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研究生：	張禎 Chang, Chen
論文名稱：	探討 RGD loop 在 Echistatin 中對於辨識整合蛋白所扮演的角色 The role of the RGD loop of Echistatin in the recognition 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：	2023
畢業學年度：	111
語文別：	英文
論文頁數：	102
中文關鍵詞：	整合蛋白、去整合蛋白、鋸鱗蝰素、RGD 迴圈、抗癌藥物
外文關鍵詞：	integrin, disintegrin, echistatin, RGD loop, anticancer drug
相關次數：	點閱：154 下載：0
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整合蛋白是 αβ 異二聚體受體，可以介導細胞與細胞和細胞外基質 (ECM) 的相互作用，並參與細胞粘附、遷移和信號轉導。去整合素是強效的整合蛋白抑製劑，可阻斷整合蛋白與ECM的相互作用，從而達到抑制腫瘤生長的作用。 Rhodostomin (Rho) 和 Echistatin (Ech) 是具有不同 RGD 環序列的去整合素，Rho是PRGDMP ，而Ech是ARGDDM，以及不同的雙硫鍵模式。在我們之前的研究中，我們發現與 RGD環相鄰的殘基會影響Rho和整合蛋白結合的特異性和親和力。例如，ARGDNP 和 ARGDWP 突變體對整合素α5β1表現出高親和力。此外，Echistatin對αvβ3, α5β1, and αIIbβ3整合素受體表現出優異的抑制活性，同時它是一種短鏈解整合素。因此，我們進一步研究與Ech的 RGD環相鄰的C端末端殘基在其與整合蛋白結合的特異性和親和力中的作用。本研究成功在畢赤酵母中表達了13個Ech突變體，純化後的產量為0.5-6.5 mg/L。細胞黏著抑制分析結果表明，Ech 27NM, 27NK, 27NP, 27WP, 27WN, 27DF, 27DA, 27DE, 27DK, 30DR, 21ARGRGDNP, 21RIARGDNP和21RIARGDDM突變體與Ech-WT相比，對整合蛋白α5β1的抑制活性降低了1.4-6003.1倍，對整合素αvβ3的抑制活性降低了4.3-3759.6倍，和對整合蛋白αIIbβ3有3.5-876.6倍的抑制活性下降。然而，與Ech-WT相比，Ech 27WN在抑制血小板聚集方面表現出1.4倍的增加。這些結果與過去我們在Rho的發現不一致，這表明Ech和Rho可能使用不同的區域與整合蛋白相互作用。而Ech和Rho的功能差異可以為設計整合素特異性拮抗劑提供分子基礎。

Integrins are αβ heterodimeric receptors that mediate cell to cell and extracellular matrix (ECM) interactions and involve in cellular adhesion, migration, and signal transduction. Disintegrins are potent integrin inhibitors, which block the interactions between integrin and ECM, thereby achieving the effect of inhibiting tumor growth. Rhodostomin (Rho) and echistatin (Ech) are disintegrins with different RGD loop sequences, PRGDMP in Rho and ARGDDM in Ech, and disulfide bond patterns. In our previous study we found that the residues adjacent to the RGD motif affect Rho’s integrins binding specificities and affinities. For example, the ARGDNP and ARGDWP mutants exhibit high affinity to integrin α5β1. Furthermore, Echistatin has been found to exhibit excellent inhibitory activity against the αvβ3, α5β1, and αIIbβ3 integrin receptors, and it is a short-chain disintegrin. We therefore propose to study the role of C-terminal residues adjacent to the RGD motif of Ech in its integrins binding specificities and affinities. In this study I have successfully expressed thirteen Ech mutants in Pichia pastoris, and the yields after purification were 0.5-6.5 mg/L. The results of cell adhesion analysis showed that Ech 27NM, 27NK, 27NP, 27WP, 27WN, 27DF, 27DA, 27DE, 27DK, 30DR, 21ARGRGDNP, 21RIARGDNP and 21RIARGDDM mutants exhibited 1.4-6003.1-fold decrease in inhibiting integrin α5β1, 4.3-3759.6-fold decrease in inhibiting integrin αvβ3 and 3.5-876.6-fold decrease in inhibiting integrin αIIbβ3 in comparison with those of Ech. Nevertheless, Ech 27WN exhibited 1.4-fold increases in inhibiting platelet aggregation in comparison of that of Ech. These results were not consistent with the findings from Rho, it suggested that Ech and Rho may use different regions to interact with integrins. The functional differences of Ech and Rho can provide molecular basis to design integrin-specific antagonist.

中文摘要	I
ABSTRACT	II
ACKNOWLEDGEMENT	III
TABLE OF CONTENTS	IV
LIST OF TABLES	VIII
LIST OF FIGURES	IX
LIST OF APPENDICES	X
ABBREVIATIONS	XII
CHAPTER I   INTRODUCTION	1
1.1	Background information	1
1.2	Integrins	2
1.2.1	Integrin structure	2
1.2.2	RGD-binding integrins	3
1.2.3	Integrin activation	4
1.2.4	Integrin antagonists as therapeutic agents	5
1.3	Disintegrins	6
1.3.1	Rhodostomin (Rho)	7
1.3.2	Echistatin (Ech)	8
CHAPTER II   RATIONALE AND SPECIFIC AIMS	10
CHAPTER III   MATERIALS AND METHODS	12
3.1	Materials	12
3.2	Construction of Ech mutants	12
3.3	Protein expression of Ech mutants	13
3.3.1	Pichia pastoris expression system	13
3.3.2	Small-scale protein expression of Ech mutants	14
3.3.3	Large-scale protein expression of Ech mutants	15
3.4	Protein purification of Ech mutants	16
3.4.1	Capto MMC chromatography of Ech mutants	16
3.4.2	Reverse-phase high performance liquid chromatography of Ech mutants	16
3.4.3	Mass spectrometric measurement of Ech mutants	17
3.5	Protein purification of fibronectin	18
3.6	Cell cultures	20
3.7	Cell adhesion assay	20
3.7.1	α5β1 Cell adhesion assay	21
3.7.2	αvβ3 and αIIbβ3 Cell adhesion assay	22
3.8	Platelet aggregation assay	23
CHAPTER IV   RESULTS	26
4.1	Expression, purification and mass characterization of echistatin mutants	26
4.2	The effects of Ech mutants on the inhibition of cell-expression integrins αvβ3, α5β1, and αIIbβ3 and platelet aggregation	27
4.2.1	The effects of Ech mutants on the inhibition of cell-expression integrins αvβ3, α5β1 and αIIbβ3	28
4.2.2	The effects of Ech mutants on the inhibition of platelet aggregation	28
4.3	The effects of N28X mutants on the inhibition of cell-expressing integrins αvβ3, α5β1, and αIIbβ3 and platelet aggregation	29
4.4	The effects of W28X mutants on the inhibition of cell-expressing integrins αvβ3, α5β1, and αIIbβ3 and platelet aggregation	29
4.5	The effect of 21RI mutants on the inhibition of cell-expressing integrins αvβ3, α5β1, and αIIbβ3	30
4.6	The effect of D28X mutants on the inhibition of cell-expressing integrins αvβ3 and α5β1		30
4.7	Summarizing Ech mutants with specificity on the inhibition of cell-expressing integrins and platelet aggregation	31
CHAPTER V   DISCUSSION	33
5.1	The effect of Ech mutants on the inhibition of integrins in comparison with those of Rho mutants	33
5.1.1	The effect of Ech-21KRARGDNP on the inhibition of integrins and platelet aggregation in comparison with those of Rho-46RIARGDNP	33
5.1.2	The effect of Ech-21RIARGDNP on the inhibition of integrins and platelet aggregation in comparison with those of Rho-46RIARGDNP	34
5.1.3	The effect of Ech-21KRARGDWP on the inhibition of integrins and platelet aggregation in comparison with Rho-46RIARGDWP	34
5.1.4	The effect of Ech-21KRARGDWN on the inhibition of integrins and platelet aggregation in comparison with Rho-46RIARGDWN	34
CHAPTER VI   CONCLUSIONS AND FUTURE PERSPECTIVE	35
REFERENCES	37
MATERIAL FORMULA	44
TABLES	52
FIGURES	64
APPENDIX TABLES	84
APPENDIX FIGURES	91
                                    

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