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研究生：	葉東鉞 Yeh, Tung-Yueh
論文名稱：	利用短鏈去整合蛋白 Echistatin 及第三型纖維黏設計整合蛋白專一性的拮抗劑 Design of Integrin-specific Antagonists Using Echistatin and Fibronectin Type III Domain
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：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	115
中文關鍵詞：	蛋白質工程、纖維黏蛋白、溶解度、熱穩定性、拮抗劑、整合蛋白、抗血小板藥物
外文關鍵詞：	Protein engineering, Fibronectin, Solubility, Thermostability, Antagonist, Integrin, Antiplatelet drug
相關次數：	點閱：77 下載：2
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整合蛋白為α/β組合成的異構二具體的細胞表面穿膜受體，參與了包含細胞的增生、遷移、貼附及凝血作用。在血小板凝集及血栓的形成的過程中，整合蛋白αIIbβ3 的參與是不可或缺的。整合蛋白αvβ3及α5β1在許多類型的癌症中都有廣泛的表現，並且能夠辨識呈現在細胞外基質中的RGD motif。因此，在藥理學上抑制這些整蛋白對於疾病的治療和預防具有重要意義，來自於毒蛇毒液中的去整合蛋白是具有淺力的整合蛋白的天然拮抗劑。根據胺基酸序列的長度及雙硫鍵的數目，其可被分為短、中、長鏈及雙聚體等四類。在我們過去的研究當中，我們利用中鏈的去整合蛋白來設計在抑制血小板凝集中具有非常好的活性及安全指數。”安全指數”一詞的定義建立在固定濃度的AP2作用下，去整合蛋白誘導血小板表面的整合蛋白αIIbβ3合活化所需的濃度。我們也成功地利用人類第三型纖維黏蛋白作為模板設計對於整合蛋白αvβ3及α5β1專一性的重組蛋白。在這篇研究中，我們提出利用短鏈去整合蛋白作為模板來促進對於整合蛋白αIIbβ3拮抗劑的活性，以及導入雙硫鍵到第三型纖維黏蛋白的突變株進而增加其熱穩定性及蛋白活性。Echistatin (Ech)是含有49個胺基酸及4對雙硫鍵的短鏈去整合蛋白。在這篇研究中， Ech-KKKRT、Ech-AKGDRR、Ech-PRNRLYG 及Ech-AKGDRR-PRNRLYG突變株對於抑制血小板凝集的IC50值分別為424.2、536.8、470.3及517.5 nM。相較於中鏈的去整合蛋白，短鏈去整合蛋白在抑制血小板凝集的能力傾向於保留原始的胺基酸序列。我們也發現在Ech含有AKGDRR motif對於CHO表現整合蛋白αIIbβ3和fibrinogen結合呈現差的抑制能力，推測其可能具有較好的安全指數。在第三型纖維黏蛋白的研究中，導入L9C-S95C (SL2)雙硫鍵到整合蛋白αvβ3專一性拮抗劑會增加其熱穩定性，從85℃上升到96.7℃。然而其對於整合蛋白αvβ3的抑制活性從94.0 nM 下降到163.7 nM。而在針對整合蛋白α5β1的第三型纖維黏蛋白以及導入L99C/S180C (SL2)、L2C/S81C (LS1)及D3C/P25C (DP1)雙硫鍵的突變株，熱穩定溫度分別為62.4℃、56.7℃、 62.1℃及58.4℃。對於抑制整合蛋白α5β1的活性分別為61.0、194.6、105.9及59.0 nM。這些結果顯示了透過導入雙硫鍵來增加熱穩定性可能不適用於所有第三型纖維黏蛋白的突變株。本篇研究的結果將作為設計整合蛋白αvβ3、α5β1及αIIbβ3專一性藥物的基礎。

Integrins are α/β heterodimeric cell surface receptors and are involved in cell proliferation, migration, adhesion, and hemostasis. Integrin αIIbβ3 is required for platelet aggregation that is essential for hemostasis and thrombosis. Integrins αvβ3 and α5β1 are widely expressed in different cancer types and recognize the RGD motif present in several extracellular matrix proteins. Therefore, pharmacological inhibition of these integrins is of great interest for the treatment and prevention of disease. Disintegrins are the most potent natural antagonists of integrins from viper venoms. According to the amino acid length and disulfide bonds number, disintegrins are classified into small, medium, large and dimeric disintegrins. In our previous study we have used medium disintegrin to design a mutant with good activity in inhibiting platelet aggregation and with high safety index. The term of ”safety index” value was to determine the level of antagonist-induced integrin αIIbβ3 activation by disintegrins in the presence of AP2.We also have successfully used type III domain of human fibronectin (Fn3) as the scaffold to design integrin αvβ3 and α5β1-specific Fn3 mutants. In this study, we proposed to use short disintegrin as a scaffold to improve the activity of integrin αIIbβ3 antagonist and to incorporate the disulfide bond into Fn3 mutant to improve their thermostability and activity. Echistatin (Ech) is a small disintegrin with 49 amino acids and 4 disulfide bonds. In the study of Ech-KKKRT, Ech-AKGDRR, Ech-PRNRLYG and Ech-AKGDRR-PRNRLYG mutants inhibited platelet aggregation with the IC 50 values of 424.2, 536.8, 470.3 and 517.5 nM. In contrast to medium disintegrin, short disintegrin preferred the Linker, RGD and C-terminal region with wild type sequences in inhibiting platelet aggregation. We also found Ech with AKGDRR motif exhibited low activity in inhibiting the adhesion of CHO-expressing integrin αIIbβ3 to fibrinogen, suggesting that it may have high safety index. In the study of Fn3, the incorporation of L9C-S95C (SL2) disulfide bond into integrin αvβ3-specific Fn3 mutant increased its melting temperature (apparent Tm) value 85 ℃ to 96.7 ℃. However, its inhibitory activity decreased from 94.0 nM to 163.7 nM. The Tm values of integrin α5β1-specific Fn3 mutant, and its L99C/S180C (SL2), L2C/S81C (LS1), and D3C/P25C (DP1) disulfide mutants were 62.4 ℃, 56.7 ℃, 62.1℃, and 58.4 ℃. The inhibitory activity of integrin α5β1-specific Fn3 mutant, and its L99C/S180C (SL2), L2C/S81C (LS1), and D3C/P25C (DP1) disulfide bond mutants were 61.0, 194.6, 105.9, and 59.0 nM. These results showed that the increase of thermostability using the incorporation of disulfide bond may not be applied for all of the Fn3 mutants. The results of this study will serve as the basis for the design of integrin αvβ3, α5β1, and αIIbβ3-specific drugs.

CHINESE ABSTRACT...........................................................................................................................I
ABSTRACT.................................................................................................................................III
ACKNOWLEDGEMENT............................................................................................................................V
TABLE OF CONTENTS........................................................................................................................VII
LIST OF TABLES............................................................................................................................XI
LIST OF FIGURES.........................................................................................................................XIII
ABBREVIATION.............................................................................................................................XIV
CHAPTER I INTRODUCTION.....................................................................................................................1
1    Integrin............................................................................................................................1
1.1   Integrin activation................................................................................................................1
1.2   Integrin αIIbβ3 and platelet aggregation...........................................................................................2
1.3   Integrin expression in caner.......................................................................................................3
1.3.1   Integrin α5β1 expression in caner..............................................................................................4
1.3.2   Integrin αvβ3 expression in caner................................................................................................5
2   Integrin antagonists.................................................................................................................6
2.1    Nature integrin ligand-disintegrin................................................................................................6
2.2    Integrin antibodies...............................................................................................................6
2.3   Peptidic integrin inhibitors.......................................................................................................7
2.4   Small-molecule integrin inhibitors.................................................................................................7
3   Fibronectin as the protein scaffold for drug design..................................................................................7
4   Fibronectin..........................................................................................................................7
4.1   Fibronectin type III domain (Fn3)..................................................................................................9
4.2   The ninth and/or tenth module of fibronectin type III domain (9,10Fn3 or 10Fn3)...................................................10
4.3   Design of integrin α5β1 and αvβ3 specific-antagonists using 9,10Fn3 and 10Fn3 as the scaffold.....................................11
5   Protein engineering.................................................................................................................11
5.1   Pharmaceutical protein formulations...............................................................................................12
5.2   Improvements of protein stability and solubility..................................................................................12
5.3    The incorporation of disulfide bonds into engineer proteins......................................................................13
5.3.1   Disulfide bonds in proteins.....................................................................................................14
5.3.2   Disulfide engineering...........................................................................................................15
5.3.3   Computational methods for disulfide engineering.................................................................................16
5.3.4   Disulfide by Design 2.0.........................................................................................................18
6   Platelets and cardiovascular diseases: acute coronary syndrome and percutaneous coronary intervention...............................20
7   Integrin αIIbβ3 antagonists in clinical use.........................................................................................21
8   Clinical adverse effects of integrin αIIbβ3 antagonists: bleeding and thrombocytopenia..............................................23
9   The challenges of integrin αIIbβ3 antagonists as pharmaceuticals....................................................................23
10   Disintegrins: the scaffolds for design of αIIbβ3 antagonists.......................................................................24
10.1   Trimucrin........................................................................................................................25
10.2   Rhodostomin......................................................................................................................25
10.3   Echistatin.......................................................................................................................26
CHAPTER II   RATIONALE AND SPECIFIC AIMS..................................................................................................27
CHAPTER III   MATERIALS AND METHODS.......................................................................................................29
1   Preparation of recombinant Ech, 10Fn3(CPRGDMPDC), 9,10Fn3(CRARGDNPDC) and their mutants.............................................29
1.1   Construction of Ech, 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC)mutants..............................................................29
1.2   Fast Cloning......................................................................................................................29
1.3   Ligation of inserted DNA and yeast recombination vector...........................................................................30
1.4   Transformation of plasmid into E.coli strain XL1-blue.............................................................................30
1.5   Transformation of linearized plasmid into P. pastoris strain X-33.................................................................31
1.6   Transformation of plasmid into E. coli strain BL21 and SHuffle....................................................................32
2   Expression of Ech, 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) proteins................................................................32
2.1   Small scale protein expression of Ech mutants.....................................................................................32
2.2   Large scale protein expression of Ech mutants.....................................................................................33
2.3   Small scale protein expression of 10Fn3(CPRGDMPDC)  and  9,10Fn3(CRARGDNPDC) mutants..............................................33
2.4   Large scale protein expression of 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) mutants................................................34
3   Purification of Ech, 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) proteins..............................................................34
3.1   Capto MMC and SP chromatography of Ech proteins...................................................................................34
3.2   Ni2+ Chelating chromatography of 10Fn3(CPRGDMPDC) and  9,10Fn3(CRARGDNPDC) Protein................................................35
3.3 	 Reverse-phase high performance liquid chromatography of Ech, 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC)Proteins....................35
3.4   Mass spectrometric measurement....................................................................................................36
4   Platelet aggregation assay..........................................................................................................36
4.1	 Preparation of platelet-rich and platelet-poor plasma............................................................................36
5   Platelet activation assay...........................................................................................................37
6   Cell adhesion assay.................................................................................................................37
6.1   Extracellular matrix preparation..................................................................................................38
6.1.1   Fibronectin purification........................................................................................................38
7   Differential Scanning Calorimetry (DSC).............................................................................................39
7.1   Thermostability analysis of 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) mutants in DSC...............................................39
8   Prediction of disulfide bonds in 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) by DbD2...................................................40
CHAPTER IV   RESULTS......................................................................................................................42
1   Expression, purification, and mass characterization of Ech, 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) mutants........................42
2   Prediction of disulfide bonds in 9,10Fn3(CRARGDNPDC) by bioinformatics tool DbD2....................................................42
3   The effect of introducing a disulfide bond to 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) mutants......................................43
3.1   Thermostability analysis of 10Fn3(CPRGDMPDC) and  9,10Fn3(CRARGDNPDC) by DSC......................................................43
3.2   The Effect of 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) mutants on integrin binding affinity.......................................44
3.3   The Solubility Analysis of 10Fn3(CPRGDMPDC) and 9,10Fn3(CRARGDNPDC) mutants.......................................................44
4   The effect of substituting Tmu-RR Linker, RGD and C-terminal region for Ech on platelet aggregation and affinity to integrin αIIbβ3.45
4.1   The effect of substituting Tmu-RR Linker region for Ech Linker region on platelet aggregation and cell adhesion...................45
4.2   The effect of substituting Tmu-RR RGD region for Ech RGD region on platelet aggregation and cell adhesion.........................46
4.3   The effect of substituting Tmu-RR C-terminal region for Ech RGD region on platelet aggregation and cell adhesion..................46
CHAPTER V   DISCUSSION....................................................................................................................47
1   Introduction of a disulfide bond between N-and C-terminus can increase the thermostability of 10Fn3(CPRGDMPDC)......................47
2   The role of nine module in 9,10Fn3(CRARGDNPDC) on thermostability and activity......................................................47
3   The role of RGD regions on safety index.............................................................................................48
4   The role of C-terminus regions on integrin recognition and platelet aggregation.....................................................48
5   The effects of linker region 14KKKRT sequences on difficulties in protein purification..............................................49
CHAPTER VI   CONCLUSIONS AND FUTURE PERSPECTIVE...........................................................................................50
REFERENCES................................................................................................................................52
TABLES....................................................................................................................................71
FIGURES...................................................................................................................................88
APPENDIX TABLES..........................................................................................................................100
APPENDIX FIGURES.........................................................................................................................105

                                    

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