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.

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.

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.

Immunoglobulin genes and their transcriptional control in teleosts

Hikima, J.i.,Jung, T.S.,Aoki, T. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2011 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.35 No.9

Immunoglobulin (Ig), which exists only in jawed vertebrates, is one of the most important molecules in adaptive immunity. In the last two decades, many teleost Ig genes have been identified by in silico data mining from the enormous gene and EST databases of many fish species. In this review, the organization of Ig gene segments, the expressed Ig isotypes and their transcriptional controls are discussed. The Ig heavy chain (IgH) locus in teleosts encodes the variable (V), the diversity (D), the joining (J) segments and three different isotypic constant (C) regions including Cμ, Cδ, and Cζ/τ genes, and is organized as a ''translocon'' type like the IgH loci of higher vertebrates. In contrast, the Ig light (L) chain locus is arranged in a ''multicluster'' or repeating set of VL, JL, and CL segments. The IgL chains have four isotypes; two κ L1/G and L3/F), σ (L2) and λ. The transcription of IgH genes in teleosts is regulated by a VH promoter and the Eμ3' enhancer, which both function in a B cell-specific manner. The location of the IgH locus, structure and transcriptional function of the Eμ3' enhancer are important to our understanding of the evolutional changes that have occurred in the IgH gene locus.

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.

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.

Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray

Eum, J.H.,Seo, Y.R.,Yoe, S.M.,Kang, S.W.,Han, S.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2007 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.31 No.11

In the present study, the complex gene expression responses of Plutella xylostella to microbial challenges and injury were surveyed using a newly constructed expressed sequence tag (EST) clone collection and cDNA microarray analysis. A total of 1132 P. xylostella ESTs were cloned, annotated and categorized by their putative functions; these included proteases, protease inhibitors, recognition molecules and anti-microbial peptides. GeneOntology revealed that 4% of the P. xylostella ESTs corresponded to immunity-related genes potentially involved in innate immunity. We then used microarray analysis to identify 44 genes that were differentially expressed with at least a two-fold expression difference in P. xylostella before and after pathogen challenge. Together, our EST categorization and microarray profiling analyses allowed us to identify 70 genes that should be considered candidate immune response genes, providing important new insights into the molecular events that occur during the innate immune response in P. xylostella.

Cytosolic thioredoxin from Ruditapes philippinarum: Molecular cloning, characterization, expression and DNA protection activity of the recombinant protein

Revathy, K.S.,Umasuthan, N.,Lee, Y.,Whang, I.,Kim, H.C.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.36 No.1

Thioredoxin (TRx) is a small redox protein that plays significant roles in protection against oxidative stress and in cell homeostasis by maintaining oxidized proteins in a reduced state. Here, we describe the isolation and characterization of a full-length TRx cDNA sequence from manila clam, Ruditapes philippinarum and named it as RpTRx. The full length sequence consists of 1416bp with an open reading frame of 318bp encoding for 106 amino acids. RpTRx protein harbors evolutionarily-conserved TRx active site <SUP>32</SUP>WCGPC<SUP>36</SUP>. Phylogenetic analysis revealed a close proximity of RpTRx with the orthologue in Japanese scallop, Chlamys farreri. RpTRx was found to be constitutively expressed in hemocyte, gill, mantle, foot and siphon indicating a general role in physiological processes in various tissues. With regard to a potential role in immune responses, the RpTRx mRNA was found to be up-regulated in hemocytes after bacterial (Vibrio tapetis) and lipopolysaccharide (LPS) challenge at 3h post-infection (p.i.); a wavering increase was observed up to 96h p.i. for LPS challenge and 48h p.i. for bacterial challenge. Thus, RpTRx may function as an intracellular antioxidant to protect the cells against ROS induced by LPS and bacterial challenges. Indeed, when recombinant RpTRx protein (rRpTRx) was over-expressed in Escherichiacoli Rosetta gami<SUP>TM</SUP> (DE3) cells, it was able to scavenge free radicals and protect super-coiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. In summary, RpTRx plays an essential role in cellular defense and maintenance of homeostasis in the manila clam.

Cathepsins in the kidney of olive flounder, Paralichthys olivaceus, and their responses to bacterial infection

Cha, I.S.,Kwon, J.,Mun, J.Y.,Park, S.B.,Jang, H.B.,Nho, S.W.,del Castillo, C.S.,Hikima, J.i.,Aoki, T.,Jung, T.S. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 Developmental and comparative immunology Vol.38 No.4

Cathepsin activities are responsible for mediating various pathways involved in immune response, including the apoptosis pathway, toll-like receptor (TLR) signaling, cytokine induction and activation of granule serine proteases. In the present study, we investigated cathepsin responses in the kidneys of olive flounder infected with Streptococcus parauberis, analyzing cathepsin expression using a label-free, quantitative proteomic approach in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR). In proteomic analyses, we detected cathepsin B, D, L and S proteins, noting significant decreases and increases in cathepsins B and L, respectively, with infection. Taken together with an evaluation of cathepsin B, D, F, K, L, S and X gene expression in normal and infected kidneys by qRT-PCR, our results indicate that cathepsins B, D, L and S are the dominant lysosomal proteases in the immune system of the teleostei, olive flounder. Cathepsins F, K and X were regarded as minor cathepsins.

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.