Minyoung Oh

Characterization of MIF family proteins: MIF and DDT from rock bream, Oplegnathus fasciatus

Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule playing vital roles in various signaling cascades, including cell proliferation, and activation of immune responses against infections. It is well known as a pivotal regulator of innate immunity. In this study, we have rescued and characterized two members of the MIF family, macrophage migration inhibitory factor (OfMIF) and D-Dopachrome tautomerase (OfDDT) from rock bream, Oplegnathus fasciatus. The deduced OfMIF and OfDDT protein sequences revealed the presence of the catalytic oxidoreductase (CXXC), motif. They also possessed highly conserved proline (P(2)) and lysine residues (K(33)), responsible for their isomerase and tautomerase functions. Rock bream MIF and DDT homologues shared higher identity with fish homologues and also with mammals and occupied a distinct position in the phylogenetic tree, depicting their evolutionary conservation. The spatial expression analysis revealed the highest expression of both OfMIF and OfDDT in liver, while portraying constitutive expression in other tissues. The recombinant proteins purified using the Escherichia coli system revealed potent oxidoreductase activity against insulin with both dithiothreitol and glutathione as reducing agents. Stimulation of rock bream head kidney cells with recombinant OfMIF and OfDDT proteins induced the expression of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and interleukin-1β (IL-1β). These results together suggest their involvement in rock bream immune defense and this study on the novel MIF family member DDT from rock bream will pave the way for further studies of this homologue in other teleosts and delineate its multiple functions.

Two distinct CXC chemokine receptors (CXCR3 and CXCR4) from the big-belly seahorse Hippocampus abdominalis: Molecular perspectives and immune defensive role upon pathogenic stress

&NA; CXC chemokine receptor 3 (CXCR3) and 4 (CXCR4) are members of the seven transmembrane G protein coupled receptor family, involved in pivotal physiological functions. In this study, seahorse CXCR3 and CXCR4 (designated as HaCXCR3 and HaCXCR4) cDNA sequences were identified from the transcriptome library and subsequently molecularly characterized. HaCXCR3 and HaCXCR4 encoded 363 and 373 amino acid long polypeptides, respectively. The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors, including seven transmembrane domains and a G protein coupled receptors family 1 profile with characteristic DRY motifs. Amino acid sequence comparison and phylogenetic analysis of these two CXC chemokine receptors revealed a close relationship to their corresponding teleost counterparts. Quantitative real time PCR analysis revealed that HaCXCR3 and HaCXCR4 were ubiquitously expressed in all the tested tissues, with highest expression levels in blood cells. The seahorse blood cells and kidney HaCXCR3 and HaCXCR4 mRNA expressions were differently modulated when challenged with Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid, confirming their involvement in post immune responses. HighlightsFull length coding sequences of CXC chemokine receptor 3 and 4 from Seahorse (HaCXCR3 and HaCXCR4) were identified.The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors.The HaCXCR3 and HaCXCR4 mRNA was significantly up‐regulated upon live pathogens and PAMPs in blood and kidney.

Molecular characterization and expression analysis of big-belly seahorse (Hippocampus abdominalis) interleukin-10 and analysis of its potent anti-inflammatory properties in LPS-induced murine macrophage RAW 264.7 cells

Interleukin-10 (IL-10) is a pleiotropic cytokine involved in the regulation of innate and adaptive immunity. In this study, IL-10 from big-belly seahorse (Hippocampus abdominalis) (HaIL-10) was characterized based on its molecular and functional aspects. The coding sequence of HaIL-10 is 570 bp in length and encodes a 189-amino acid residue protein (calculated molecular weight, 21.89 kDa). The deduced amino acid sequence comprises a typical signal peptide and a mature peptide domain sequence carrying four conserved Cys residues and two additional Cys residues specific to fish. Phylogenetic analysis indicated an evolutionary relationship between HaIL-10 and its counterparts in other vertebrates, with close clustering to the fish-specific homologs. Recombinant HaIL-10 (rHaIL-10) significantly reduced nitric oxide (NO) production by lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells in a concentration-dependent manner but had no effect on cell viability, suggestive of its involvement in immune response. The protein expressions of iNOS and COX-2 were significantly reduced by rHaIL-10 in LPS-induced murine macrophages RAW 264.7 cells. HaIL-10 mRNA expression was observed in all analyzed tissues, with the maximum expression being noted in the kidney and ovary. However, transcriptional levels of HaIL-10 were significantly higher in the blood, gill, and intestine upon in vivo induction with LPS, polyinosinic:polycytidylic acid [poly (I:C)], and Streptococcus iniae. To summarize, our findings help in the improved understanding of the biological functions of HaIL-10 and modulation of HaIL-10 mRNA expression in response to immune stress.

A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a pro-inflammatory chemokine

Abstract Chemokines are a family of chemotactic cytokines that regulate leukocyte migration. They are classified into four groups namely, CXC, CC, C and CX3C, based on the formation of a disulfide bridge. Among these, CXC chemokines have been identified as the largest group of chemokines in humans. In this study, we identified and functionally characterized a homolog of CXC chemokine from the big‐belly seahorse, Hippocampus abdominalis, and designated it as ShCXCL. The cDNA of ShCXCL composed of a 342‐bp open reading frame encoding 113 amino acids (aa). The CXC family‐specific small cytokine domain (SCY) was identified from the mature peptide region, which comprised of a conserved CXC motif. As ShCXCL lacks an ELR (Glutamic acid‐Leucine‐Arginine) motif, it belongs to ELR− subfamily. The recombinant ShCXCL protein strongly induced the nitric oxide (NO) production in macrophage cells (RAW 264.7 cell line) and showed the chemotactic effect on flounder peripheral blood leukocytes. Tissue profiling showed a ubiquitous expression pattern in all examined tissues, with a high abundance in spleen. The up‐regulated mRNA expression pattern of ShCXCL was observed in blood and kidney tissues after immune stimulation by live bacteria, such as Streptococcus iniae and Edwardsiella tarda, and mitogens, such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly I:C), suggesting its important role in host immune defense against microbial infection. HighlightsA CXC chemokine gene (ShCXCL) with CXC family features was identified.ShCXCL transcripts were constitutively expressed with highest expression in spleen.Modulated transcription of ShCXCL upon challenges revealed its role in host immunity.rShCXCL strongly induced the NO production in RAW 264.7 cells.rShCXCL showed an effective chemotactic activity towards leukocytes.

Molecular Genetic Characterization and Analysis of Glucocorticoid Receptor Expression in the Big-belly Seahorse Hippocampus abdominalis

스트레스란 다양한 자극에 대하여 나타나는 체내의 비특이적 반응이다(Selye, 1973). 어류는 상처와 포식활동, 질병, 오염물 질과 같은 다양한 스트레스 요인에 노출되어 있으며, 과거부터 스트레스가 어류에 미치는 영향에 대한 많은 연구가 보고되었 다(Barton and Iwama, 1991; Gamperl et al., 1994; Reid et al., 1998; Tort, 2011). 스트레스가 중추신경계에 전달되면 부신 피 질자극호르몬 방출호르몬(corticotrophin-releasing hormone, CRH)이 뇌하수체에서 부신 피질자극호르몬(adrenocorticotropic hormone, ACTH)을 분비시키고, ACTH는 간신세포에 서 글루코코르티코이드(glucocorticoid)의 방출을 촉진시킨다. 글루코코르티코이드는 지질성분으로 세포 막을 자유롭게 통과 하다 글루코코르티코이드 수용체(glucocorticoid receptor)와 결합함으로써 음성 또는 양성 조절되며 스트레스에 대항하여 생체의 항상성을 유지하는데 중요한 역할을 한다(Mommsen et al., 1999). 에너지원인 혈당량을 증가시키고, 면역체계에서는 염증 관련 유전자의 전사를 억제하며 그 결과 염증부위로의 백 혈구 이동, 사이토카인 유전자 및 고착분자의 발현이 억제되어 항 염증 작용을 나타낸다(Cato and Wade, 1996; De Bosscher et al., 2000). 그뿐만 아니라 염류세포와 Na+, K+-ATPase 활성 에 관여해 이온과 삼투압을 조절하며, 성장과 번식을 억제하는 빅벨리해마(Hippocampus abdominalis) 글루코코르티코이드 수용체의 분자 유전학적 동정과 발현 분석

Characterization of Mitochondrial Heat Shock Protein 75 (mtHSP75) of the Big-belly Seahorse Hippocampus abdominalis

Mitochondrial heat shock protein 75 (mtHSP75) is a member of the HSP90 family and plays essential roles in refolding proteins of the mitochondrial matrix. Mitochondria provide energy in the form of ATP and generate reactive oxygen species (ROS). Heat shock proteins (HSPs) are activated in response to stress, and protect cells. In this study, we characterized the mtHSP75 of the big-belly seahorse Hippocampus abdominalis. The protein (BsmtHSP75) is encoded by an open reading frame (ORF) of 2,157 nucleotides, has 719 amino acids (aa), and is of molecular mass 82 kDa. BsmtHSP75 has two functional domains, a histidine kinase-like ATPase (HATPase_c) domain (123–276 aa) and an HSP90 family domain (302–718 aa). BsmtHSP75 was expressed in all tested tissues of healthy seahorses. The ovary contained the highest transcription level, followed (in order) by the blood, brain, and muscle. Pouch tissue showed the lowest expression level. The expression of BsmtHSP75 was significantly (P<0.05) up-regulated on viral or bacterial challenge, suggesting that BsmtHSP75 plays a role in the immune defense against bacterial and viral pathogens.