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Innate Immune Responses And Host Defense Mechanisms By Two Thioredoxin Like Proteins In Big-Belly Seahorse (Hippocampus abdominalis) And Terminal Complement Complexes In Redlip Mullet (Liza haematocheila)

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Abstract
Thioredoxin is a highly conserved protein found in both prokaryotes and eukaryotes. Reactive oxygen species (ROS) are produced in response to metabolic processes, radiation, metal oxidation, and pathological infections. High levels of ROS lead to cell death via autophagy. However, thioredoxin acts as an active regulatory enzyme in response to excessive ROS. Here, we performed in-silico analysis, immune challenge experiments, and functional assays of seahorse thioredoxin-like protein 1 (HaTXNL1) and thioredoxin domain-containing protein 17 (HaTXNDC17). Evolutionary identification showed that HaTXNL1and HaTXNDC17 proteins belongs to the thioredoxin superfamily comprising 289 and 123 amino acids respectively. HaTXNL1 possesses an N-terminal active thioredoxin domain and C-terminal proteasome-interacting thioredoxin domain (PITH) of TXNL1 which is a component of 26S proteasome and binds to the matrix or cell. HaTXNDC17 possesses only thioredoxin domain. Pairwise alignment results showed 99.0 % identity and similarity with Hippocampus comes. Similar to the other thioredoxins, conserved thiol-disulfide cysteine residue containing Cys-X-X-Cys motif can be found in TXNL1 and TXNDC17 sequences. HaTXNL1 comprised two N-linked glycosylation sites at 72NISA75 and 139NESD142 and HaTXNDC17 has no sites. According to the quantitative real-time polymerase chain reaction analysis from healthy seahorses, highest HaTXNL1 mRNA expression was observed in muscle, followed by ovary, brain, gill, and blood tissues. The highest spatial mRNA expressions of HaTXNDC17 were observed in the muscle, brain, and intestine. Moreover, significant temporal expression of HaTXNL1 and HaTXNDC17 was observed in blood, gill and kidney tissues after bacterial stimuli. The DPPH assay showed that the radical scavenging activity varies in a concentration-dependent manner. The insulin reduction assay demonstrated a significant logarithmic relationship with the concentration of rHaTXNL1 and rHaTXNDC17. Moreover, FHM cells treated with recombinant HaTXNDC17 significantly enhanced cellular viability under oxidative stress. Together, these results show that HaTXNL1 and HaTXNDC17 function is important for maintaining cellular redox homeostasis and that it is also involved in the immune mechanism in seahorses.
The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were detected in the TCC components, but the CCP and FIMAC domains were absent in MuC8β and MuC9. Expression analysis of four TCC genes from healthy redlip mullets showed the highest expression levels in the liver, whereas limited expression was observed in other tissues; immune-induced expression in the head kidney and spleen revealed significant responses against Lactococcus garvieae and poly I:C injection, suggesting their involvement in MAC formation in response to harmful pathogenic infections. Furthermore, the response to poly I:C may suggest the role of TCC components in the breakdown of the membrane of enveloped viruses. These findings may help to elucidate the mechanisms behind the complement system of the teleosts innate immunity.
Author(s)
Dileepa Sripal Liyanage
Issued Date
2019
Awarded Date
2019. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000008877
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
Acknowledgement III
SUMMARY . V
List of Figures . VII
List of Tables . VIII
CHAPTER 1 . 1
1. Introduction . 2
2. Materials and Methods 5
2.1. Identification of big-belly seahorse thioredoxin-like sequences 5
2.2. In silico analysis of sequences . 5
2.3. Tissue distribution and immune challenge/ Rearing of seahorses and tissue collection 7
2.4. cDNA library construction . 8
2.5. Spatial and temporal transcriptional analysis . 8
2.6. Recombinant plasmid construction 9
2.7. Protein expression and purification 10
2.8. Functional assays . 11
2.8.1. DPPH radical-scavenging assay 11
2.8.2. Insulin disulfide reduction assay . 11
2.8.3. Protective effect on the cultured cells under oxidative stress . 12
3. Results . 13
3.1. Molecular characterization . 13
3.1.1. Homology and phylogenetic analysis . 16
3.1.2. Protein structure 22
3.1.3. Analysis of mRNA expression 24
3.1.4. Expression and purification of rHaTXNL1 and rHaTXNDC17 . 28
3.2. Functional assays . 29
3.2.1. DPPH assay . 29
3.2.2. Insulin disulfide reduction assay . 31
3.2.3. The protective effect on the cultured cells under oxidative stress 32
4. Discussion . 33
CHAPTER 2 . 39
1. Introduction . 40
2. Materials and Methods 42
2.1. Database construction and isolation of the MuC6, MuC7, MuC8β, and MuC9 sequences . 42
2.2. Sequence characterization 43
2.3. Fish rearing, immune challenge, and collection of tissues . 44
2.4. RNA isolation and cDNA library construction 44
2.5. Tissue distribution and Immune challenge expression analysis of TCC genes . 45
3. Results . 46
3.1. Isolation and sequence analysis of full-length TCC genes from mullet cDNA . 46
3.2. Alignment of TCC amino acid sequences and phylogenetic analysis . 53
3.3. Spatial mRNA expression of TCC . 55
3.4. Induced expression of TCC genes after infection with LPS, Poly I:C, and L. garvieae 57
4. Discussion . 59
References . 64
Degree
Master
Publisher
제주대학교 대학원
Citation
Dileepa Sripal Liyanage. (2019). Innate Immune Responses And Host Defense Mechanisms By Two Thioredoxin Like Proteins In Big-Belly Seahorse (Hippocampus abdominalis) And Terminal Complement Complexes In Redlip Mullet (Liza haematocheila)
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General Graduate School > Marine Life Sciences
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