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Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP-like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP-like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aquaculture industry in Korea has grown rapidly since the 1960s, and it is a major food source. However, the expansion of aquaculture systems has increased the chances of infectious disease outbreaks, and vaccination plays an important role in commercial fish farming. This is the first comprehensive review of commercial fish vaccines in Korea. It not only provides an overview of commercially available fish vaccines and their associated approval processes and laws, but also some perspectives on research advances regarding fish vaccines in Korea. In Korea, fish vaccines are approved only after their safety and effectiveness have been verified according to the Pharmaceutical Affairs Act, and after approval, each vaccine lot must pass the national evaluation criteria. As of the end of 2019, 29 vaccines were approved for 10 fish pathogens, including both single and combination vaccines containing more than two inactivated pathogens. The approved fish vaccines consist of 2 immersion vaccines, as well as 1 intramuscular and 26 intraperitoneal vaccines, which require syringe injection. All the 29 vaccines are manufactured as formalin-inactivated vaccines; 1 is an adjuvant vaccine and 28 are non-adjuvant vaccines; 25 are bacterial vaccines, 2 are viral vaccines, 1 is a parasite vaccine, and 1 is a parasite and bacterial vaccine. In terms of the target fish species, 27 vaccines are used in the olive flounder (Paralichthys olivaceus), 1 in the starry flounder (Platichthys stellatus), and 1 in the red seabream (Pagrus major), striped beakfish (Oplegnathus fasciatus), and amberjack (Seriola quinqueradiata). This imbalance exists mostly because the olive flounder is the main farmed fish species in Korea. In 2018, 67.71 million vaccine doses were distributed following satisfactory performance in the national evaluation. They were used to vaccinate approximately 80.6% of farmed olive flounders., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Atypical chemokine receptor 4 (ACKR4) is regulated by cytokines, binds chemokines and regulates the chemokine gradient. We verified the cDNA sequence by confirming ACKR4 from red sea bream (PmACKR4) by next generation sequencing (NGS) and analysed the molecular characteristics and gene expression profile. In the analysis using the predicted amino acid sequence of PmACKR4, a highly conserved G protein-coupled receptor 1 region and two cysteine residues were identified and included in the ACKR4 teleost cluster in the phylogenetic analysis. In healthy red sea bream, PmACKR4 mRNA was expressed at the highest levels in head kidney and was upregulated in all immune -related tissues used in the experiment after challenges with Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV). These results suggest that ACKR4 is highly conserved in red sea bream and may play an important role in the immune system as previously reported. It is thought that ACKR4 acts as a regulator of immune -related cells via immune reactions after pathogenic infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Peptidoglycan recognition proteins are members of the family of pattern recognition receptors (PRRs), that play important roles in the recognition of peptidoglycan and various biological processes. In this study, we have characterized peptidoglycan recognition protein-SC2 (PGRP-SC2) in rock bream (Oplegnathus fasciatus) (RbPGRP-SC2) and analysed its expression in various tissues after pathogen challenge. A sequence alignment revealed that the residues essential to zinc binding of the deduced protein were highly conserved among all the organisms. Phylogenetic analysis revealed that RbPGRP-SC2 is most closely related to the large yellow croaker PGRP-SC2. RbPGRP-SC2 was ubiquitously expressed in all tissues analysed, predominantly distributed in muscle and skin. After challenge with microbial pathogens (Edwardsiella piscicida), Streptococcus iniae or red seabream iridovirus [RSIV]), RbPGRP-SC2 was up-regulated in all the tissues examined, especially in liver. We produced recombinant RbPGRP-SC2 (rRbPGRP-SC2) using an Escherichia coli expression system. The rRbPGRP-SC2 had agglutination activity towards both Gram-negative (E. piscicida) and Gram-positive bacteria (S. iniae). In addition, rRbPGRP-SC2 induced leukocyte apoptosis and promoted leukocyte phagocytosis. These results suggest that the RbPGRP-SC2 plays an important role in the immune system and in maintaining cellular homeostasis of rock bream., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

CD2 is expressed on the surfaces of virtually all T cells and natural killer (NK) cells. In mammals, the CD2 molecule is 50 kDa. The cytoplasmic tail of CD2 interacts with CD2-associated protein (CD2AP), which plays an important role in mediating the trigger signal in outer magnetic pole cells. In this study, we identified CD2AP from rock bream and investigated its gene expression. The ORF of CD2AP (1950 bp) encodes 650 amino acids (aa). CD2AP has a Src homology 3 (SH3) domain. Quantitative real-time PCR analysis revealed that CD2AP shows higher expression in the gills and skin. Under experimental challenge, CD2AP gene expression was increased as relative to the control after 7 days. This result will improve our understanding of blood vessels in teleost fish, and will provide a basis for the study of CD2-related genes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

CD28 is a co-stimulatory receptor that provides a critical second signal alongside T cell receptors for the activation of naive T cells. We characterized the CD28 gene of rock bream, which has a deduced amino acid sequence of 221 residues with an extracellular Ig-superfamily V domain, transmembrane region, and cytoplasmic tail. The conservation in domain structures and other motifs shows that it is highly likely that RbCD28 is a homologue of mammalian CD28 and may have related co-stimulatory functions. RbCD28 is constitutively expressed in most tissues that were analysed, with a relatively higher expression in teleost lymphoid organs, such as spleens, gills, trunk kidneys and skin. Unlike human CD28, RbCD28 is highly expressed in skin and gill-associated lymphoid organs. Although gills showed constitutive expression of RbCD28 in control animals, after a pathogen challenge, induction of CD28 was low, particularly in RSIV and E. tarda infection. Whereas induction of RbCD28 was observed in kidney during E. tarda and S. iniae infection, downregulation was observed during RSIV infection. In the case of the liver, E. tarda caused an initial upregulation of RbCD28. RbCD28 activation of T cells in the spleen was limited to S. iniae infection. Activation of RbCD28 observed in lymphoid organs during infection of various pathogens shows its key role as a co-stimulatory receptor of T cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Granulocyte colony stimulating factor (GCSF) has a key role in the production of neutrophilic granulocytes during normal hematopoietic development and release of neutrophils into the blood circulation. In this study we have identified and characterized two paralogs of GCSF (RbGCSF) in rock bream. Although RbGCSF-1 and RbGCSF-2 share low sequence conservation, its domains and protein structure still share significant similarity. Basal levels of RbGCSF-1 gene expression was high in peripheral blood leukocytes (PBLs), spleen and intestine whereas the RbGCSF-2 was highly expressed in PBLs and kidney, of healthy animals. A significant induction of RbGCSFs were observed after the challenge with Streptococcus iniae in kidney, spleen and gills during initial hours of infection. Whereas Edwarsiella tarda infection caused a reasonable expression in kidney. Red seabream iridovirus caused induction of RbGCSF-1 transcription only in gills during initial hours. This higher expression of RbGCSF in early hours may be its response to induce emergency hematopoiesis, due to shortage of neutrophils to combat the surge in pathogens. The difference in induction of RbGCSF paralogs during infection may constitute to its different roles or overlapping functions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The rapid haemostasis of fish prevents bleeding or infection that could be caused by physical properties of the aquatic environment. Additionally, the innate immune system is the first line of defence against infection and is responsible for the recognition of pathogen-associated molecular patterns, which are important for the activation of acquired immune responses. Coagulation factor II (CFII) is an important factor in the coagulation system and is involved in recognition and interaction with various bacterial and extracellular proteins. In this study, we identified and characterised the gene encoding CFII in rock bream (Oplegnathus fasciatus) (RbCFII) and analysed its expression in various tissues after a pathogen challenge. The full-length RbCFII cDNA (2079 bp) contained an open reading frame of 1854 bp encoding 617 amino acids. Alignment analysis revealed that a gamma-carboxyglutamic acid-rich domain, two kringle domains, and a trypsin-like serine protease domain of the deduced protein were well conserved. RbCFII was ubiquitously expressed in all tissues examined but, predominantly detected in the liver and skin. RbCFII expression was dramatically up-regulated in the kidney, spleen and liver after infection with Edwardsiella tarda, Streptococcus iniae, or red seabream iridovirus. The recombinant protein RbCFII (rRbCFII) produced using an Escherichia coli expression system was able to bind all examined bacteria. Interestingly, rRbCFII has agglutination activities towards E. coli and E. tarda, while no agglutination was shown toward Vibrio ordalii and S. iniae. These findings indicate that rRbCFII performs an immunological function in the immune response, and might be involved in innate immunity as well as blood coagulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lectins are carbohydrate-binding proteins that play important roles in the recognition and elimination of pathogens via the innate immune system. Pentraxins (PTX) are humoral lectins, which are multifunctional proteins in vertebrates. Pentraxins can be divided into two groups based on their primary structure: short (C-reactive protein and serum amyloid P [SAP]) and long pentraxins (PTX3 and neuronal pentraxins). Previously, SAP was shown to have Ca(2+)-dependent binding specificity for various ligands and to be a major acute phase protein. In this study, we identified and characterised the gene encoding SAP isoform 1 in rock bream (Oplegnathus fasciatus) (RbSAP1) and analysed its expression in various tissues after a pathogen challenge. An alignment analysis conducted based on the deduced amino acid sequence of RbSAP1 (1918 bp full-length cDNA with a 699 bp open reading frame encoding 232 amino acids) and SAPs and PTXs isolated from other organisms, revealed that the pentraxin domain and cysteine residues of the deduced protein are conserved. RbSAP1, which was ubiquitously expressed in all tissues examined, was predominantly detected in head kidney, trunk kidney, peripheral blood leukocytes, and gills. RbSAP1 expression was dramatically up-regulated in the kidney and liver after infection with Edwardsiella tarda, Streptococcus iniae, or red seabream iridovirus. Purified rRbSAP1 was able to bind Gram-negative bacteria, Gram-positive bacteria, and pathogen-associated molecular patterns. Interestingly, rRbSAP1 aggregated Gram-negative bacteria in the presence of Ca(2+). The anti-pathogen activity of rRbSAP1 suggests that SAP functions in innate immunity in the rock bream., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Adjuvant is an immune enhancer commonly used during vaccination to enhance the host immune response. In the present study, we analysed the recombinant protein produced from rock bream thioredoxin 1 cDNA (rRbTRx1). To verify the immune-stimulatory effect of this recombinant protein, changes in the expression level of several genes were investigated using the cDNA microarray chips in rock bream peripheral blood leukocytes stimulated with rRbTRx1. Furthermore, the immune responses of rock bream to Streptococcus iniae FKC (formalin-killed cell) vaccination alone or in combination with recombinant proteins were analysed after an experimental challenge with living S. iniae. Microarray analysis showed that 237 unique genes were upregulated more than two-fold after rRbTRx1 stimulation. Serum agglutination titres were relatively low; however, the FKC vaccine still conferred protection against S. iniae. Moreover, the adverse effects showed no statistically significant difference between fish injected with a concentration and non-injected fish. After experimental challenge to the rock bream by injection with living bacteria (S. iniae), the relative percent survival in the vaccinated groups with FKC + rRbTRx1 was significantly higher than that of the vaccinated groups with FKC alone, which were 85.9% and 68.2%, respectively. This indicated that the recombinant protein as an adjuvant showed synergism with the injected vaccine in rock bream., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cathepsins are lysosomal cysteine proteases belonging to the papain family, whose members play important roles in normal metabolism for the maintenance of cellular homeostasis. Rock bream (Oplegnathus fasciatus) cathepsin H (RbCTSH) cDNAs were identified by expressed sequence tag analysis of a lipopolysaccharide-stimulated rock bream liver cDNA library. The full-length RbCTSH cDNA (1326 bp) contained an open reading frame of 978 bp encoding 325 amino acids. The presence of an ERFNIN-like motif was predicted in the propeptide region of RbCTSH. Furthermore, multiple alignments showed that the EPQNCSAT region was well conserved among other cathepsin H sequences. Phylogenetic analysis revealed that RbCTSH is most closely related to Nile tilapia cathepsin H. RbCTSH was expressed significantly in the intestine, spleen, head kidney and stomach. RbCTSH mRNA expression was also examined in several tissues under conditions of bacterial and viral challenge. All examined tissues of fish infected with Edwardsiella tarda, Streptococcus iniae and red sea bream iridovirus (RSIV) showed significant increases in RbCTSH expression compared to the control. In the kidney and spleen, RbCTSH mRNA expression was upregulated markedly following infection with bacterial pathogens. These findings indicate that RbCTSH plays an important role in the innate immune response of rock bream. Furthermore, these results provide important information for the identification of other cathepsin H genes in various fish species., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Thioredoxins (TRxs) are a family of small evolutionarily conserved proteins that are essential for the maintenance of cellular homeostasis. Two TRx homologue cDNAs were isolated from a black rockfish concanavalin A (Con A)/phorbol myristate acetate (PMA)-stimulated leucocyte cDNA library and named BrTPx1-1 and BrTPx1-2. As compared with other known TRx peptide sequences, the most conserved regions of both BrTRx1-1 and BrTRx1-2 peptides were found to be the redox-active site Trp-Cys-X-X-Cys (WCXXC). The TRx present in most species is a TRx1-2 protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in the larger 13 kDa BrTRx1-1 protein, a Cys-Pro-Pro-Cys (CPPC) active site was identified. Here, we report the identification of a new member of the TRx protein family from the teleost black rockfish, which defines a new subclass of 13-kDa TRx1-1 proteins. Phylogenetic analysis indicated that both BrTRx1-1 and BrTRx1-2 were grouped with other vertebrate TRx1 peptides. BrTRx1-1 expression was strongly induced in peripheral blood leucocytes (PBLs) 12-24 h following Con A/PMA stimulation, with peak expression at 24 h post-stimulation. BrTRx1-2 was induced in PBLs after stimulation with lipopolysaccharide (LPS), Con A/PMA, or poly I:C at 24 h. The BrTRx1-1 gene was predominantly expressed in the liver and gills, while BrTRx1-2 was expressed in PBLs and gills. After treatment with a high concentration (10 μg/mL) of rBrTRx1-1 or rBrTRx1-2, kidney leucocytes exhibited increased cell proliferation and viability under oxidative stress., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cathepsins are lysosomal cysteine proteases belonging to the papain family, members of which play important roles in normal metabolism for maintenance of cellular homeostasis. Rock bream (Oplegnathus fasciatus) cathepsin O and S (RbCTSO and RbCTSS, respectively) cDNAs were identified by expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCTSO cDNA (1698 bp) contained an open reading frame (ORF) of 1017 bp encoding 338 amino acids. The full-length RbCTSS cDNA was 1401 bp in length and contained an ORF of 1014 bp encoding 337 amino acids. RbCTSO was significantly expressed in the liver, peripheral blood lymphocytes (PBLs) and spleen. On the other hand, RbCTSS showed significant expression in the liver, trunk kidney, muscle and gills. Real-time RT-PCR was used to examine RbCTSO and RbCTSS mRNA expression in several tissues (kidney, spleen, liver and gill) under conditions of bacterial and viral challenge. Experimental infection of rock bream with Streptococcus iniae and red sea bream iridovirus (RSIV) resulted in significant increases in RbCTSO and RbCTSS mRNA levels in the tissues., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Thioredoxins (TRxs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. TRx1, which contains a conserved redox-active site, Cys-Gly-Pro-Cys, is a proinflammatory cytokine, B cell growth factor, macrophage migration inhibiting factor (MIF), and an immune regulatory cytokine. The TRx1 homologue cDNA was isolated from the rock bream LPS-stimulated liver cDNA library, RbTRx1. RbTRx1 consists of 730 bp full-length cDNA with a 324 bp open reading frame encoding 108 amino acids. When compared with other known TRx1 peptide sequences, the most conserved region of the RbTRx1 peptide was the redox-active site Cys-Gly-Pro-Cys. Phylogenetic analysis grouped the RbTRx1 with other vertebrate TRx1 peptides. Quantitative real-time PCR analysis revealed the presence of RbTRx1 transcripts in liver, gill, kidney, and muscle. The expression of RbTRx1 mRNA in kidney leukocytes was upregulated after bacterial and viral challenge. The kidney leukocytes were treated with a high concentration of rRbTRx1, which significantly enhanced cell proliferation (1 μg/ml and 10 μg/ml) and viability under oxidative stress (10 μg/ml)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)