology | Year: 2014

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. Invitro 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 invivo 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. © 2014 Elsevier Ltd.

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