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Abalone is an important and highly priced species in world wide aquaculture. Inorder to understand the physiological and biological functions of the abalone, we have forcused on polysaccharide degrading (digestive), calcium regulatory and immune response genes in this study.
In the first part of this study, we identified and functionally characterized the 6 EST clones of polysaccharide degrading enzymes denoted as PDEs from disk abalone Haliotis discus discus cDNA library. The genes that responsible for polysaccharide degrading are endo β-1,4-glucanase (HdEndg), α-amylase (HdAmy), alginate lyase (HdAlgl), β-mannanase (HdMann), laminarinase (HdLmn) and arylsulfatase (HdArys). Sequence characterization of the abalone PDEs showed that those genes were matched with other known poly saccharide degrading enzymes and respective motifs. The recombinant enzymes of selected PDEs were purified using pMAL fusion protein purification system. Recombinat PDEs were characterized for their specific function and other factors such as optimal temperature, pH and thermal stability. The HdEndg comprises 1824 bp ORF coding for 608 amino acids. The purified HdEndg enzyme showed the 0.3 U/mg specific activity at 40 oC optimal temperature and pH 4.5. HdAmy has 1536 bp ORF coding for 511 amino acids. The optimum temperature and pH of HdAmy were 50 oC, 7.5, respectively. Alginate lyase is responsible for the degradation of alginate, which is considered as the main structural component of brown seaweed. HdAlgl consists of 897 bp ORF codes for 298 amino acids. The purified enzyme showed 2 Unit/mg activity towards sodium alginate at 40 oC optimum temperature. HdMann cDNA contains an ORF of 1125 bp that encodes 375 amino acids, of which the first 18 residues comprise the secretion signal peptide. According to searches for the NCBI, GenBank databases, the amino-acid sequence of HdMann was found to show 49% and 48% identity values to Mitilus edulis and H. discus hannai mananase, respectively.
Sulfatases are responsible for hydrolyzing the sulphate ester bonds from variety of substrates such as glycosaminoglycans, proteoglycans, steroids and glycolipids. The sequence characterization results revealed that HdArys encodes the main characteristic motifs and functional amino acid residues of the sulfatase family. HdArys cDNA contains 1449 bp ORF that encodes 481 amino acids. The HdArys amino acid sequence consists of a sulfatase domain (24-433 aa) and characteristic sulfatase signature motif was present at 72CSPSR76. Also, functionally important residues involved in substrate binding, activation, metal coordination and enzyme stabilization were observed in the sequence. The HdArys showed the highest level amino acid sequence identity (45%) with Helix pomatia sulfatase I precursor while it shared 41% identity with Rattus norvegicus, Bos taurus and Homo sapiens arylsulfatase B.
For the in vivo expression analysis of PDEs in disk abalone, the animals were starved for 8 weeks and after that re-fed the animals continuously for 28 days. Semi quantitative RT-PCR was carried out to determine the mRNA expression analysis in the digestive tract and hepatopancreas tissues. All the PDEs were strongly expressed in hepatopancreas than digestive tract, suggesting that key organ which encountered digestive enzyme is hepatopancreas. Also, expression level was varies in different genes. Following the starvation period, maximum weight loss was 18.6%. During 8 weeks of starvation, all polysaccharide degrading enzymes were significantly (p<0.05) decreased compare to fed animals (control). During 2 weeks of re-feeding, gradual increase of mRNA transcription was observed, suggesting that abalones can survive for 8 week of starvation.
In the second part of study we have characterized the calcium regulatory regucalcin gene and its mRNA expression analysis. Calcium ion acts as a second messenger, in the regulation of many cellular and physiological functions such as muscle contraction, neuronal activation, cell differentiation and cell death in many organisms. Abalone regucalcin (HdReg) ORF consists of 918 nucleotides encoding 305 amino acids. The HdReg amino acid sequence did not contain the EF-hand motif as a Ca2+ binding domain, suggesting a novel class of Ca2+ binding protein. According to searches for the NCBI, GenBank databases, it showed 45% identity to chicken and zebrafish, and 44% to rat and mouse regucalcin in deduced amino acid level. In vivo regucalcin tissue specific expression was investigated in gill, mantle, digestive tract, abductor muscle by semi quantitative RT-PCR. Abalones were injected with 0.5 mg CaCl2/g of animal intramuscularly and determined the Ca regulatory role of HdReg. HdReg mRNA was expressed in gill, mantle, digestive tract, and abductor muscle. Semi-quantitative RT-PCR results showed that an intramuscular administration of CaCl2 could significantly induce regucalcin mRNA in abductor muscle after 30 min of administration and reached maximum after 1 h. Subsequently, the expression level was decreased after 2 h. This indicates that the expression of regucalcin mRNA is constitutive, and specifically up regulated in abalone abductor muscle by Ca2+ administration.
In the third part of this study was focused on immune related pattern recognition gene of disk abalone with respect to mRNA expression analysis after immune challenge by diffent immune modulators. Pattern recognition molecules play an important role in innate immunity by recognizing common epitopes on microorganisms surface and initiate the host immune response, when binding to non self pathogen associated molecular patterns (PAMPs).
Pattern recognition protein (PRP) was isolated from a disk abalone, H. discus discus, whole body cDNA library. The HdPRP ORF consists of 1260 bp encodes 420 amino acids with 20 aa of a signal peptide sequence. The deduced aa sequence of HdPRP showed high identity to β-glucan recognition protein (BGRP) of the fresh water snail Biomphalaria glabrata (50%). Characteristic motifs such as potential polysaccharide binding, putative cell adhesion, and glucanase motifs were found in HdPRP with slight modifications to other invertebrate PRP motifs. To evaluate in vivo PRP mRNA expression, abalones were intra muscularly injected with Vibrio alginolyticus (150 l/animal, OD600=1.0), lipopolysaccharide (4 g/g animal) and β-1,3-glucan (6 g/g animal). RT-PCR results showed that HdPRP was constitutively expressed in gill, mantle, digestive tract, hepatopancreas and hemocytes. Animals injected with V. alginolyticus showed that the expression was increased initially within 12 h post injection in gill and it was increased until 48 h. These data indicated that the abalone PRP was constitutively expressed in all the selected tissues and it acts as an acute inducible protein that could play an important role in abalone immune defense mechanism.
Finally, in this study demonstrated that disk abalone has functionally active different polysaccharide degrading enzymes, which are expressed mainly in hepatopancreas. Also, abalone has regucalcin that is important for efficient Ca2+ balance in the body. Also, constitutive expression of PRP in abalone could support to maintain innate immune defense to protect from different microorganisms.
전복은 전세계적으로 많이 양식되는 고부가가치의 패류로 알려져 있다. 본 연구에서는 전복의 생리학적, 생물학적 기능들을 이해하기 위해 다당류분해 (소화), 칼슘 조절 및 면역에 관련된 유전자들에 초점을 맞추었다.
본 연구의 첫번째로 까막전복의 cDNA library로부터 6가지 다당류 분해 효소들(PDEs)에 대해 EST 클론들을 선택하여 확인하고 기능적 특성을 분석하였다. 다당류 분해와 관련된 그 유전자들은 endo β-1,4-glucanase (HdEndg), α-amylase (HdAmy), alginate lyase (HdAlgl), β- mannanase (HdMann), laminarinase (HdLmn)와 arylsulfatase (HdArys)이다.
까막전복의 PDEs염기서열들은 기존에 알려진 다당류 분해 효소의 서열과 비교 분석되어졌고, motif들이 확인되어졌다. 그 염기서열들을 토대로 pMAL fusion protein purification system을 이용하여 재조합 단백질을 생산하였고, 재조합된 PDEs의 기능 분석 및 최적온도, 최적pH, 열안정성과 같은 특성을 분석하였다.
HdEndg을 코딩하는 염기서열은 1824 bp (608 aa)로 분석되었다. HdEndg 효소는 40 oC에서 최적 온도를 나타냈고, pH 4.5에서 최적pH를 나타냈으며 이들 조건하에서 0.3 U/mg의 특이활성을 나타냈다. HdAmy는 1536 bp (511 aa)의 코딩 염기 서열을 가지고 있고 50 oC에서 최적 온도를 나타냈으며, 최적 pH는 7.5에서 나타났다. Alginate lyase는 갈조류의 주된 구조 성분인 alginate를 분해하는 효소로, HdAlgl을 코딩하는 염기서열은 897 bp (298 aa)로 분석되었다. 그 정제된 재조합 단백질은 최적 온도인 40 oC에서 sodium alginate에 대해 2 U/mg의 활성을 나타냈다. HdMann 를 코딩하는 염기서열은 1125 bp (375 aa)로 분석되었고, 처음 18개의 아미노산 잔기는 세포외로 분비시키는 신호서열로 확인되었다. 유전자 데이터베이스 (NCBI)로부터 아미노산 서열을 비교 분석한 결과 Mitilus edulis와 H. discus hannai mananase에 각각 49%, 48%의 유사성을 나타내었다. Sulfatase는 glycosaminoglycans, proteoglycans, steroids, glycolipids와 같은 다양한 기질로부터 sulfate ester bond를 가수분해 한다. 그 서열 특성을 분석한 결과 모티브 및 기능적 아미노산 잔기들이 sulfatase family의 주된 특성을 나타내는 것으로 확인 되었다. HdArys 를 코딩하는 염기서열은 1449 bp (481 aa)로 분석되었고, HdArys의 아미노산 구성 중 24-433 aa 부분에 sulfatase domain을 포함하고 있었다. 그리고 72CSPSR76에는 sulfatase signature motif가 보여졌고, 기질과 결합, 활성, 금속 조절, 효소 안정화 부위 등이 포함되어 있었다. HdArys는 Helix pomatia sulfatase I precursor에 가장 높은 유사성(45%)를 나타냈고 Rattus norvegicus, Bos Taurus, Homo sapiens arylsulfatase B 와도 41%의 유사성을 보였다.
까막전복에서 PDEs의 in vivo 발현 분석을 위해, 초기 8주 동안 먹이를 공급하지 않았다가 그 후 28일 동안 먹이를 공급하였다. 아가미, 맨틀, 근육, 소화관, 간췌장 조직으로부터 mRNA 발현 분석을 위해 Semi quantitative RT-PCR를 수행 하였다. 그 결과 모든 PDEs들이 소화관에서보다 간췌장에서 강하게 발현되었음을 확인하였으며 이것은 소화효소들이 간췌장에 많이 존재하고 있기 때문으로 생각된다. 또한 발현 수준이 서로 다른 유전자들에서 다양하게 보여졌다. 먹이를 공급하지 않은 기간동안 체중이 최대 18.6%까지 소실되었다. 먹이를 공급한 8주 동안은 모든 다당류 분해 효소들이 대조구에 비해 의미있는 감소를 보였다. 먹이를 주고나서 2주 동안은 mRNA 전사 수준이 점차적으로 증가하는 것을 확인하였고 먹이를 준 8주 동안 전복들은 살아남을 수 있었다.
본 연구의 두 번째 파트에서는 칼슘 조절에 관여하는 regucalcin 유전자에 대한 분석과 mRNA 발현 수준을 분석하였다. 칼슘 이온은 많은 세포질과 생리적 기능들에서 근육 수축, 신경 세포 신호 전달 활성화, 세포 분열 및 세포 죽음 등에 2차 전달자로서 관여를 한다. 전복의 regucalcin (HdReg)를 코딩하는 염기서열은 918 bp (305 aa)로 확인되었다. 그 HdReg 아미노산 서열은 기존의 Ca2+ binding domain처럼 EF-hand motif를 포함하지 않았기에, 새로운 클래스의 Ca2+ binding protein으로 제안한다. 유전자 데이터베이스로부터 HdReg의 아미노산 서열을 비교 분석한 결과, 닭과 제브라피쉬의 regucalcin과 45%의 유사성을 나타냈으며, rat과 mouse의 regucalcin과는 44%의 유사성을 보였다. In vivo에서 아가미, 맨틀, 소화관, 근육으로부터 regucalcin의 조직 특이적 발현 분석을 위해 semi quantitative RT-PCR을 수행하였다. 0.5 mg CaCl2/g를 전복의 근육 안쪽에 주사하였고 HdReg의 칼슘 조절 역할을 진단하였다. HdReg mRNA는 아가미, 맨틀, 소화관, 근육 모두에서 발현 되어졌다.
Semi-quantitative RT-PCR 결과 CaCl2 를 근육 안쪽에 투입한 그룹에서 30분 후 근육에서 regucalcin의 mRNA가 의미있게 유도되어졌고, 1시간 후 발현 수준이 최고치에 도달했다. 2시간 째에는 발현 레벨이 감소하는 경향을 보였다. 전체적으로 볼 때 칼슘을 주입 후 근육에서 regucalcin의 발현 레벨이 특이적으로 증가함을 나타냈다.
본 연구의 마지막 파트는 면역에 관계된 pattern recognition 유전자에 초점을 맞추었고, 전복에서 다른 면역 조절인자들에 의한 면역 첼린져 후 면역관련 유전자들의 mRNA 발현 레벨을 확인하였다. pattern recognition protein은 미생물들의 표면상에 공통적인 epitope들의 인지에 의한 초기 면역 반응에서 중요한 역할을 하고 비자기 병원성 분자 패턴들이 붙었을 때 숙주 면역 반응을 시작한다.
까막전복의 whole body cDNA library로부터 Pattern recognition protein (PRP) 유전자가 선택되어졌고 분석 결과 HdPRP를 코딩하는 염기서열은 20 aa의 아미노산 서열을 포함하는 1260 bp (420 aa)로 분석됬다. 그 아미노산 서열은 민물 달팽이인 Biomphalaria glabrata 의 glucan recognition protein (BGRP) 에 50%의 유사성을 나타내었다. Potential polysaccharide binding, putative cell adhesion 그리고 glucanase motifs과 같은 motif 분석 결과 기존의 무척추동물 PRP motif에 조금 변화된 형태의 motif가 발견되었다.
In vivo에서 PRP의 mRNA 발현 수준을 확인하기 위해, 전복에 Vibrio alginolyticus (150 l/animal, OD600=1.0), lipopolysaccharide (4 g/g animal) 와 β-1,3-glucan (6 g/g animal)를 근육 안쪽으로 주입하였고, RT-PCR 결과 HdPRP가 아가미, 맨틀, 소화관, 간췌장, 혈구에서 발현됨을 확인하였다. V. alginolyticus 를 주입하였을 때는 12시간 후 아가미에서 발현이 증가하였고 48시간까지 증가함을 나타냈다. 이들 데이터는 전복의 PRP가 모든 선택된 조직들에서 지속적으로 발현되고 전복 방어 메커니즘에도 중요한 역할을 하는 것으로 판단된다.
결론적으로 본 연구에서 보여지듯이 까막전복에서 기능적으로 활성을 갖는 다양한 다당류 분해 효소들은 주로 간췌장에서 발현되었다. 또한 regucalcin이 체내에서 충분한 칼슘 조절을 위해 중요한 것으로 보여졌다. 그리고 전복에서 PRP의 발현은 다른 미생물들로부터 자신을 보호하기 위한 초기 면역 반응을 주로 도와줄 수 있는 것으로 보여진다.
Chamilani Nikapitiya
Issued Date
Awarded Date
2008. 8
대학원 수산생명의학과
Jehee Lee
Table Of Contents
PART 1: Molecular Characterization and Expression Analysis of Polysaccharide Degrading Enzymes from Disk Abalone, Haliotis discus discus 1
Identification, cloning and sequence characterization of disk abalone PDEs 13
Identification and cloning of PDE related genes from disk abalone cDNA library 13
Sequence characterization of disk abalone PDEs 13
In vivo PDE mRNA expression analysis during starvation and re-feeding 14
Animals 14
Starvation treatment and isolation of abalone tissues 14
Determination of weight loss % during starvation 16
RNA extraction and cDNA synthesis 16
Tissue specific mRNA analysis by RT-PCR 16
Data analysis 17
Functional characterization of PDE in disk abalone 17
Cloning of PDE coding sequences into the pMAL-c2x expression vector 17
Over-expression and purification of recombinant proteins 18
Biochemical characterization of recombinant PDEs 19
Endoglucanase activity assay 19
Alpha amylase activity assay 20
HdAlgl activity assay 20
Sulfatase activity assay 21
Sequence characterization of PDEs 22
Sequence characterization of disk abalone endoglucanase (HdEndg) 22
Phylogenetic analysis of HdEndg 23
Sequence characterization of disk abalone alpha amylase (HdAmy) 31
Phylogenetic analysis of HdAmy 31
Sequence characterization of disk abalone alginate lyase (HdAlgl) 39
Phylogenetic analysis of HdAlgl 39
Sequence characterization of disk abalone β-mannanase (HdMann) 43
Phylogenetic analysis of HdMann 43
Sequence characterization of HdLms 49
Sequence characterization of disk abalone arylsulfatase (HdArys) 51
Phylogenetic analysis of HdArys 52
3D-Structure analysis of HdEndg, HdAmy, HdMann and HdArys 59
In vivo PDE mRNA expression analysis during starvation and re-feeding 62
Determination of weight loss % 62
Tissue specific mRNA expression analysis 62
The mRNA expression of PDEs in abalone hepatopancreas during starvation 65
RT-PCR analysis of antioxidant CuZnSOD expression during starvation 68
Functional characterization of abalone PDEs 70
Purification of recombinant PDEs by pMAL protein purification system 70
Enzymatic activity and biochemical properties of recombinant HdEndg 72
Enzymatic activity and biochemical properties of recombinant HdAmy 75
Enzymatic activity and biochemical properties of recombinant HdAlgl 78
Enzymatic activity and biochemical properties of recombinant HdArys 80

PART 2: Molecular Characterization and Expression Analysis of Calcium Regulatory Protein Regucalcin from Disk Abalone Haliotis discus discus 96
Cloning and sequencing of abalone regucalcin cDNA 101
Abalones 101
CaCl2 administration and tissue collection 101
RNA extraction and cDNA construction 102
Semi-quantitative RT-PCR analysis 102
Sequence analysis 103
Sequence characterization of regucalcin 105
Expression of regucalcin in different tissues 106
Response of intramuscular CaCl2 injection 114

Part 3: Molecular Characterization and Expression Analysis of Pattern Recognition Protein from Disk Abalone Haliotis discus discus 120
Cloning and sequencing of abalone PRP cDNA 125
Animals 125
Bacterial challenge, LPS, β-1,3-glucan injection and tissue collection 126
RNA isolation, cDNA construction and semi quantitative RT-PCR analysis 126
Statistical analysis 127
Sequence characterization of HdPRP full length cDNA 129
Phylogenetic analysis of HdPRP 130
Tissue expression analysis of HdPRP mRNA 137
HdPRP mRNA expression analysis after V. alginolyticus, LPS and β-1,3-glucan induction 137
HdSOD mRNA expression analysis after V. alginolygticus, LPS and β-1,3- glucan induction 138

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