Functional insights into two potent constituents in molluscan physiology, identified from Disk abalone (Haliotis discus discus) deciphering their putative significance in host defense
- 해양 생물은 그들의 생리학적 메커니즘에 영향을 주는 다양한 환경요인에 노출되어있으며 이러한 요인에 저항하기 위해서 다양한 방어기전을 수행해야 한다. 전복은 주요 양식 진미품으로서 상업적으로 매우 가치가 크다. 또한, 해양 무척추 연체동물 패류에 속하는 종으로 병원균 감염에 대해 독특한 면역 매커니즘과 생체 내 불균형을 일으키는 다양한 요인에 저항하는 생화학적 메커니즘을 가진다.
최근 전복의 유전현상에 관한 연구는 많이 진행되고 있지만, 다양한 스트레스 환경에서의 숙주 방어 능력에 관한 연구는 많지 않다. 따라서 이 연구에서는 전복의 방어체계에서 중요한 분자 구성성분인 박테리아로부터 숙주를 보호하는transferrin, 항독성 기능을하는glutathione reductase유전자를 분자학적 및 기능학적으로 동정하였다.
Transferrin의 기본적인 기능은 3가 철이온과 결합하는것으로 대사과정이 일어나는 장소로 그들을 운반하는 역할을 하며, 면역반응에도 관여한다. 이러한 이유로transferrin은 숙주 면역에있어 중요한 급성 단백질로 알려졌다. 이 연구에서 둥근전복 transferrin like 유전자 (AbTrf)를 분자학적 및 기능학적으로 동정하였다. AbTrf는 728개의 아미노산을 암호화하는 2189 bp의ORF로 구성되었으며, 아미노산의N-말단 부분과C-말단 부분의transferrin like domain, 철이온과의 결합을 위한 active site, 보존된 시스테인 잔기가 존재하였다. qPCR을 수행하여AbTrf의 조직별 발현을 분석한 결과, 맨틀과 근육에서 높은 발현을 보였다. Vibrio parahaemolyticus, Listeria monocytogenes, LPS로 인위감염시킨 전복의 아가미와 혈구에서AbTrf mRNA의 발현이 증가하였다. Transferrin N -말단 도메인의 재조합 단백질은 chrome azurol S (CAS) assay 통해 단백질 농도에 따라 철 3가 이온의 결합능이 비례함을 보였다. 그리고 철에 의존적인 Escherichia coli의 성장은 억제하였지만Lactobacillus plantarum 의 성장을 억제하지는 못했다. 실혐결과들을 종합해보면, AbTrf는 병원균으로부터 철이온을 뺏음으로써 숙주의 선천면역에 중요한 역할을 하는것으로 생각된다.
Glutathione reductase은 산화형 glutathione을 환원형으로의 반응을 촉매하는 효소이다. 환원형이 과산화수소에대한 항독성에 중요한 기능을 하기 때문에 산화형과 환원형의 비율은 중요하며, 이를 위해 glutathione reductase은 필수적이다. 이 연구에서는 둥근전복 glutathione reductase (AbGSR) 의 분자학적 및 기능학적으로 동정하였다. 909개의 아미노산을 암호화하는 2325 bp 의ORF로 구성되었으며, 아미노산에 pyridine nucleotide – disulphide oxidoreductase domain, pyridine nucleotide-disulphide oxidoreductase dimerization domain, rossmann-fold NAD(P)(+)- binding proteins superfamily signature 가 존재하였다. 4개의 기능적인 잔기인FAD binding site, glutathione binding site, NADPH binding motif 와 assembly domain도 잘 보존된것을 확인하였다. glutathione 재조합 단백질로glutathione reductase활성 실험을 수행하였다. 최적의 pH와 온도는 7.0 pH와 50℃로 측정되었으며, 각각 배지의 이온강도에 대해 어떤 효과도 나타내지 않았고, 효소활성은Cu+2 과 Cd+2 이온에 의해 억제되었다. Disk diffusion assay 결과 세포 보호 효과를 보여주었고, MTT assay와 flow cytometry를 통해서도 상당한 세포 보호 효과를 나타냈다. AbGSR은 전복의 다양한 조직에서 발현되었으며, Vibrio parahaemolyticus, Listeria monocytogenes , LPS로 인위감염시킨 경우 AbGSR의 발현이 유도되었다. 종합적으로, AbGSR은 숙주 방어체제를 중재하는 항독성에서 매우 중요한 역할을 하는것으로 보인다.
The marine species including fish and shellfish are frequently encountered with different environmental factors which affect the proper functionality of normal physiological mechanisms. To defy these environmental conditions, marine organisms need to carry different kind of host defense mechanisms and strong resistance to extreme environmental conditions. When it comes to shellfish aqua farming, abalone is identified as one of the main delicacy which can exclusively be obtained by aquaculture methods. Abalone as a "marine invertebrate molluscan shellfish species", needs to be equipped with unique types of host defense mechanisms against pathogenic infections and a range of biochemical mechanisms to be resisted against different environmental factors that possibly can imbalance the normal physiology of animal.
Even though currently undergoing researches on abalone physiology focus only on the general phenomena, not much studies are undergoing on the uncommon mechanisms which can provide outstanding host defensive abilities and survival capabilities under septic and different stress conditions, and yet to be properly investigated in order to reveal the valuable piece of hidden knowledge. In the presence of so called background, we design the current study to molecularly identify and functionally annotate the uncommon physiological molecular components which are not yet identified from abalone. As a preliminary approach to start this worthful contrive, in this thesis I have extensively characterized a transferrin homolog identified from our disk abalone (Haliotis discus discus) database which capacities an indirect but significant involvement in an antibacterial host defense mechanisms. Moreover, I direct the current study to identify a homolog of glutathione reductase from our database which is not extensively studied in molluscan family, in order to investigate the essence of having antioxidants defense systems in shellfish species such as abalones.
The basic function of transferrin is to bind iron (III) ions in the medium and to deliver them to the locations where they are required for metabolic processes. It also takes part in the host immune defense mainly via its ability to bind to iron (III) ions. Hence, transferrin is also identified as an important acute-phase protein in host immunity. Since transferrin is known to be a major player in innate immunity, in the present study we sought to identify, and molecularly and functionally characterize a transferrin-like gene from disk abalone (Haliotis discus discus) named as AbTrf. AbTrf consisted of a 2187-bp open reading frame (ORF) which encodes a 728 amino acid (aa) protein. The putative amino acid sequence of AbTrf harbored N- and C-terminal transferrin-like domains, active sites for iron binding, and conserved cysteine residues. A constitutive tissue specific AbTrf expression pattern was detected by qPCR in abalones where mantle and muscle showed high AbTrf expression levels. Three immune challenge experiments were conducted using Vibrio parahaemolyticus, Listeria monocytogenes and LPS as stimuli and, subsequently, AbTrf mRNA expression levels were quantified in gill and hemocytes in a time-course manner. The mRNA expression was greatly induced in both tissues in response to both challenges. Evidencing the functional property of transferrins, recombinant AbTrf N-terminal domain (AbTrf-N) showed dose-dependent iron (III) binding activity detected by chrome azurol S (CAS) assay system. Moreover, recombinant AbTrf-N could significantly inhibit the growth of iron-dependent bacterium, Escherichia coli in a dose-dependent manner. However, AbTrf-N was unable to show any detectable bacteriostatic activity against iron-independent bacterium Lactobacillus plantarum (L. plantarum) even at its highest concentration. Collectively, our results suggest that AbTrf might play a significant role in the host innate immunity, possibly by withholding iron from pathogens.
Glutathione reductase (GSR) as an enzyme catalyzes the biochemical conversion of glutathione oxidized form (GSSG) into reduced form (GSH). Since ratio between two forms of glutathione (GSH/GSSG) is important for the proper functioning of GSH to act as an antioxidant against H2O2, contribution of GSR is essential. We conducted the current study to reveal the molecular and functional properties of GSR similitude identified from disk abalones (Haliotis discus discus). The identified cDNA sequence (2325 bp) bears a 1356 bp long ORF, coding for a 909 bp long amino acid sequence which harbored a pyridine nucleotide – disulphide oxidoreductase domain (171-246 aa), a Pyridine nucleotide-disulphide oxidoreductase dimerization domain and a Rossmann-fold NAD(P)(+)- binding proteins superfamily signature. Four functional residues; FAD binding site, glutathione binding site, NADPH binding motif and assembly domain were identified to be conserved among the other species. The recombinant AbGSR (rAbGSR) exhibited a detectable activity with standard glutathione reductase activity assay. The optimum pH and optimum temperature for the reaction were detected at 7.0 pH and 50 °C, respectively while showing no effect of the ionic strength of the medium. The enzymatic reaction was vastly inhibited by Cu+2 and Cd+2 ions. A considerable cell protective effect was detected with disk diffusion assay conducted with rAbGSR. Moreover, with MTT assay and flow cytometry, significance of cell protectiveness provided by rAbGSR was confirmed. Moreover, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression exhibited the potent inductions in three immune challenges conducted with Vibrio parahaemolyticus, Listeria monocytogenes and LPS predicting its possible involvements in host defense mechanisms under pathogenic infections. Taken together, results of current study suggest that AbGSR might play an important role in antioxidant mediated host defense mechanisms while providing insights into immunological contribution of AbGSR.
- Herath Mudiyanselage Lalinka
- Issued Date
- Awarded Date
- 2016. 2
- Alternative Author(s)
- 헤라스 무디얀세라게 라린카
- 대학원 해양생명과학과
- Table Of Contents
- Introduction 1
CHAPTER I: Molecular insights into a molluscan transferrin homolog identified from disk abalone (Haliotis discus discus) evidencing its detectable role in host antibacterial defense 13
1. Abstract 13
2. Material and Methodology 14
2.1. Identification of AbTrf cDNA from a transcriptome database 14
2.2. Sequence characterization and evolutionary analysis 14
2.3. Overexpression of the recombinant abalone transferrin N- terminal domain (rAbTrf-N) 15
2.4. Iron-binding assay of rAbTrf-N protein (chrome azurol S assay, CAS) 17
2.5. Shellfish rearing for the immune challenge experiment 17
2.6. Immune challenge experiment, RNA extraction, and cDNA synthesis 18
2.7. Quantitative real time PCR (qPCR) analysis of mRNA expression levels 19
2.8. Antibacterial assay for rAbTrf-N 20
3. Result and Discussion 21
3.1. Identification and bioinformatics characterization of AbTrf cDNA sequence and the derived amino acid sequence 21
3.2. Sequence homology and phylogenetic analysis of abalone transferrin amino acid sequence 25
3.3. Putative functional prediction of AbTrf based on the construction of the 3D structure 29
3.4. Functional assessment of the putative AbTrf-N protein in Fe3+ binding using purified recombinant AbTrf-N protein 31
3.5 Tissue specific expression of AbTrf mRNA 34
3.6. Modulation of AbTrf mRNA profile upon pathogenic challenge conditions 35
3.7. Investigation of antimicrobial property of AbTrf-N 38
CHAPTER II: Molecular identification and functional delineation of a glutathione reductase similitude from Disk abalone (Haliotis discus discus): insights as a potent player in host antioxidant defense 43
1. Abstract 43
2. Material and Methodology 44
2.1. Identification of AbGSR cDNA from cDNA database 44
2.2. Sequence characterization, evolutionary relationships and structural prediction of AbGSR 45
2.3 Recombinant expression and purification of putative AbGSR protein 46
2.4. GSR specific enzymatic assay, assessment of enzymatic properties of rAbGSR and inhibitory effect of metal ions 48
2.4.1. Glutathione reductase activity assay 48
2.4.2. Enzymatic properties determination 48
2.4.3. Effect of heavy metals on rAbGSR activity 49
2.5. Antioxidant properties of rAbGSR 49
2.5.1. Disk diffusion assay 49
2.5.2. Cell culture and cell viability assays 50
18.104.22.168. MTT assay 50
22.214.171.124. Flow cytometry 51
2.6.Investigation of immune related mRNA expression pattern of AbGSR 51
2.6.1. Animal rearing 51
2.6.2. Immune challenge experiment, RNA extraction and cDNA synthesis 52
2.6.3. Quantitative real time PCR (qPCR) analysis of mRNA expression levels 53
2.7.Statistical analysis 54
3. Results and Discussion 54
3.1. Sequence identification and Bioinformatics analysis of AbGSR cDNA and predicted amino acid sequences 54
3.2. Sequence homology and evolutionary analysis 55
3.3. The functional interpretation of AbGSR based on 3D structural features 60
3.4. Overexpression and functional properties confirmation of recombinant AbGSR protein 61
3.5. Enzymatic properties validation and effect of heavy metals on rAbGSR activity 64
3.6. Involvement of AbGSR in antioxidant defense mechanisms 67
3.7. qPCR analysis of AbGSR tissue specific mRNA expression and immune related expression patterns 72
3.8. Conclusion. 75
- 제주대학교 대학원
- Herath Mudiyanselage Lalinka. (2015). Functional insights into two potent constituents in molluscan physiology, identified from Disk abalone (Haliotis discus discus) deciphering their putative significance in host defense
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