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研究生：	陳柔瑞 Chin, Jo-Rhui
論文名稱：	去整合蛋白的KGD迴圈、C端區域以及二聚體對於整合蛋白交互作用所扮演的角色 The Roles of the KGD Loop, C-terminus, and Dimeric Forms of Disintegrins in the Interactions of Integrins
指導教授：	莊偉哲 Chuang, Woei-Jer
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	128
中文關鍵詞：	整合蛋白、馬來蝮蛇蛇毒蛋白、龜殼花蛇毒蛋白、二聚體
外文關鍵詞：	Integrin, Rhodostomin, Trimucrin, dimer
相關次數：	點閱：151 下載：6
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在蛇毒所發現的去整合蛋白（disintegrin）是個有效的整合蛋白（integrin）抑製劑家族，由47至84個氨基酸以及4至7對雙硫鍵所組成。然而，他們抑制很多整合蛋白β1和β3家族並且會造成血小板低下。在本研究中，利用馬來蝮蛇（Rhodostomin, Rho）和龜殼花（Trimucrin, Tmu）蛇毒去整合蛋白作為骨架來探討：（1）二聚體形式的去整合蛋白對於抑制整合蛋白活性的影響；（2）RGD迴圈、連接區域以及C端區域對於結合至整合蛋白所扮演的角色;（3）馬來蝮蛇蛇毒蛋白的C端區域（65PDLX）對於整合蛋白αvβ3和α5β1交互作用所扮演的角色，同時設計對整合蛋白αIIbβ3具有高安全指數的拮抗劑。細胞黏附實驗證實了二聚體蛋白相較於單體蛋白可以提升對整合蛋白的親和力，並且這些整合蛋白相對的提升幅度是αIIbβ3 > αvβ3 ~ α5β1。例如，Rho二聚體蛋白抑制整合蛋白αIIbβ3的活性有12.5倍的提升，IC50為4.7 nM。Rho、KG 和ARLDDL二聚體蛋白對於抑制整合蛋白αvβ3的活性有3.7至4.9倍的提升，IC50為2.9至11.5 nM。有趣的是，KG 二聚體對於抑制整合蛋白α5β1的活性具有4.7倍的提升，IC50為6.6 nM。並且，KG二聚體可以抑制由血管內皮生長因子所誘導的人臍靜脈內皮細胞的增生（VEGF-induced HUVEC proliferation），IC50為141.8 nM，其活性相較於野生型的馬來蝮蛇蛇毒蛋白有78倍的提升。我們發現C端區域由NGLYG突變成NRLYG會增加對整合蛋白αIIbβ3的親和力。相反的，C端區域突變株，Y68E、Y68G和Y68K的分析結果得知他們對於整合蛋白αvβ3和α5β1的活性並沒有顯著的差異。AKGDWN序列的龜殼花蛇毒蛋白突變株抑制血小板凝集的活性高，IC50約51至125 nM，但是具有3至4的低安全指數值。相反的，連接區域為IEEGT序列的突變株具有25至35的高安全指數值，但是活性會低於1000 nM。KGD突變株相對的安全指數值為KGDNP > KGDRP > KGDFP > KGDWN。這些結果證實AKGDRP突變株可能是個有潛力的抗血栓藥物，因為它抑制血小板凝集的活性高達154至192 nM，同時具有12至15的中等安全指數值。另外，我們發現39KKKRT48AKGDRP67PRNGLYG突變株在CHO所表現的αIIbβ3系統中，它抑制由錳離子活化的整合蛋白αIIbβ3相較於靜止狀態的整合蛋白αIIbβ3有五倍的提升。本項研究結果能夠提供未來針對具專一性的整合蛋白藥物設計的基礎。

Disintegrins are a family of potent integrin inhibitors found in snake venoms that contain 47 to 84 amino acids with 4-7 disulfide bonds. However, they inhibit many integrins of the β1 and β3 classes and caused thrombocytopenia. In this study, snake venom disintegrins, Rhodostomin (Rho) and Trimucrin (Tmu), were used as protein scaffolds to study: (1) the effect of dimeric forms on the inhibition of integrins; (2) the roles of RGD loop, linker and C-terminal regions in the binding to integrins; and (3) the role of Rho C-terminal region (65PDLX) in the interactions of integrins αvβ3 and α5β1, as well as to design integrin αIIbβ3-specific antagonist with high safety index. Cell adhesion analysis showed that dimeric proteins in comparison with their corresponding monomers exhibited higher integrin affinity, and the relative increases were integrins αIIbβ3 > αvβ3 ~ α5β1. For example, Rho dimer exhibited 12.5-fold increase in inhibiting integrin αIIbβ3 with the IC50 value of 4.7 nM. Rho, KG (a pan-integrins mutant), and ARLDDL (an αvβ3-specific mutant) dimers caused 3.7-4.9-fold increases in inhibiting integrin αvβ3 with the IC50 values of 2.9-11.5 nM. Interestingly, KG dimer exhibited a 4.7-fold increase in inhibiting integrin α5β1 with the IC50 value of 6.6 nM. It inhibited VEGF-induced HUVEC proliferation with IC50 value of 141.8 nM, causing a 78-fold increase in activity as compared with wild-type Rho. We found that the C-terminal mutation of NGLYG into NRLYG increased its integrin αIIbβ3 binding affinity. In contrast, the analysis of C-terminal mutants Y68E, Y68G, and Y68K showed no significant effect on their integrins αvβ3 and α5β1 activity. Tmu mutants with an AKGDWN motif retained high integrin activity with the IC50 values of 51-125 nM but had low safety index values of 3-4. In contrast, the linker mutants with IEEGT sequence exhibited high safety index values of 25-35 and had lower activity with the IC50 values of ~1000 nM. The analysis of the KGD mutants showed that the relative safety indexes were KGDNP > KGDRP > KGDFP > KGDWN. These results suggest that the AKGDRP mutants may serve as potential antithrombotic drug lead due to high activity in inhibiting platelet aggregation of 154-192 nM and medium safety index values of 12-15. In particular, we found that the 39KKKRT-48AKGDRP-67PRNGLYG mutant exhibited a 5-fold increase in the inhibitory activity toward Mn2+-activated integrin αIIbβ3 as compared to resting form in CHO-expressing αIIbβ3 system. The results of this study will serve as the basis for the design of integrin-specific drugs.

Tables of Contents
CHINESE ABSTRACT	I
ABSTRACT	II
ACKNOWLEDGMENT	IV
TABLE OF CONTENTS	V
LIST OF TABLES	X
LIST OF FIGURES	XII 
ABBREVIATION	XIV
CHAPTER 1   INTRODUCTION	1
1 RATIONALE	1
2 INTEGRINS	2
2.1 Overview of Integrins	2
2.2 Integrins and Ligands Binding	2
2.3 Integrin clustering and Multivalent ligands	4
2.4 Integrins and Ligands Complex Structures	4
2.5 Integrins and Diseases	5
2.6 Integrins and Thromboembolic Disorders	6
2.7 Integrins and Cancers	7
3 DISINTEGRINS	8
3.1 Overview of Disintegrins	8
3.2 Functional Regions of Disintegrins	10
3.2.1 RGD Loop	10
3.2.2 Linker Region	10
3.2.3 C-terminal Region	11
3.3 Biomedical Applications of Disintegrins	11
4 SCAFFOLD FOR DRUG DESIGN	12
4.1 Rhodostomin (Rho)	12
4.2 Trimucrin (Tmu)	13
5 THE CHALLENGES OF INTEGRIN ANTAGONISTS AS PHARMACEUTICALS	13
6 MONOCLONAL ANTIBODY AP2 AS THE DETECTION OF INTEGRIN ACTIVATION	14
7 THE REGULATION OF INTEGRIN ACTIVATION BY MN2+	15
8 MOLECULAR DOCKING	15
CHAPTER 2   SPECIFIC AIMS AND STRATEGIES	17
1 THE EFFECT OF DIMERIC FORMS OF DISINTEGRINS ON THE INHIBITION OF INTEGRINS	17
2 αvβx- AND α5β1-SPECIFIC RHO MUTANTS ON THE INHIBITION OF CELL PROLIFERATION	18 
3 THE ROLES OF RGD LOOP, LINKER AND C-TERMINAL REGIONS OF TMU ON PLATELET AGGREGATION AND SAFETY INDEX VALUE	18
4 THE ROLE OF RHO C-TERMINAL REGION (65PDLX) IN THE INTERACTIONS OF INTEGRINS αvβ3 AND α5β1	20
CHAPTER 3 MATERIALS AND METHODS	21
1 PREPARATION OF RECOMBINANT RHO, TMU, AND THEIR MUTANTS	21
1.1 Construction of Rho and Tmu Mutants	21
1.1.1 List of Strains, Vector and Culture Media Recipes	21
1.1.2 Overlap Extension PCR	23
1.1.3 Ligation of Inserted DNA and Yeast Recombination Vector	24
1.1.4 Transformation of Plasmid into E. coli Strain XL1-Blue	24
1.1.5 Transformation of Linearized plasmid into Pichia strain X-33	25
1.2 Expression of Rho and Tmu proteins	26
1.2.1 List of Culture Media and Solution Recipes	26
1.2.2 Small Scale Expression of Rho and Tmu proteins	27
1.2.3 Large Scale Expression of Rho and Tmu proteins	27
1.3 Purification of Rho and Tmu proteins	28
1.3.1 List of Buffer Recipes	28
1.3.2 Capto MMC Chromatography of Rho and Tmu Proteins	30
1.3.3 Reverse-phase High Performance liquid Chromatography of Rho and Tmu Proteins	31
2 MASS SPECTROMETRIC MEASUREMENT	32
3 PLATELET AGGREGATION ASSAY	32
3.1 List of Solution Recipes	32
3.2 Preparation of Platelet-rich and Platelet-poor plasma	33
3.3 Functional Analysis of Rho and Tmu Proteins in Inhibiting Platelet Aggregation	34
4 PLATELET ACTIVATION ASSAY	35
5 CELL ADHESION ASSAY	36
5.1 List of Cell Lines and Culture Medium	36
5.2 Extracellular matrix preparation	37
5.2.1 List of Solution Recipes	37
5.2.2 Fibronectin Purification	40
5.2.3 Vitronectin Purification	40
5.3 Cell Cultures	42
5.4 Functional Analysis of Rho and Tmu Proteins in Inhibiting Cell Adhesion	42
5.5 Tmu Proteins in Inhibiting Mn2+-activated CHO-αIIbβ3 Adhesion	44
6 CELL PROLIFERATION ASSAY	44
6.1 List of Cell Line and Culture Medium	44
6.2 Functional Analysis of Rho Proteins in Inhibiting Cell Proliferation	46
7 MOLECULAR DOCKING	47
7.1 Molecular Docking of Tmu Mutants into Integrin αIIbβ3	47
CHAPTER 4 RESULTS	48
1 EXPRESSION, PURIFICATION AND MASS CHARACTERIZATION OF RHO AND TMU MUTANTS	48
2 THE EFFECT OF DIMERIC DISINTEGRINS ON INTEGRIN RECOGNITION	49
2.1 The Effect of Dimeric Disintegrins on Platelet Aggregation	49
2.2 The Effect of Dimeric Disintegrins on Cell Adhesion	50
3 THE EFFECT OF KG DIMER ON CELL PROLIFERATION	50
4 THE EFFECT OF RGD LOOP, LINKER, AND C-TERMINAL REGIONS ON PLATELET AGGREGATION AND SAFETY INDEX VALUE	51
4.1 The Effect of KGD Loop (Residues 50-55) on Platelet Aggregation and Safety Index Value	51
4.2 The Effect of Linker Region (Residues 41-55) on Platelet Aggregation and Safety Index Value	52
4.3 The Effect of C-terminal Region (Residues 67-73) on Platelet Aggregation and Safety Index Value	53
5 THE EFFECTS OF RHO AND TMU MUTANTS ON MN2+-ACTIVATED AND RESTING INTEGRIN αIIbβ3 RECOGNITION	54
6 THE EFFECT OF RHO C-TERMINAL REGION (65PDLX) ON INTEGRIN RECOGNITION	55
CHAPTER 5 DISCUSSION	56
1 THE EFFECT OF DIMERIC FORMS OF DISINTEGRINS ON THE INHIBITION OF INTEGRINS	56
1.1 The Ways in which Dimeric Proteins Increase its Binding Affinity to Integrins	56
1.2 The Difference in the Binding Affinity of Dimeric Disintegrins on Integrins αvβ3 and α5β1	57
2 THE ROLES OF RGD LOOP, LINKER AND C-TERMINAL REGIONS OF TMU ON PLATELET AGGREGATION AND SAFETY INDEX VALUE	58
2.1 The Role of R70 residue on Integrin αIIbβ3 Recognition	58
2.2 The Effect of C-terminal Region with 67PRNS Amino Acid Sequence on Safety Index Value	59
2.3 The Effect of Residue 54 with Hydrophobic Amino Acid on Safety Index Value	59
2.4 The Improvement of Tmu Mutants with Higher Activity and Safety Index Value	60
CHAPTER 6 CONCLUSIONS	61
CHAPTER 7 FUTURE PERSPECTIVE	63
REFERENCES	64
TABLES	72
FIGURES	91
APPENDIX TABLES	101
APPENDIX FIGURES	106

 

                                    

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