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MOLECULAR CHARACTERIZATION AND IDENTIFICATION OF C-TYPE LECTINS FROM BIG BELLY SEAHORSE (Hippocampus abdominalis) AND BLACK ROCKFISH (Sebastes schlegeli)

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Abstract
빅벨리 해마와 조피볼락은 한국에서 가치있는 양식 어종이다. 하지만 집약식 양식에서 병원균 감염에 의한 어류의 치사가 발생하므로 양식생물의 면역 기전을 이해하는것이 지속가능한 양식산업을 유지하기위해 필요하다. 생물은 항상 병원균에 노출되어있으며 감염으로부터 숙주를 보호하기 위한 면역을 지닌다. 선천면역의 중요한 역할은 미생물의 구성성분으로 그들의 생활사에 중요한 기능을 하는 병원체 연관분자 패턴을 인지하는것이다. 패턴 인지 수용체는 이러한 병원체 연관 분자패턴을 인지함으로써 옵소닌화, 식세포작용, 항균 펩타이드 및 염증사이토가인의 전사 등의 다양한 면역반응을 일으킨다. C-type lectin은 패턴 인지 수용체의 한 종류로, Ca2+ 이온에 의존적인 탄수화물 결합 렉틴으로 명명되었으며 하나의 탄수화물인지 도메인을 가지고 있고 그들의 구조에따라 여러개의 그룹으로 구분할 수 있다. 그 중 Dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN)은 수지상세포의 이동, 항원 섭취 및 제시, T-세포 활성화와 같은 수지상세포의 생활사에 관여하고, Macrophage-inducible C-type lectin (Mincle) 은병원체 연관 분자패턴뿐만 아니라 손상된 세포에서 특별히 발현되는 패턴(damage-associated molecular patterns, DAMPs)을 인지함으로써 염증반응을 일으킨다.
이 연구에서는 빅벨리해마로부터 DC-SING 유전자, 조피볼락으로부터 Mincle 유전자를 동정하였다. ShDCS와 RfMIN의 cDNA및 아미노산 서열을 다양한 생물정보학 프로그램을 사용하여 분석하였다. ExPASy PROSITE 과 SMART program을 통해 특징적인 도메인을 확인하였고, Clustal 및EMBOSS needle program사용하여multiple sequence alignment과 pairwise sequence alignment를 수행하였다. 계통발생학적 분석은 Mega5의 neighbour-joining (NJ) method를 이용 하였으며, qPCR을 이용하여 조직별 mRNA 발현과 면역자극 후의 발현 변화를 확인하였다. ShDCS와 RfMIN의 탄수화물인지도메인 부분을 발현벡터에 클로닝하여 단백질의 발현을 유도하였다. 정제된 단백질로 응집반응 수행하였으며 ShDCS재조합 단백질로 당 특이적 결합 활성을 측정하였다.
해마의 DC-SIGN (ShDCS)과 조피볼락의 Mincle (RfMIN)은 각각 462 개의 아미노산을 암호화하는 1386 bp의 ORF 및254 개의 아미노산을 암호화하는 765 bp로 구성되었다. ShDCS와 RfMIN 의 단백질에는transmembrane domain 과carbohydrate recognition domain (CRD)이 존재하였으며 CRD에는 2개의 이황화결합을 형성하는 4개의 시스테인 잔기가 보존되었다. 그리고 ShDCS에는 탄수화물 결합과 관련있는 motif인 QPN 과 WND motif 가 존재하였고, RfMIN에는 EPN 과 WNF motif가 존재하였다. ShDCS는 Oplegnathus fasciatus 와 61.6 %로 높은 아미노산 상동성을 보였고, RfMIN은Larimichthys crocea 와 79.3 %의 상동성을 보였으며, 계통수 분석결과, ShDCS와 RfMIN 모두 어류 cluster에 속하는것을 확인하였다. ShDCS의 조직별 발현양상은 신장과 아가미, RfMIN은 간과 비장에서 높은 발현 양상을 보였다. LPS , Edwardsiella tarda, Streptococcus iniae로 감염시킨 해마에서 ShDCS의 발현의 변동이 나타났으며, LPS, Streptococcus iniae, Poly I:C로 감염시킨 조피볼락에서는 RfMIN의 발현이 증가하였다. ShDCS와 RfMIN 재조합단백질은 박테리아에 응집반응을 나타냈으며, ShDCS재조합 단백질은Sucrose 와 Lactose에 특이적인 결합을 보였다. 이러한 결과를 토대로, ShDCS 와 RfMIN은 C-type lectin의 특징을 가지며, 병원균 인지 수용체의 기능을 담당하는것으로 생각된다.
maintain a sustainable aquaculture industry. Organisms always are exposed to pathogens and have immunity to defense against infections. Important role of the innate immunity is detecting pathogen associated molecular patterns (PAMPs) which are microbial components and crucial for their life. Pattern recognition receptors (PRRs) arise various immune responses including opsonization, phagocytosis, and transcription of antimicrobial peptides and inflammatory cytokines by recognizing PAMPs. C-type lectin receptors (CLRs) which are one of the PRRs, are originally named as Ca2+-dependent (C-type) carbohydrate-binding lectins and contain at least one carbohydrate-recognition domain (CRD) which binds carbohydrate and modulates binding activity in Ca2+ ion depended manner. CLRs can divided into several groups according to their structure.
Dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) is a kind of C-type lectins which involve in the events of life cycle of the dendritic cells (DC) such as DC migration, antigen capture and presentation along with T cell priming. Macrophage-inducible C-type lectin (Mincle) is a member of the macrophage receptors of CLRs recognize damage-associated molecular patterns (DAMPs) as well as PAMPs. Binding ligands Mincle cause the inflammatory response to defense host or repair tissues.
In this study, DC-SIGN from big belly seahorse (Hippocampus abdominalis) and Mincle from black rockfish (Sebastes schlegeli) were characterized. Derived amino acid sequences of ShDCS and RfMIN were analyzed using bioinformatics tools. Characteristic domain and motifs were predicted by ExPASy PROSITE and SMART program. The Clustal program was used to create the multiple sequence alignment and pairwise sequence alignment was performed by EMBOSS needle program. The phylogenetic tree was constructed using the neighbor-joining algorithm using Mega 5 program. The tissue specific mRNA expression profile and their transcriptional modulation after immune challenges were determined using qPCR. Furthermore, two genes were over expressed and purified (rShDCS and rRfMIN, respectively). Agglutination ass ay was conducted with both rShDCS and rRfMIN while sugar specific binding test was conducted with rShDCS.
The open reading frame (ORF) of ShDCS and RfMIN were consisted of 1386 bp and 765 bp, encoding for 462 amino acids 254 amino acids and respectively. The ShDCS and RfMIN contain a transmembrane domain and carbohydrate recognition domain (CRD), in which 4 cysteine residues which form two disulfide bonds. ShDCS had two conserved motifs; QPN and WND motif in Ca2+ binding site which is involved in carbohydrate binding, whereas RfMIN harbored the EPN and WNF motif. ShDCS shared highest amino acid identity (61.6 %) with Oplegnathus fasciatus and RfMIN exhibited identity (79.3 %) with Larimichthys crocea. Phylogenetic analysis indicated that both ShDCS and RfMIN were clustered with the fish clade. The tissue distribution of ShDCS expression was transcribed universally in all the tissues examined, but with high abundance in kidney and gill. RfMIN expression was detected in all the tested tissues, with the highest expression level in liver and spleen. According to the immune challenge experiments carried out using lipopolysaccharides (LPS), Edwardsiella tarda (E. tarda) and Streptococcus iniae (S. iniae) as stimuli, ShDCS mRNA expression level was fluctuated. RfMIN mRNA level was up-regulated upon immune challenge with LPS, S. iniae and Polyinosinic:polycytidylic (Poly I:C). rShDCS and rRfMIN showed different agglutination activity against different bacteria species and rShDCS showed significant affinity to Sucrose and Lactose. Collectively, this study suggests that ShDCS and RfMIN were harbored common features of C-type lectin and they may function as pattern recognition receptors against pathogens to alarm the septic condition to the host immune system.
Author(s)
조은영
Issued Date
2015
Awarded Date
2016. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007415
Alternative Author(s)
Jo, Eunyoung
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
INTRODUCTION 1
Chapter I 9
Molecular characterization and identification of DC-SIGN like gene from big belly seahorse (Hippocampus abdominalis) 9
1. ABSTRACT 9
2. MATERIALS AND METHODS 10
2.1. Sequence characterization of ShDCS 10
2.2. Fish and tissue collection 11
2.3. Immune challenge experiment 11
2.4. RNA isolation and cDNA synthesis 11
2.5. Expression analysis by quantitative real-time PCR (qPCR) 12
2.6. Cloning of ShDCS carbohydrate recognition domain (CRD) 13
2.7. Overexpression and purification of recombinant ShDCS CRD (rShDCS) 13
2.8. Agglutination assay 14
2.9. Sugar specific binding assay 15
3. RESULTS AND DISCUSSION 16
3.1 Sequence characterization of ShDCS 16
3.2. Tissue-specific expression of ShDCS 20
3.3. The temporal expression profile of ShDCS following bacteria challenge. 22
3.4. Overexpression and purification of recombinant ShDCS CRD (rShDCS) 26
3.5. Agglutination assay 27
3.6. Sugar specific binding assay 28
Chapter Ⅱ
Molecular characterization and identification of Mincle gene from black rockfish (Sebastes schlegeli) 30
1. ABSTRACT 30
2. MATERIALS AND METHODS 31
2.1. Black rockfish cDNA database 31
2.2. Sequence characterization of RfMIN 31
2.3. Fish and tissue collection 32
2.4. Immune challenge experiment 32
2.5. RNA isolation and cDNA synthesis 32
2.6. Expression analysis by quantitative real-time PCR (qPCR) 33
2.7. Cloning of RfMIN carbohydrate recognition domain (CRD) 34
2.8. Overexpression and purification of recombinant RfMIN CRD (rRfMIN) 34
2.9. Agglutination assay 35
3. RESULTS AND DISCUSSION 36
3.1. Sequence characterization of RfMIN 36
3.2. Tissue-specific expression of RfMIN 40
3.3. The temporal expression profile of RfMIN following pathogenic injection. 41
3.4. Overexpression and purification of recombinant RfMIN CRD (rRfMIN) 44
3.5. Agglutination assay 45
REFERENCES 46
ACKNOWLEDGEMENT 52
Degree
Master
Publisher
제주대학교 대학원
Citation
조은영. (2015). MOLECULAR CHARACTERIZATION AND IDENTIFICATION OF C-TYPE LECTINS FROM BIG BELLY SEAHORSE (Hippocampus abdominalis) AND BLACK ROCKFISH (Sebastes schlegeli)
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General Graduate School > Marine Life Sciences
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