Ju-Won Kim

Molecular identification and expression analysis of two distinct BPI/LBPs (bactericidal permeability-increasing protein/LPS-binding protein) from rock bream, Oplegnathus fasciatus

We identified two cDNAs designated as RbBPI/LBP-1 and RbBPI/LBP2, respectively, which were identified by expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The two cDNA displayed 36.9% identity at the translated amino acid level. Despite the low level of identity between the two genes, high conservation was seen in the BPI/LBP/CETP N-terminal, LPS-binding, the proline-rich central and the BPI/LBP/CETP C-terminal domains. The full-length RbBPI/LBP-1 cDNA (1945 bp) contained an open reading frame (ORF) of 1431 bp encoding 476 amino acids. The full-length RbBPI/LBP-2 cDNA was 2652 bp in length and contained an ORF of 1422 bp encoding 473 amino acids. RbBPI/LBP-1 was significantly expressed in the spleen, liver, intestine and gill. On the other hand, RbBPI/LBP-2 showed significant expression in the kidney, peripheral blood leukocytes, and spleen. Real-time RT-PCR was used to examine RbBPI/LBP-1 and RbBPI/LBP-2 mRNA expression in kidney under conditions of bacterial and viral challenge. Experimental infection of rock bream with Streptococcus iniae, Edwardsiella tarda, and red sea bream iridovirus resulted in significant increases in RbBPI/LBP-1 and RbBPI/LBP-2 mRNA levels in the kidneys, however, the increases in transcription was seen at different time points.

Molecular characterisation and expression analysis of a fish-egg lectin in rock bream, and its response to bacterial or viral infection

A fish-egg lectin, RbFEL, was identified from rock bream (Oplegnathus fasciatus) and its expression analysed. In both vertebrates and invertebrates, carbohydrate-binding proteins (lectins) play an important role in innate immunity against microbial invasion. Here, we report the cloning of a fish-egg lectin from rock bream using a combination of expression sequence tag (EST) analyses. The full-length cDNA of RbFEL is composed of 1512-bp with a 780-bp ORF that encodes 259 amino acid residues. The deduced polypeptide exhibits six conserved residues of the FEL family. All cysteine positions in each domain were completely conserved. Reverse transcription quantitative real-time PCR analysis of the tissues revealed that RbFEL mRNA was abundantly expressed in liver, moderately expressed in head kidney and rarely expressed in other tissues. Expression analyses of time series sampled fertilised eggs showed that expression gradually increased 1, 3, 12, 24 and 36 h after fertilisation. In addition, RbFEL expression was significantly up-regulated in rock bream challenged with Edwardsiella tarda, Streptococcus iniae and red sea bream iridovirus (RSIV). These results suggest that RbFEL is a member of the egg-lectin family and is involved in the innate immune response in rock bream.

Molecular characterization, expression, and functional analysis of two thioredoxins in the black rockfish (Sebastes schlegelii).

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.

Molecular characterisation and biological activity of a novel CXC chemokine gene in rock bream (Oplegnathus fasciatus)

Chemokines are chemoattractant cytokines defined by the presence of four conserved cysteine residues. In mammals, these cytokines can be divided into four subfamilies depending on the arrangement of the first two conserved cysteines in the sequence, and include the CXC(α), CC(β), C(γ), and CX3C(δ) classes. We identified CXC chemokine cDNA, designated RbCXC, isolated using expressed sequence tag analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCXC cDNA (742 bp) contained an open reading frame of 342 bp encoding 114 amino acids. Results from phylogenetic analysis showed that RbCXC was strictly separated into a distinct clade compared to other known CXC chemokine subgroups. RbCXC was significantly expressed in the trunk kidney, liver, spleen, gill, peripheral blood leukocytes (PBLs), and head kidney. Rock bream PBLs were stimulated with several mitogens, including LPS and polyinosinic-polycytidylic acid (poly I:C), which significantly induced the expression of RbCXC mRNA. RbCXC mRNA expression was examined in several tissues under conditions of bacterial and viral challenge. Experimental challenges revealed that all examined tissues from fish infected with Edwardsiella tarda and red sea bream iridovirus showed significant increases in RbCXC expression compared to the control. In the case of Streptococcus iniae infection, RbCXC mRNA expression was markedly upregulated in the kidney, spleen, and liver. In addition, a maltose binding protein fusion recombinant RbCXC (~53 kDa) was produced in an Escherichia coli expression system and purified. Subsequently, the addition of purified recombinant RbCXC (rRbCXC) to kidney leukocytes was examined to investigate the impact of proliferative and chemotactic activity. The rRbCXC induced significant kidney leukocyte proliferation and attraction at concentrations ranging from 10 to 300 μg/mL, suggesting that it can be utilised as an immune stimulant and/or molecular adjuvant to enhance the immunological effects of vaccines.

Cloning, characterisation, and expression analysis of the cathepsin D gene from rock bream (Oplegnathus fasciatus).

Cathepsins are lysosomal cysteine proteases belonging to the papain family, members of which play important roles in normal metabolism for the maintenance of cellular homeostasis. Rock bream (Oplegnathus fasciatus) cathepsin D (RbCTSD) cDNAs were identified by expressed sequence tag analysis of a lipopolysaccharide-stimulated rock bream liver cDNA library. The full-length RbCTSD cDNA (1644 bp) contained an open reading frame of 1191 bp encoding 396 amino acids. Alignment analysis revealed that the active sites and N-glycosylation sites of the deduced protein were well conserved. Phylogenetic analysis revealed that RbCTSD is most closely related to the Mi-iuy croaker (Miichthys miiuy) cathepsin D. RbCTSD was ubiquitously expressed in all the examined tissues, predominantly in muscle and kidneys. RbCTSD mRNA expression was also examined in several tissues under conditions of bacterial and viral challenge. All examined tissues of fish infected with Edwardsiella tarda (E. tarda), Streptococcus iniae (S. iniae), and red sea bream iridovirus (RSIV) showed significant increases in RbCTSD expression compared with the control. In the kidney and spleen, RbCTSD mRNA expression was markedly upregulated following infection with all tested pathogens. These findings indicate that RbCTSD plays an important role in the innate immune response of rock bream. Furthermore, these results provide important information for the identification of other cathepsin D genes in various fish species.

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

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 1204 bp with a 945-bp open reading frame (ORF) that encoded a 314 amino-acid protein, while that of RbFTL-2 consisted of 1614 bp 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.

Expression analysis and biological activity of moronecidin from rock bream, Oplegnathus fasciatus.

The piscidin-family, one of antimicrobial peptides (AMPs) mainly distributed in fish, is crucial effectors of fish innate immune response. Piscidin-family typically has broad-spectrum antimicrobial activity and the ability to modulate the immune response. In this study, we identified moronecidin (Rbmoro) included in piscidin-family from rock bream and investigated its gene expression using quantitative real-time PCR and biological activity (including antimicrobial and cytotoxic activity). The coding region of Rbmoro was 204 bp encoding 67 amino acid residues. Tertiary structure prediction of Rbmoro showed an amphipathic α-helical structure. Rbmoro gene was widely expressed in different tissues of healthy fish. Additionally, Rbmoro gene expression was induced in all tested tissues after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. We synthesized mature peptide of Rbmoro based on amino acid sequence of its AMP 12 domain, and the synthetic peptide appeared broad-spectrum antimicrobial activity to various bacteria. However, the synthetic peptide has weak haemolytic activity against fish erythrocytes. These results suggest that Rbmoro might play an important role in innate immune response of rock bream.

Piscidin: Antimicrobial peptide of rock bream, Oplegnathus fasciatus

The piscidin family consists of antimicrobial peptides (AMPs) that are mainly found in fish and are crucial effectors of fish innate immune responses. The piscidin family typically has broad-spectrum antimicrobial activity and can modulate immune responses. In this study, we cloned rock bream piscidin (Rbpisc) and investigated its gene expression and biological activity (including antimicrobial and cytotoxic activities). The coding region of Rbpisc consisted of 213 base pairs (bp) encoding 70 amino acid residues. The tertiary structure predicted for Rbpisc includes an amphipathic helix-loop-helix structure. The Rbpisc gene was highly expressed in the gills of healthy fish. The gene expression of Rbpisc increased in the gills after pathogen infection, while the expression was down-regulated in other tissues. A synthetic peptide based on the AMP 12 domain amino acid sequence of Rbpisc appeared to have broad-spectrum antimicrobial activity against various bacteria. However, the synthetic peptide exhibited weak haemolytic activity against fish erythrocytes. These results suggest that Rbpisc might play an important role in the innate immune responses of rock bream.

Functional characterisation and expression analysis of recombinant serum amyloid P isoform 1 (RbSAP1) from rock bream (Oplegnathus fasciatus).

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.

Microarray analysis of gene expression in peripheral blood leucocytes from rock bream (Oplegnathus fasciatus) after stimulation by LPS, ConA/PMA, and poly I:C

The responses of peripheral blood leucocytes (PBLs) from the rock bream Oplegnathus fasciatus stimulated with lipopolysaccharides (LPS), concanavalin A/phorbol myristate acetate (Con A/PMA), and/or polyriboinosinic polyribocytidylic acid (poly I:C) were investigated using a cDNA microarray consisting of 2,400 clones. A total of 336 unique genes were more than twofold up-regulated: 169 in LPS-stimulated PBLs, 209 in Con A/PMA-stimulated PBLs, and 146 in poly I:C-stimulated PBLs. Moreover, other genes were similarly up-regulated with combinations of stimulants: 85 genes in PBLs stimulated with both LPS and Con A/PMA, 79 in PBLs stimulated with both LPS and poly I:C, and 66 in PBLs stimulated with both Con A/PMA and poly I:C. Furthermore, 42 other genes were more than twofold up-regulated in PBLs stimulated with all tested mitogens. Other genes were not significantly induced. Overall, these results suggest that certain genes in rock bream play important roles in physiological and pathological processes. Furthermore, the microarray analysis showed promise as a tool for investigating immune mechanisms in teleost fish.