Ji-Min Jeong

Molecular cloning and expression analysis of a thioredoxin from rock bream, Oplegnathus fasciatus, and biological activity of the recombinant protein

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).

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 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.

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.

First description of programmed cell death10 (PDCD10) in rock bream (Oplegnathus fasciatus): Potential relations to the regulation of apoptosis by several pathogens

In this study, we isolated and characterized programmed cell death10 (PDCD10), which is known to be related to apoptosis, from rock bream (Oplegnathus fasciatus). The full-length rock bream PDCD10 (RbPDCD10) cDNA (1459 bp) contains an open reading frame of 633 bp that encodes 210 amino acids. Furthermore, multiple alignments revealed that the six of the α-helix bundles were well conserved among the other PDCD10 sequences tested. RbPDCD10 was significantly expressed in the liver, RBC (red blood cell), gill, intestine, trunk kidney and spleen. RbPDCD10 gene expression was also examined in several tissues, including the kidney, spleen, liver, and gill, under bacterial and viral challenges. Generally, all of the examined tissues from the fish that were infected with Edwardsiella tarda and the red sea bream iridovirus (RSIV) exhibited significant up-regulations of RbPDCD10 expression compared to the controls. However, RbPDCD10 expression exhibited dramatic down-regulations in all of the examined tissues following injections of Streptococcus iniae, which is major bacterial pathogen that is responsible for mass mortality in rock bream. Our results revealed that rock bream PDCD10 may be involved in the apoptotic regulation of rock bream immune responses.

Molecular characterization of a T cell co-stimulatory receptor, CD28 of rock bream (Oplegnathus fasciatus): Transcriptional expression during bacterial and viral stimulation

Abstract 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. HighlightsCD28 a T cell costimulatory receptor is identified in Rock bream.Conservation in domain and motifs shows that RbCD28 is a homolog of mammalian CD28.It was highly expressed in various teleost lymphoid organs including gill and skin.E. tarda, S. iniae and RSIV infection caused differential expression of RbCD28 in lymphoid organs.Activation of RbCD28 in lymphoid organs during infection shows its key role as a costimulatory receptor of T cells.

First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus.

CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

The first report of siglec-3/CD33 gene in a teleost (rock bream, Oplegnathus fasciatus ): An analysis of its spatial expression during stimulation to red seabream iridovirus (RSIV) and two bacterial pathogens

&NA; Siglec‐3/CD33 is a myeloid‐specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG‐like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine‐based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real‐time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells.

Protective responses of two paralogs of granulocyte colony stimulating factor (GCSF) in rock bream, Oplegnathus fasciatus during bacterial and viral infection

&NA; 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. HighlightsTwo GCSF paralogs of rock bream were identified and characterized.Basal levels of GCSF‐1 and GCSF‐2 expression were high in tissues with immuno‐hematopoietic related functions.High induction of GCSF paralogs in kidney, spleen and gills of rock bream after the challenge with Streptococcus iniae.Possibilities of difference in roles or overlapping functions for GCSF paralogs were observed.

Cooperation of erythrocytes with leukocytes in immune response of a teleost Oplegnathus fasciatus

The most abundant cell type in the blood of mammals and fish is erythrocyte. Unlike mammalian erythrocyte, fish erythrocyte is nucleated. The functional differentiation of teleost erythrocyte is insufficient compared with that of mammals. Therefore, fish erythrocyte may have different functions from that of mammals. Functional interaction between erythrocyte and leukocyte was confirmed by the cDNA microarray newly constructed in this study to investigate characterization of rock bream erythrocyte. In this study, different immune related genes of erythrocytes were annotated by microarray analysis. Microarray analysis showed that a total of 338 genes were up-regulated in co-cultured erythrocytes with leukocytes by LPS stimulation when comparing to erythrocytes stimulated LPS. Many genes in erythrocyte of rock bream were up-regulated in presence of leukocytes, suggesting that erythrocytes interact with leukocytes to trigger expression of various genes associated with the immune system. Our results provide valuable information that direct and indirect immunological function of fish erythrocyte.