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Molecular dissection of Cystatin B and C from big-belly seahorse and Cyclooxygenase 2 from red-spotted grouper reveals their implications in host immune defense mechanisms

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빅벨리해마의 Cystatin B 및 C와 붉바리의 Cyclooxygenase 2의 분자유전학적 분석을 통한 숙주 면역 방어 메커니즘에 미치는 영향 연구
Aquaculture has emerged as a vital fish production practice, overtaking the captive fisheries due to insufficient wild capture to meet the rising demand for fish over the past few decades. It contributes to a substantial impact on global food security as the cheapest source of human nutrition and providing livelihood. Nevertheless, aquaculture has been combatted due to frequent outbreaks of pathogen infections. Therefore, it is important to have a deep insight into fish immune defense mechanisms, and the overall objective of the current study is to perceive the immune defense mechanisms of teleosts and thereby foster sustainable aquaculture through disease prevention. In the present study, I have identified and characterized two Cystatin homologs from big-belly seahorse and Cyclooxygenase 2 from red-spotted grouper and elucidated their roles in the innate immune system of teleost.
Big-belly seahorse (Hippocampus abdominalis) is the largest seahorse species, naturally inhabited in shallow coastal areas, such as seagrass beds and natural estuaries. They have good commercial value for the aquarium trade and Chinese traditional medicine, albeit they are highly vulnerable 0to their natural habitat loss and overexploitation due to many human interventions. Therefore, seahorse aquaculture has emerged as a good solution to overcome consumer demand while conserving the wild population. However, there are some shortcomings in seahorse aquaculture such as their high sensitivity towards the varying culture conditions and the frequent outbreak of pathogen infections.
Red spotted grouper (Epinephelus akaara) is an ideal research model and a perfect candidate for extensive aquaculture owing to its high nutritional value, rapid growth, and high market demand. However, the frequent outbreak of viral diseases, especially, Nervous Necrosis Virus, are the major drawbacks in grouper aquaculture that has been creating a huge economic loss annually.
Cystatins are natural inhibitors of lysosomal cysteine proteases, including cathepsins B, L, H, and S. Cystatin B (CSTB) is a member of the type 1 cystatin family and found to be involved in numerous pathophysiological conditions. Cystatin C (CSTC) is a member of the type 2 cystatin family and is an essential biomarker in the prognosis of several diseases. In this study, the 297-bp cystatin B (HaCSTB) and 390-bp cystatin C (HaCSTC) cDNA from big-belly seahorse were cloned and characterized by screening the pre-established cDNA library. Based on similarities in sequence, HaCSTB is a homolog of the teleostean type 1 cystatin family. HaCSTC is a homolog of the teleost type 2 cystatin family with putative catalytic cystatin domains, signal peptides, and disulfide bonds. HaCSTB and HaCSTC transcripts were ubiquitously expressed in all tested big-belly seahorse tissues, with the highest expression in blood and ovaries respectively. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae caused significant upregulation in both HaCSTB and HaCSTC transcript levels. Using a pMAL-c5X expression vector, the 10.80-kDa of recombinant HaCSTB protein (rHaCSTB) and 14.29-kDa of recombinant HaCSTC protein (rHaCSTC) were expressed in Escherichia coli BL21 (DE3), and its protease inhibitory activity against papain cysteine protease was determined with the aid of a protease substrate. Papain was competitively blocked by both rHaCSTB and rHaCSTC in a dose-dependent manner. In response to viral hemorrhagic septicemia virus (VHSV) infection, both HaCSTB and HaCSTC overexpression significantly (p < 0.05) decreased the expression of VHSV transcripts, pro-inflammatory cytokines, and pro-apoptotic genes; while increasing the expression of anti-apoptotic genes in fathead minnow (FHM) cells. Furthermore, both HaCSTB and HaCSTC overexpression protected VHSV-infected FHM cells against VHSV-induced apoptosis and increased cell viability.
Cyclooxygenases (COXs) are major biosynthetic enzymes of prostaglandins, which play a key role in the generation of inflammatory response by acting on specific cellular receptors. There are two types of COX isomers, COX-1, and COX-2. COX-1 is constitutively expressed as house-keeping gene whereas COX-2 is highly inducible in response to various external stimuli. COX-2 is the most comprehensively studied mammalian dioxygenase and commonly used in anti-inflammatory drug therapies. The cDNA sequence of COX-2 with 1827 bp length, encoding 608 amino acid sequence, was cloned from Epinephelus akaara and designated as EaCOX-2. According to in silico analysis EaCOX-2 is a teleostean homolog of myeloperoxidase superfamily with prostaglandin endoperoxide synthase domain and a membrane-binding domain. The 3D structure of EaCOX-2 was a homodimer with heme binding sites at the center of the catalytic domains. The results of pairwise alignment disclosed that EaCOX-2 possessed 99% of the highest identity and 99% of similarity with Epinephelus lanceolatus. EaCOX-2 transcripts were ubiquitously expressed in all tested, red-spotted grouper tissues, with significantly (p<0.05) the highest expression in gills. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, and nervous necrosis virus caused significant (p<0.05) upregulation in EaCOX-2 transcript levels at 12 h and 24 h post-infection in gills. The overexpression of EaCOX-2 exhibited pro-inflammatory effect through reducing FHM cell viability, activation of classical M1 type macrophage polarization and increased nitric oxide (NO) production in murine macrophage cells (RAW264.7). Collectively, our findings imply the profound roles of Cystatins and Cyclooxygenase 2 against pathogen infections in teleosts by modulating fish immune responses.
코다고다 야샤라 카빈디
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Kodagoda Yasara Kavindi
Graduate School Jeju National University
대학원 해양생명과학과
LeeJe hee
Table Of Contents
1.1. Aquaculture 13
1.2. Big-belly seahorse (Hippocampus abdominalis) 14
1.3. Red-spotted grouper (Epinephelus akaara) 17
2.1. Introduction 20
2.2. Materials and methodology 24
2.2.1. Identification and bioinformatics analysis of HaCSTB and HaCSTC 24
2.2.2. Experimental fish, tissue distribution, and immune challenge experiments 25
2.2.3. Total RNA isolation and cDNA synthesis 26
2.2.4. Analysis of spatial and temporal expression by quantitative real-time PCR (qPCR) 27
2.2.5. Cloning and construction of HaCSTB and HaCSTC expression vectors 28
2.2.6. Overexpression and purification of recombinant HaCSTB (rHaCSTB) and HaCSTC (rHaCSTC) proteins 33
2.2.7. Maintenance of FHM cell line and transfection 34
2.2.8 Subcellular localization of HaCSTB 34
2.2.9. Functional assays 35 Flow cytometry analysis of apoptotic cell percentage 38
2.3. Results 39
2.3.1. Identification and bioinformatics analysis of HaCSTB and HaCSTC 39
2.3.2. Spatial and temporal expression analysis of HaCSTB and HaCSTC 45
2.3.3. Overexpression and protein purification 48
2.3.4. Subcellular localization of HaCSTB 50
2.3.5. Functional assays 50 Papain inhibitory activity assay 50 Effect of pH and temperature effect on papain inhibitory activity 52 HaCSTB and HaCSTC overexpression protects FHM cells against VHSV infection 52 HaCSTB and HaCSTC overexpression suppresses VHSV gene expression 53 HaCSTB and HaCSTC overexpression reduces VHSV-induced apoptosis 56
2.4. Discussion 60
2.5. Conclusion 70
3.1. Introduction 73
3.2. Materials and methods 75
3.2.1. Identification and in-silico analysis of EaCOX2 75
3.2.2. Fish husbandry 76
3.2.3. Immune challenge experiment and tissue collection 76
3.2.4. RNA isolation and cDNA synthesis 77
3.2.5. EaCOX2 transcription analysis by quantitative real-time PCR (qPCR) 77
3.2.6. Construction of recombinant EaCOX-2 expression vectors, cell culture, and transfection 78
3.2.7. Functional assays 80 Determination of cell viability by WST-1 assay 80 Determination of macrophage polarization 81
3. 3. Results 83
3.3.1. Identification and sequence characterization of EaCOX2 83
3.3.2. Spatial expression pattern of EaCOX2 87
3.3.3. Temporal expression of EaCOX2 after immune challenge 88
3.3.4. the effect of EaCOX2 overexpression on FHM cell viability 89
3.3.5. The effect of EaCOX2 overexpression on apoptosis 90
3.3.6. The effect of EaCOX2 overexpression on macrophage polarization 92
3.3.7. The effect of EaCOX2 overexpression on NO production 92
3.4. Discussion 94
3.5. Conclusion 97
References 98
Graduate School Jeju National University
코다고다 야샤라 카빈디. (2023). Molecular dissection of Cystatin B and C from big-belly seahorse and Cyclooxygenase 2 from red-spotted grouper reveals their implications in host immune defense mechanisms.
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General Graduate School > Marine Life Sciences
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