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Structural and functional delineation of two glutathione S-Transferases from Disk abalone (Haliotis discus discus) AND Two phospholipid scramblase 1–related proteins from Red lip mullet (Liza haematocheila); deciphering their transcriptional responses against immune stimulants

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Abstract
Disk abalone (Haliotis discus discus) and redlip mullet (Liza haematocheila) are two species which are very important in the aquaculture industry, especially in South Korea. However, both these key species are highly vulnerable to the spreading of infectious diseases caused by bacteria, viruses and parasites. Relatively high stock densities and poor environmental conditions in the culture farms resulted with mass mortalities ending with financial losses. Therefore, understanding the immune responses against infectious diseases in abalones and redlip mullets is essential for a sustainable development of the aquaculture industry. Therefore, in this study we have identified and studied two types of genes: Glutathione S- transferases and phospholipid scramblases from disk abalone and redlip mullet for the understanding of their characteristic features and behaviors in line with innate immune system.
Glutathione S-transferases (GSTs) are a superfamily of detoxification enzymes that primarily catalyze the nucleophilic addition of reduced glutathione to both endogenous and exogenous electrophiles. GSTs can convert toxic substances into less reactive and more hydrophilic products to facilitate their excretion. Currently, based on their primary and tertiary structures, substrate/inhibitor specificity and immunological cross-reactivity, cytosolic GSTs have been grouped into more than ten classes, which have been designated as classes α, β, δ, ζ, θ, μ, ρ, π, σ, τ, ϕ, ɛ and Ω. In this study we are dealing with GST theta (θ) and kappa (κ) revealing their molecular and transcriptional properties. Characterization of AbGST-θ revealed with 226 amino acids, 26.6 kDa of predicted molecular mass and 8.9 of theoretical isoelectric point. As illustrated in the multiple sequence alignment, eight glutathione binding sites (G-sites) and ten substrate binding sites (H-sites) were identified in the well-distinct N-terminal and C-terminal domains of AbGST-θ, respectively. AbGST-θ exhibited its highest sequence identity with Mizuhopecten yessoensis (59.1%) and the phylogenetic tree clearly positioned AbGST-θ with pre-defined GST-θ molluscan homologues. The AbGST-θ was highly expressed in digestive tract of un-challenged abalones. Upon challenge experiment, AbGST-θ showed significant modulations in their transcriptional levels depending on the time and the tissue type. The biochemical properties of AbGST-θ were identified to be 37 ℃ of optimum temperature and 7.5 of optimum pH. The determined enzyme kinetic parameters of AbGST-θ showed low affinity towards 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. Nonetheless with Cibacron blue, IC50 (half maximal inhibitory concentration) was calculated to be 0.08 μM while observing 100% inhibition with 100 μM. Furthermore, AbGST-θ resulted with significant protection ability towards H2O2, CdCl2, CuSO4 and ZnCl2 in the disk diffusion assay.
The predicted AbGSTκ protein consists of 227 amino acids, with a predicted molecular weight of 25.6 kDa and a theoretical isoelectric point (pI) of 7.78. In silico analysis reveals that AbGSTκ is a disulfide bond formation protein A (DsbA), consisting of a thioredoxin domain, GSH binding sites (G-sites), and a catalytic residue. In contrast, no hydrophobic ligand binding site (H-site), or signal peptides, were detected. AbGSTκ showed the highest sequence identity with the orthologue from pufferfish (Takifugu obscurus) (60.0%). In a phylogenetic tree, AbGSTκ clustered closely together with other fish GSTκs, and was evolutionarily distanced from other cytosolic GSTs. The predicted three-dimensional structure clearly demonstrates that the dimer adopts a butterfly-like shape. A tissue distribution analysis revealed that GSTκ was highly expressed in the digestive tract, suggesting it has detoxification ability. Depending on the tissue and time, AbGSTκ showed different expression patterns, and levels of expression, following challenge of the abalone with immune stimulants. Enzyme kinetics of the purified recombinant proteins demonstrated its conjugating ability using 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates, and suggested it has a low affinity for both substrates. The optimum temperature and pH for the rAbGSTκ GSH: CDNB conjugating activity were found to be 35°C and pH 8, respectively indicating that the abalone is well adapted to a wide range of environmental conditions. Cibacron blue (100 μM) was capable of completely inhibiting rAbGSTκ (100%) with an IC50 (half maximal inhibitory concentration) of 0.05 μM. A disk diffusion assay revealed that rAbGSTκ could significantly protect cells from H2O2, CdCl2, and ZnCl2.
Phospholipid scramblases (PLSCRs) are a family of transmembrane proteins known to be responsible for Ca2+-mediated bidirectional phospholipid translocation in the plasma membrane. Apart from the scrambling activity of PLSCRs, recent studies revealed their diverse other roles, including antiviral defense, tumorigenesis, protein– DNA interactions, apoptosis regulation, and cell activation. Nonetheless, the biological and transcriptional functions of PLSCRs in fish have not been discovered to date. Therefore, in this study, two new members related to the PLSCR1 family were identified in the red lip mullet (Liza haematocheila) as MuPLSCR1like-a and MuPLSCR1like-b, and their characteristics were studied at molecular and transcriptional levels.
Sequence analysis revealed that MuPLSCR1like-a and MuPLSCR1like-b are composed of 245 and 228 amino acid residues (aa) with the predicted molecular weights of 27.82 and 25.74 kDa, respectively. A constructed phylogenetic tree showed that MuPLSCR1like-a and MuPLSCR1like-b are clustered together with other known PLSCR1 and -2 orthologues, thus pointing to the relatedness to both PLSCR1 and PLSCR2 families. Two-dimensional (2D) and 3D graphical representations illustrated the well-known 12-stranded β-barrel structure of MuPLSCR1like-a and MuPLSCR1like-b with transmembrane orientation toward the phospholipid bilayer. In analysis of tissue-specific expression, the highest expression of MuPLSCR1like-a was observed in the intestine, whereas MuPLSCR1like-b was highly expressed in the brain, indicating isoform specificity. Of note, we found that the transcription of MuPLSCR1like-a and MuPLSCR1like-b was significantly upregulated when the fish were stimulated with poly(I:C), suggesting that such immune responses target viral infections. Overall, this study provides the first experimental insight into the characteristics and immune-system relevance of PLSCR1-related genes in red lip mullets.
Author(s)
Wijamunige Gayashani Sandamalika
Issued Date
2019
Awarded Date
2019. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000008878
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
Acknowledgement i
SUMMARY . iv
List of Figures . viii
List of Tables . x
CHAPTER 1: . 1
Structural and functional delineation of two glutathione S-Transferases from Disk abalone (Haliotis discus discus). 1
1. Introduction 2
2. Materials and Methods 7
2.1 Experimental animals and tissue collection . 7
2.2 Chemicals & reagents 7
2.3 Identification & sequence analysis of GSTs from Disk Abalone 7
2.4 Tissue isolation and immune challenge experiment 9
2.5 RNA extraction and first-strand cDNA synthesis . 9
2.6 Transcriptional analysis Quantitative real-time PCR 10
2.7 Cloning of GSTs into the pMAL-c5X expression vector 11
2.8 Overexpression and purification of recombinant AbGST proteins . 12
2.9 GST enzyme assay 12
2.10 Michaelis-Menten Kinetics . 13
2.11 The effect of temperature, pH and inhibitors on rAbGSTs activity 13
2.12 Disk diffusion assay 13
2.13 Statistical Analysis 14
3. Results . 15
3.1 Molecular characterization of AbGSTs . 15
3.2 Homology analysis and phylogenetic relationship 16
3.3 Tertiary structural model of AbGSTs 21
3.4 Tissue-specific expression of AbGSTs 24
3.5 AbGSTs expressions against pathogenic stress . 26
3.6 Construction of recombinant AbGST proteins and purification 35
3.7 Specific activity and kinetic assay . 36
3.8 Effect of temperature, pH, and an inhibitor on GSTs activity . 38
3.9 Disk diffusion assay 42
4. Discussion . 44
CHAPTER 2: . 60
Two phospholipid scramblase 1related proteins (PLSCR1like-a & -b) from Liza haematocheila: Molecular and transcriptional features and expression analysis after immune stimulation 60
1. Introduction . 61
2. Materials and methods . 65
2.1. Experimental fish rearing and tissue collection 65
2.2. RNA extraction and cDNA synthesis . 66
2.3. Identification and sequence analysis of MuPLSCR1like proteins . 66
2.4. Transcriptional analysis by Quantitative real time PCR (qPCR) . 67
3. Results . 69
3.1. Identification and characterization of MuPLSCR1like-a and b . 69
3.2. Homology analysis of MuPLSCR1like-a and -b 71
3.3. The 2D and 3D structural analysis of MuPLSCR1like-a & -b . 73
3.4. Construction of the phylogenetic tree . 75
3.5. Tissue distribution analysis of MuPLSCR1like-a and -b . 76
3.6. Expression analysis of MuPLSCR1like proteins after immune stimulation 78
4. Discussion . 80
5. Conclusion . 88
References 89
Degree
Master
Publisher
제주대학교 대학원
Citation
Wijamunige Gayashani Sandamalika. (2019). Structural and functional delineation of two glutathione S-Transferases from Disk abalone (Haliotis discus discus) AND Two phospholipid scramblase 1–related proteins from Red lip mullet (Liza haematocheila); deciphering their transcriptional responses against immune stimulants
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General Graduate School > Marine Life Sciences
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