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Identification and molecular characterization of two peroxiredoxin counterparts from Japanese eel (Anguilla japonica): Revealing their potent antioxidative properties and putative immune relevancy

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Abstract
Peroxiredoxins (Prdx)는 유해한 과산화수소수를 무독한 산물로 감소시킴으로써 세포 산화 스트에 중요한 역할을 하는 thiol specific antioxidant enzymes의 large family에 속한다. Peroxiredoxin superfamily는 Prdx1, Prdx2, Prdx3, Prdx4, Prdx5 and Prdx6라고 알려진 6개의 isoform으로 구성되어있다. Prdx의 6개는 촉매활성부위에 cysteine residues의 수에 따라 3개의 subfamily (typical-2Cys Prdx, atypical-2 Cys Prdx and 1- Cys Prdx) 로 더욱 세분화 된다. Prdxs 사이에서 Prdx4는 Prdx superfamily에서 유일하게 분비 단백질인 typical-2Cys Prdx subgroup에 속한다. Prdx4는 산화적 스트레스 대응하여 숙주 세포를 보호하거나, 종양 성장의 enhancer로 작용하고 그리고 신호 전달의 조절과 같은 다양한 범위의 생물학적 과정에서 풍부한 것으로 알려져 있다. One- Cys Pdx subgroup에 유일하게 포함되어있는 prdx6 isoform은 possesses peroxidase와 phospholipase A2 activity와 같은 두 가지 기능을 가진 세포 내 효소이다. 그러므로 뱀장어(Anguilla japonica)와 같은 경제적으로 중요한 수산양식생물 종에서 면역학적 측면으로 구조적, 기능적 특성에 대한 Prdx4 와 Prx6의 역할을 이해하는 것이 중요하다.
이 연구에서는, 이전 cDNA library를 사용하여 뱀장어(Anguilla japonica)에서 Prdx4 와 Prdx6을 동정하였다. Prdx4 와 Prdx6의 derived amino acid sequences 는 ClustalW2 multiple sequence alignment와 EMBOSS needle pairwise sequence alignment를 사용하여 다른 taxonomic classes에서 유래한 Prdx4 와 Prdx6 서열을 비교하였다. Orthologous 사이에 관계를 확립하기 위해 계통수 분석은 모든 보고된 Prdx isoforms를 고려하여 수행되었다. 구조적인고 기능적인 관계를 설명하기 위해서, I-TASSER server 와 PyMOL molecular graphic software를 사용하여 Prdx4 와Prdx6의 컴퓨터 기반 3D 구조적 상동성 모델를 수행하였다. Prdx4 와 Pdx6 의 open reading frame은 pMAL-c2X expression vector에 클로닝을 하였다. 그리고 단백질의 항산화적 특성을 결정하기 위해서 정제된 재조합 단백질은 MTT assay를 하였다. 마지막으로 실시간 PCR(quantitative real time PCR)은 건강한 어류에서 조직 특이적 mRNA 발현과 세균 병원체와 자극제에 의한 유도 후에 전사적인 조절에 대하여 실시하였다.
뱀장어의 Prdx4 (AjPrdx4) 와Prdx6 (AjPrdx6) 전체 서열은 각각 786bp와 669bp이고 262와 223 아미노산을 암호화하고 있었다. AjPrdx4의 단백질은 N-terminus (111FTFVCPTEI120) and C-terminus (233GEVCPAGW240) catalytic active sites을 포함하는 characteristic typical 2-Cys Prdx domain architecture가 있었다. 반면에, AjPrdx6은 with N-terminus conserved catalytic active site (PVCTTE)를 가진 1-Cys Prdx sub family에 속한 domain architecture와 비슷했다. The AjPrdx4 와AjPrdx6 모두 Salmo salar 와 매우 높은 identity와 similarity을 보여주었다. 계통수 분석에서 AjPrdx4와AjPrdx6는 각각 전형적인 2-Cys Prdx ,1-CysPrdx의 주요 clades에 속해있었다. AjPrdx4의 컴퓨터 모의 3D 모델은 핵심에 7개의 β-sheet와 측면에 9개의 α helix로 구성되어있었다. 반면에, AjPrdx6는 핵심에 9개의 β-sheet와 측면에 7개의 α-helixes로 되어있었다. 게다가 앞선 모델들은 기능적인 특성에 대한 AjPrdx4 와 AjPrdx6의3D globular arrangement도 정확한 방향인 것으로 확인되었다.
AjPrdx4 와 AjPrdx6의 항산화적 특성을 알기 위해서 재조합 단백질을 발현시키고 순수 분리하였다. 재조합 단백질은 산화스트레스와 연계된 H2O2에 대응하여 뚜렷한 세포 보호 효과를 보여주었다. 산화스트레스에서 AjPrdx4의 재조합 단백질에서 80% 이상의 cell viability를 확인하였고AjPrdx6의 재조합 단백질에서는 50% 이상의 cell viability를 관찰하였다. 여기에 우리는 실험한 모든 조직에서 AjPrdx4 와 AjPrdx6 mRNA전사가 고르게 분포하는 것을 관찰하였다. AjPrdx4 와 AjPrdx6의 mRNA 전사는 세균(Edwardsiella tarda), lipopolysaccharide (LPS) 와 Polyinosinic:polycytidylic (Poly I:C)를 공격 실험하였을 때 간과 비장을 포함한 유력한 면역조직에서 상당히 상향 조절되었다.
종합적으로, 뱀장어의 Prdx4 와Prdx6 분석한 본 연구는 알려진 Prdx4 와Prdx6에서 보존된 전형적인 구조적, 기능적 특성을 보여주었다. 따라서 이 연구는 면역학적 측면에서 뱀장어의 항산화적 특성을 이해하는데 상당한 기여와 가치가 있을 것이다
Peroxiredoxins (Prdx) are a large family of thiol specific antioxidant enzymes in eukaryotes and prokaryotes which play a critical role in cellular oxidative stress by reducing harmful peroxide compounds into nontoxic products. The peroxiredoxin superfamily is consisted of six different isoforms, known as Prdx1, Prdx2, Prdx3, Prdx4, Prdx5 and Prdx6. The six members of Prdxs are further categorized into three sub families (typical-2Cys Prdx, atypical-2 Cys Prdx and 1- Cys Prdx) depending on number of cysteine residues present in their catalytic active sites. Among Prdxs the Prdx4 is a member of typical-2Cys Prdx subgroup which is the only a secretory protein in Prdx superfamily. The Prdx4 is known to be involved in plenty of biological process in diverse range, such as protect the host cells against the oxidative stress, and act as an enhancer of tumor growth and regulation of signal transduction. One- Cys Prdx subgroup contains only Prdx6 isoform, is an intracellular bi-functional enzyme which possesses peroxidase and phospholipase A2 activity. Hence, it is important to understand the role of Prdx4 and Prdx6 in economically important aquaculture fish species like Japanese eel (Anguilla japonica) with respect to its structural and functional properties in immunological prospective.
In this study, Prdx4 and Prdx6 genes were identified from Japanese eel (Anguilla japonica) using previously constructed cDNA library. Derived amino acid sequences of Prdx4 and Prdx6 from Japanese eel were compared with known Prdx4 and Prdx6 sequences from different taxonomic classes using ClustalW2 multiple sequence alignment and EMBOSS needle pairwise sequence alignment. The phylogenetic trees were constructed considering all reported Prdx isoforms in order to establish the relationship between orthologous. In order to elucidate the structural and functional relationship, computer based 3D structural homology models of Prdx4 and Prdx6 were performed using I-TASSER server and PyMOL molecular graphic software. The open reading frames of both Prdx4 and Prdx6 were cloned into pMAL-c2X expression vector and the purified recombinant proteins were subjected to MTT assay to determine the antioxidant properties of each protein. Finally quantitative real time PCR was performed to examine the tissue specific mRNA expression of these peroxiredoxins in healthy fish and their transcriptional modulation after induction of fish with chemical stimulants and a live bacterial pathogen.
The full length coding sequences of Anguilla japonica Prdx4 (AjPrdx4) and Prdx6 (AjPrdx6) were 786 bp and 669 bp in length which encodes for a 262 and 223 amino acid polypeptides, respectively. The protein of AjPrdx4 exhibited characteristic typical 2-Cys Prdx domain architecture including N-terminus (111FTFVCPTEI120) and C-terminus (233GEVCPAGW240) catalytic active sites, whereas AjPrdx6 resembled the domain architecture belonging to the 1-Cys Prdx sub family with N-terminus conserved catalytic active site (44PVCTTE49). The AjPrdx4 and AjPrdx6 exhibited highest amino acids identity and similarity to that of Salmo salar. Phylogenetic analysis indicated clustering of AjPrdx4 and AjPrdx6 in the main clades of typical 2-Cys Prdx and 1-Cys Prdx respectively. The computer simulated 3D modal of AjPrdx4 composed of seven core stranded β sheets flanked by nine α helixes, whereas AjPrdx6 consisted of nine core stranded β-sheets and seven α-helixes. Furthermore generated models affirm that correct orientation of the 3D globular arrangements of AjPrdx4 and AjPrdx6 with respect to their functional properties.
In order to characterize the antioxidant properties of AjPrdx4 and AjPrdx6 recombinant proteins were expressed and purified. The recombinant proteins showed remarkable cell protection against H2O2 mediated oxidative stress. Over 80% of cell viability was detected with recombinant AjPrdx4, while over 50% cell viability was observed with recombinant AjPrdx6 under oxidative stress. Herein we observed ubiquitous distribution of AjPrdx4 and AjPrdx6 mRNA transcripts in all the tested tissues. The mRNA transcripts of AjPRdx4 and AjPRdx6 were significantly up-regulated in potent immune tissues including liver and spleen upon immune challenge with live bacteria (Edwardsiella tarda), lipopolysaccharide (LPS) and Polyinosinic:polycytidylic (Poly I:C).
Collectively, the present study in analysis of Prdx4 and Prdx6 in Japanese eel demonstrated common structural and functional features were conserved with known Prdx4 and Prdx6 counterparts. Hence this study will be valuable and make significant contribution to understand the antioxidant properties of Japanese eel in immunological prospective.
Author(s)
Thanthrige Thiunuwan Priyathilaka
Issued Date
2014
Awarded Date
2015. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007007
Department
대학원 해양생명과학과
Table Of Contents
1. INTRODUCTION 1
2. MATERIALS AND METHODS 9
2.1. Identification of AjPrdx4 and AjPrdx6 cDNA sequences 9
2.2. insilico analysis 9
2.3 Experimental animals 10
2.4 Tissue collection 10
2.5 Immune challenge experiment 10
2.6 RNA extraction 11
2.7 AjPrdx4 and AjPrdx6 mRNA expression analysis by quantitative real time Polymerase Chain Reaction (qPCR) 12
2.8 Cloning of AjPrdx4 and AjPrdx6 coding sequences 13
2.9 Overexpression and purification of recombinant AjPrdx4 and AjPrdx6 14
2.10 Cell cultures 15
3. RESULTS AND DISCUSSION 17
3.1. PART 1 17
3.1.1 Sequence characterization 17
3.1.2. Phylogenetic analysis 21
3.1.3. Predicted 3D homology modal of AjPrdx6 22
3.1.4. Tissue distribution analysis of AjPrdx6 25
3.1.5. Expression profile of AjPrdx6 upon E. tarda, LPS and Poly I:C challenge 26
3.1.6. Over expression and purification of recombinant AjPrdx6 34
3.1.7. Protective effects of recombinant AjPrdx6 (rAjPrdx6) on cultured cells under oxidative stress 34
3.2. PART-2 37
3.2.1. Sequence characterization 37
3.2.2. Phylogenetic analysis 41
3.2.3. Modeled tertiary structure of AjPrdx4 42
3.2.4. Tissue distribution analysis of AjPrdx4 45
3.2.5. Regulation of AjPrdx4 expression in response to pathogenic infection 46
3.2.6. Over expression of recombinant AjPrdx4 (rAjPrdx4) 51
3.2.7. Protective effects of recombinant rAjPrdx4 on cultured cells under oxidative stress 52
CONCLUSIONS 55
REFERENCES 56
ACKNOWLEDGEMENT 60
Degree
Master
Publisher
제주대학교 대학원
Citation
Thanthrige Thiunuwan Priyathilaka. (2014). Identification and molecular characterization of two peroxiredoxin counterparts from Japanese eel (Anguilla japonica): Revealing their potent antioxidative properties and putative immune relevancy
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General Graduate School > Marine Life Sciences
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