Seasonal variation and comparative analysis of non-specific humoral immune substances in the skin mucus of olive flounder (Paralichthys olivaceus)

Jung, T.S.,del Castillo, C.S.,Javaregowda, P.K.,Dalvi, R.S.,Nho, S.W.,Park, S.B.,Jang, H.B.,Cha, I.S.,Sung, H.W.,Hikima, J.i.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.38 No.2

The epidermal secretion of fish contains various non-specific immune substances that act as the first line of defense against invading pathogens. The present study investigated the level of mucosal antibodies, the activities of hemagglutinin and protease, and other enzymes in the skin mucus of farm reared olive flounder (Paralichthys olivaceus) for 1year, in order to gain an insight into the relationship between these mucosal immune substances and their seasonal variation. These levels varied significantly during different months of sample collection. The present study showed a positive correlation between water temperature and the level of mucosal antibodies, and an inverse relationship between the level of mucosal antibodies and the activity of mucosal hemagglutinin and protease, but no relationship between lysozyme activity and other innate immune substances. This relationship is thought to be a compensatory response in olive flounder to protect itself against pathogenic microorganisms which are inherently present in the aquatic environment.

Characterization and antiviral function of a cytosolic sensor gene, MDA5, in Japanese flounder, Paralichthys olivaceus

Ohtani, M.,Hikima, J.i.,Kondo, H.,Hirono, I.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.5

Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24-75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.

Innate immune response in the hemolymph of an ascidian, Halocynthia roretzi, showing soft tunic syndrome, using label-free quantitative proteomics

Cha, I.S.,Castillo, C.S.d.,Nho, S.W.,Hikima, J.i.,Aoki, T.,Jung, T.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 Developmental and comparative immunology Vol.35 No.8

Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome.

Molecular cloning and expression analysis of two distinct F-type lectins from the rock bream, Oplegnathus fasciatus

Park, H.J.,Kim, J.W.,Kim, E.G.,Kim, H.N.,Chae, Y.S.,Jeong, J.M.,Kim, D.H.,Park, C.I. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 Developmental and comparative immunology Vol.36 No.1

Several lectin families characterized by distinct signature sequence motifs and structural folds, such as C-type, peptidoglycan recognition protein, ficolin, pentraxins, and most recently galectins, have been implicated in immune surveillance. In this study, two distinct F-type lectins RbFTL-1 and RbFTL-2, from the rock bream (Oplegnathus fasciatus), were identified and their expression was analyzed. The full-length cDNA of RbFTL-1 was composed of 1204bp with a 945-bp open reading frame (ORF) that encoded a 314 amino-acid protein, while that of RbFTL-2 consisted of 1614bp with a 951-bp ORF encoding a 316 amino-acid protein. RbFTL-1 and RbFTL-2 mRNAs were predominately expressed in the head-kidney and in the liver, respectively. Levels of the RbFTL-1 mRNA transcript increased up to 5.0- and 2.8-fold in the head-kidney and trunk-kidney compared to the muscle, respectively, while those of the RbFTL-2 mRNA transcript increased up to 12.0-fold in liver. The expression of RbFTL-1 and RbFTL-2 were differentially up-regulated in rock bream challenged with Edwardsiella tarda, Streptococcus iniae, and RSIV, with significant increases at 1 and 3h post-challenge compared to the controls.

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.

Genomic characterization and expression analysis of complement component 8α and 8β in rock bream (Oplegnathus fasciatus)

Wickramaarachchi, W.D.N.,Whang, I.,Wan, Q.,Bathige, S.D.N.K.,De Zoysa, M.,Lim, B.S.,Yeo, S.Y.,Park, M.A.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2013 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.39 No.3

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 64bp of 5'-UTR, open reading frame (ORF) of 1794bp, which encodes a polypeptide of 598 amino acids, 212bp of 3'-UTR. The Rb-C8β gene contains 5'-UTR of 27bp, open reading frame (ORF) of 1761bp, which encodes a polypeptide of 587 amino acids, 3'-UTR of 164bp. 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.

A novel C-type lectin from abalone, Haliotis discus discus, agglutinates Vibrio alginolyticus

Wang, N.,Whang, I.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2008 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.32 No.9

Owing to its specific binding to carbohydrates, lectins play important roles in pathogen recognition and clearance in invertebrate animals. In this study, a novel C-type lectin (designated CLHd) gene was isolated from abalone, Haliotis discus discus, cDNA library. The complete cDNA sequence of the CLHd gene is 508 base pairs in length, and encodes 151 amino acids. CLHd shares a highly conserved carbohydrate recognition domain with C-type lectins from mollusk and fish. The mRNA expressions of CLHd in healthy and bacterial-challenged abalones were examined using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). CLHd mRNA transcription was up-regulated by Vibrio alginolyticus challenge and reached the maximum expression at 24h after the bacterial injection. To understand its biological activity, the recombinant CLHd gene was constructed and expressed in Escherichia coli. The recombinant CLHd specifically agglutinated V. alginolyticus at a concentration of 50μg/ml in a calcium-dependant way. Both the gene expression analysis and recombinant protein activity assay suggest that CLHd is an important immune gene involved in the recognition and elimination of pathogens in abalones.

Bombyx mori transferrin: Genomic structure, expression and antimicrobial activity of recombinant protein

Yun, E.Y.,Lee, J.K.,Kwon, O.Y.,Hwang, J.S.,Kim, I.,Kang, S.W.,Lee, W.J.,Ding, J.L.,You, K.H.,Goo, T.W. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2009 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.33 No.10

Transferrin (Tf) is a multifunctional, iron binding protein found in both vertebrates and invertebrates. Although transferrin has been suggested to play a role in innate immunity, its immunological function during infection has not been characterized. In this study, we identified and characterized Bombyx mori transferrin (BmTf). The promoter region of BmTf has numerous putative NF-κB binding sites, suggesting its possible function in innate immunity. Analysis of BmTf gene expression shows that it is highly inducible in response to a wide variety of pathogens including bacteria, fungus, and viruses. Recombinant BmTf protein produced in a baculovirus system exhibits iron binding capacity and antibacterial activity against various Gram-positive and -negative bacteria. Taken together, our results indicate that BmTf is an inducible immune effector molecule that may play an important role in pathogen clearance of insect innate immunity.

Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus)

Premachandra, H.K.A.,Wan, Q.,Elvitigala, D.A.S.,De Zoysa, M.,Choi, C.Y.,Whang, I.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.38 No.4

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone.

Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: Sequence analysis, expression profile, and a possible biological role in host immunosuppression

Ibrahim, A.M.A.,Choi, J.Y.,Je, Y.H.,Kim, Y. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2007 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.31 No.10

A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). On this process, several putative open reading frames have been proposed, among which there was a large gene family coding for protein tyrosine phosphatases (PTPs). This study analyzed the deduced amino acid sequences of 14 CpBV-PTPs in terms of conserved domains with other known polydnaviral PTPs and determined their expression patterns in diamondback moth, Plutella xylostella, parasitized by C. plutellae. The analyzed CpBV-PTPs share the common 10 motifs with classical type of PTPs. However, there are variations among CpBV-PTPs in active site sequence and phosphorylation sites. Quantitative real-time polymerase chain reaction (PCR) indicated that most PTPs in the parasitized P. xylostella were expressed from the first day of parasitization and increased the expression levels during parasitization. All 14 PTPs were expressed in both immune-associated tissues of fat body and hemocytes in the parasitized host. During last instar, the PTP enzyme activity of the parasitized P. xylostella was significantly lower than that of the nonparasitized. The reduction of the PTP activity was observed in cytosolic fraction, but not in membrane fraction. The hemocytes of parasitized P. xylostella markedly lost their spreading ability in response to a cytokine (PSP1: plasmatocyte-spreading peptide 1). The functional link between the reduced PTP activity and the suppressed hemocytic behavior was evidenced by the inhibitory effect of sodium orthovanadate (a specific PTP inhibitor) on hemocyte-spreading behavior of nonparasitized P. xylostella. These results suggest that CpBV-PTPs are expressed in the parasitized P. xylostella and affect cellular PTP activity, which may be associated with host immunosuppression.