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研究生：	許維哲 Hsu, Wei-Che
論文名稱：	優化馬來蝮蛇蛇毒蛋白的連接區域，RLD迴圈及C端來改善對整合蛋白αvβ3的活性、選擇性及安全性 Optimization of the linker region, RLD loop and C-terminus of rhodostomin to improve integrin αvβ3 activity, selectivity and safety
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：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	90
中文關鍵詞：	整合蛋白αvβ3 、馬來蝮蛇蛇毒蛋白、連接區域、RGD迴圈、C端
外文關鍵詞：	Integrin αvβ3, Rhodostomin, Linker region, RGD loop, C-terminus
相關次數：	點閱：49 下載：0
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整合蛋白是α/β異構雙體穿膜蛋白，作為細胞外基質的配體。他們調控著很多細胞途徑。在血管新生中，整合蛋白對上皮細胞的移動、增生和生存是重要的。過去研究證實整合蛋白功能的抑制對於壓抑腫瘤的血管新生及生長是有所功用的。因此，整合蛋白可能作為藥物設計的目標來治療許多癌症。在哺乳動物中24種整合蛋白的其中8種會辨識細胞外基質蛋白裡的RGD迴圈，而整合蛋白αvβ3及αvβ5會參與腫瘤的血管新生作用。馬來蝮蛇蛇毒蛋白(Rhodostomin, Rho)屬於去整合蛋白，也是其中一種有效能的整合蛋白抑制劑。在我們實驗室過去的研究已經成功地利用馬來蝮蛇蛇毒蛋白去設計一個具有48ARLDDL53迴圈，並對整合蛋白αvβ3有專一性的去整合蛋白。我們也發現在馬來蝮蛇蛇毒蛋白上，除了RGD區域以外，連接區域和C端對它們本身的活性及選擇性是重要的。因此我們將同時突變這些區域來改善對整合蛋白αvβ3有專一性的馬來蝮蛇蛇毒蛋白突變物。在我的研究中我已經成功地在Pichia pastoris表現17個馬來蝮蛇蛇毒蛋白突變物並純化出蛋白。突變物39KKART-46RRARLDDP-66RYH、39KKART-46RRARLDDP-66GLYG、39KKART-46ARARLDDP-66GLYG、39KKART-46ARARLDDL-66GLYG、39KKART-46EEARLDDP-66RYH、39KKART-46DDARLDDL-66GLYG和39KKART-46DDARLDDP-66GLYG對整合蛋白αvβ3的抑制能力分別為36.3、75.2、89.4、177.4、988.6、5761.0和8635.4 nM，這些結果顯示在第46-47殘基進行突變會導致1.1-114倍活性的改變。我們還發現突變物39KKART-46EEARLDDP-66RYH和39KKART-46DDARLDDP-66GLYG對整合蛋白αvβ3的抑制能力分別為988.6和8635.4 nM，顯示C端的重要性。突變物39KKART-46RRARLD52DR-66GLYG、39KKART-46RIARLD52RR-66GLYG、39KKART-46RIARLD52DR-66GLYG和39KKART-46EDARLD52DR-66GLYG對整合蛋白αvβ3的抑制能力分別為454.9、533.8、1190.7和 >17679.5 nM，顯示第46-47殘基以及第52-53殘基對它們本身的活性有重要性。突變物39KKART-46RR48ARLDDP-66RYH、39KKART-46RR48NRLDDP-66RYH和39KKART-46RR48ERLDDP-66RYH對整合蛋白αvβ3的抑制能力分別為36.3、283.5和1018.7 nM，這些結果顯示在第48殘基進行突變會導致3.5-28倍活性的改變。突變物39KKART-46RRARLDD53P-66RYH、39KKART-46RRARLDD53Y-66RYH、39KKART-46RRARLDD53P-66GLYG、39KKART-46ARARLDD53P-66GLYG、39KKART-46ARARLDD53L-66GLYG和39KKART-46RRARLDD53R-66GLYG對整合蛋白αvβ3的抑制能力分別為36.3、72.8、75.2、89.4、177.4和454.9 nM，這些結果顯示在第53殘基進行突變會導致1.9-6倍活性的改變。突變物39KKART-46RRARLDDP54DD-66RYH和39KKART-46RRARLDDP54YD-66RYH對整合蛋白αvβ3的抑制能力分別為36.3和2207.3 nM，顯示第54-55殘基對它們本身的活性有重要性。突變物39KKART-46RRARLDDP-66RNPWNG、39KKART-46RRARLDDP-66RNRFH、39KKART-46RRARLDDP-66RYH、39KKART-46RRARLDDP-66GLYG、39KKART-46EEARLDDP-66RYH和39KKART-46DDARLDDP-66GLYG對整合蛋白αvβ3的抑制能力分別為29.0、30.5、36.3、75.2、988.6和8635.4 nM，這些結果顯示在C端進行突變會導致1-8.7倍活性的改變。以上對整合蛋白αvβ3抑制能力較好的突變物對整合蛋白αvβ3也表現較高的選擇性。這些研究的結果將用於改善設計對整合蛋白αvβ3有專一性的拮抗劑來作為癌症的治療藥物。

Integrins are αβ heterodimeric transmembrane receptors for extracellular matrix ligands. They modulate many cellular processes. During angiogenesis, integrins are essential for endothelial cell migration, proliferation, and survival. Inhibition of integrin function was found to exhibit various activities in suppressing angiogenesis and tumor growth. Thus, integrins can serve as potential therapeutic targets in treating many cancers. Eight of 24 integrins recognize the tripeptide motif Arg-Gly-Asp (RGD) within extracellular matrix (ECM) proteins, and integrins αvβ3 and αvβ5 are involved in tumor angiogenesis. Rhodostomin (Rho) belongs to the disintegrin family and is one of the most potent integrin inhibitors. In our previous study we have successfully used Rho to design an integrin αvβ3-specific disintegrin with a 48ARLDDL53 motif. We also found that not only the RGD region but also the linker region and the C-terminal region of Rho were important for their activity and selectivity. We therefore proposed to mutate these regions at the same time for improving integrin αvβ3-specific Rho mutant. In this study I have successfully expressed seventeen Rho mutants in Pichia pastoris and purified them to homogeneity. The inhibitory αvβ3 activities of Rho 39KKART-46RRARLDDP-66RYH, 39KKART-46RRARLDDP-66GLYG, 39KKART-46ARARLDDP-66GLYG, 39KKART-46ARARLDDL-66GLYG, 39KKART-46EEARLDDP-66RYH, 39KKART-46DDARLDDL-66GLYG and 39KKART-46DDARLDDP-66GLYG mutants were 36.3, 75.2, 89.4, 177.4, 988.6, 5761.0, and 8635.4 nM. These results showed that the mutations on residues 46-47 caused 1.1-114-fold changes in activity. We also found that the inhibitory αvβ3 activities of 39KKART-46EEARLDDP-66RYH and 39KKART-46DDARLDDP-66GLYG mutants were 988.6 and 8635.4 nM, showing an important role of C-terminal region. The inhibitory αvβ3 activities of 39KKART-46RRARLD52DR-66GLYG, 39KKART-46RIARLD52RR-66GLYG, 39KKART-46RIARLD52DR-66GLYG and 39KKART-46EDARLD52DR-66GLYG mutants were 454.9, 533.8, 1190.7, and >17679.5 nM, showing that the residues 46-47 and 52-53 are important for their activities. The inhibitory αvβ3 activities of 39KKART-46RR48ARLDDP-66RYH, 39KKART-46RR48NRLDDP-66RYH and 39KKART-46RR48ERLDDP-66RYH mutants were 36.3, 283.5 and 1018.7 nM. These results demonstrated that the mutations on residues 48 caused 3.5-28-fold changes in activity. The inhibitory αvβ3 activities of 39KKART-46RRARLDD53P-66RYH, 39KKART-46RRARLDD53Y-66RYH, 39KKART-46RRARLDD53P-66GLYG, 39KKART-46ARARLDD53P-66GLYG, 39KKART-46ARARLDD53L-66GLYG and 39KKART-46RRARLDD53R-66GLYG mutants were 36.3, 72.8, 75.2, 89.4, 177.4 and 454.9 nM. These results explained that the mutations on residues 53 caused 1.9-6-fold changes in activity. The inhibitory αvβ3 activities of 39KKART-46RRARLDDP54DD-66RYH and 39KKART-46RRARLDDP54YD-66RYH mutants were 36.3 and 2207.3 nM, showing that the residues 54-55 are important for their activities. The inhibitory αvβ3 activities of 39KKART-46RRARLDDP-66RNPWNG, 39KKART-46RRARLDDP-66RNRFH, 39KKART-46RRARLDDP-66RYH, 39KKART-46RRARLDDP-66GLYG, 39KKART-46EEARLDDP-66RYH and 39KKART-46DDARLDDP-66GLYG mutants were 29.0, 30.5, 36.3, 75.2, 988.6 and 8635.4 nM. These results illustrated that the mutations on C-terminal region caused 1-8.7-fold changes in activity. Rho mutants that exhibited higher activity in inhibiting integrin αvβ3 demonstrated higher selectivity for integrin αvβ3. The results of this study will be used to improve the design of integrin αvβ3-specific antagonist for cancer therapy.

CHINESE ABSTRACT........ I
ABSTRACT.......... III
ACKNOWLEDGEMENT........ V
TABLE OF CONTENTS........ VI
LIST OF TABLES........ IX
LIST OF FIGURES......... X
ABBREVIATIONS........... XI
CHAPTER I INTRODUCTION....... 1
1.1 Integrins............. 1
1.2 The roles of RGD-binding integrins in angiogenesis ..... 2
1.3 Integrin αvβ3 antagonists in clinical trails for cancer therapy........ 3
1.4 Disintegrins............ 4
1.4.1 Rhodostomin (Rho).........6
1.5 Pichia pastoris (P. pastoris) expression system........ 7
1.6 A potent integrin αvβ3-specific rhodostomin mutant...... 8
CHAPTER II RATIONALE AND SPECIFIC AIMS..... 9
CHAPTER III MATERIALS AND METHODS..... 11
3.1 Construction of rhodostomin mutants.......... 11
3.1.1 Lists of host cells and vector......... 11
3.1.2 FastCloning.......... 11
3.1.3 Escherichia coli XL1-Blue strain competent cell transformation...... 12
3.1.4 Pichia pastoris X-33 strain competent cell transformation..... 13
3.2 Expression of rhodostomin mutants........ 14
3.2.1 Lists of culture media and buffer recipes....... 14
3.2.2 Small scale protein expression of rhodostomin mutants....... 15
3.2.3 Large scale protein expression of rhodostomin mutants....... 16
3.3 Purification of rhodostomin mutants........ 16
3.3.1 List of buffer recipes.......... 16
3.3.2 Purification of rhodostomin mutants by nickel chelating chromatography.. 19
3.3.3 Purification of rhodostomin mutants by reverse-phase high performance 
liquid chromatography (RP-HPLC)....... 19
3.4 Mass spectrometric measurement.......... 20
3.5 Cell adhesion competition assay........ 20
3.5.1 Lists of cell lines and culture media....... 20
3.5.1.1 Methods for cell culture......... 22
3.5.2 Extracellular matrix preparation........ 23
3.5.2.1 List of buffer recipes......... 23
3.5.2.2 Fibronectin Purification......... 23
3.5.3 Functional analysis of rhodostomin mutants in inhibiting cell adhesion... 24
CHAPTER IV RESULTS....... 26
4.1 Expression, purification and mass characterization of rhodostomin mutants.... 26
4.2 The effects of linker region, residues adjacent to RLD motif and C-terminal region
of rhodostomin mutants on integrin αvβ3 activities...... 26
  4.2.1 The effects of N-terminal residues adjacent to RLD motif of rhodostomin
mutants on integrin αvβ3 activities....... 27
  4.2.2 The effects of C-terminal residues adjacent to RLD motif of rhodostomin
mutants on integrin αvβ3 activities............ 29
  4.2.3 The effects of C-terminal region of rhodostomin mutants on integrin αvβ3
activities........... 31
4.3 The effects of linker region, residues adjacent to RLD motif and C-terminal region
of rhodostomin mutants on integrin α5β1 activities ............... 31
4.4 The effects of linker region, residues adjacent to RLD motif and C-terminal region
of rhodostomin mutants on integrin αvβ5 activities ............. 32
4.5 The effects of linker region, residues adjacent to RLD motif and C-terminal region
of rhodostomin mutants on integrin αvβ6 activities .............. 33
4.6 The effects of linker region, residues adjacent to RLD motif and C-terminal region
of rhodostomin mutants on integrin αIIbβ3 activities...... 34
CHAPTER V DISCUSSION........ 36
5.1 The relationship between the yields of rhodostomin mutants and the amino acid
patterns around disulfide bonds......... 36
5.2 The effects of residues adjacent to RLD motif of rhodostomin mutants on integrin
αvβ3 recognition............ 36
5.3 The improvement of rhodostomin mutants with higher activity in inhibiting integrin 
αvβ3........... 37
CHAPTER VI CONCLUSIONS AND FUTURE PERSPECTIVE... 39
REFERENCES......... 42
TABLES......... 47
FIGURES.......... 58
APPENDIX TABLES........ 72
APPENDIX FIGURES........ 82
                                    

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Chun-Hao Huang and Woei-Jer Chuang. Development of selective and potent integrin αvβ3- and/or α5β1-specific disintegrins for cancer therapy. National Cheng Kung University, 2015.

Fang-Chi Shen and Woei-Jer Chuang. The role of the XRGD motif of rhodostomin in integrins recognition. National Cheng Kung University, 2010.

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