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系統識別號 U0026-0908201213041500
論文名稱(中文) 利用馬來腹蛇蛇毒蛋白探討去整合蛋白C端序列對於整合蛋白的辨識及受質誘導結合位的表現
論文名稱(英文) The Use of Rhodostomin to Study the Effect of C-terminal Region of Disintegrin on Recognition and Ligand-Induced Binding Site of Integrins
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 100
學期 2
出版年 101
研究生(中文) 梁天豪
研究生(英文) Tien-Hao Liang
學號 S16994051
學位類別 碩士
語文別 中文
論文頁數 125頁
口試委員 指導教授-莊偉哲
口試委員-王淑鶯
口試委員-羅玉枝
中文關鍵字 馬來腹蛇蛇毒蛋白  C端序列  整合蛋白  去整合蛋白 
英文關鍵字 Rhodostomin  C-terminal region  integrin  disintegrin 
學科別分類
中文摘要 整合蛋白由兩個異質次單元所構成,會調控細胞和細胞以及細胞或胞外間質之間的交互作用。在哺乳類動物中參與像是細胞黏著、分離、凋亡及訊息傳遞等重要的生理機制。在已被發現的24種整合蛋白中,有8種為辨識受質上具有的RGD motif而進行結合。去整合蛋白為含有RGD/KGD motif的總稱,其對整合蛋白有高度的親和性而被視為有潛力的拮抗劑。在過去的研究中,發現去整合蛋白C端區域對於整合蛋白的辨識有高度的影響力。舉例來說,去整合蛋白Echistatin對於整合蛋白αvβ3及αIIbβ3有高度的親和力,並會造成整合蛋白的結構進行改變而暴露出受質誘導結合位抗原(Ligand-induced binding site epitope)。而在對去整合蛋白Echistatin C端的9個胺基酸序列做剔除,發現對於整合蛋白αIIbβ3的抑制能力降低,而經由轉殖表現的整合蛋白αIIbβ3及αvβ3其也沒有產生LIBS的表現,但還是保有抑制整合蛋白αvβ3的能力。因此,在本篇研究中,我們利用馬來蝮蛇毒液粹取出的蛇毒蛋白Rhodostomin(Rho)做為模式蛋白探討去整合蛋白C端序列對於整合蛋白的辨識所扮演的角色。在我的研究中,在對所有中等大小的蛇毒蛋白序列做分析比較,選出常見於C端的六個序列,-P65RWNDL, -P65RNRFH, -P65RNPFHA, -P65RNPWNG, -P65RNGLYG, 以及-P65GLYG,,搭配RGD motif之序列為野生型的P48RGDMP、專一抑制整合蛋白αvβ3的A48RLDDL以及專一抑制整合蛋白αIIbβ3的A48KGDWN。首先,我們成功的利用P. pastoris系統表現出18個不同活化區間序列搭配不同C端序列的突變株。在活性區間序列為野生型搭配不同C端序列的突變株對不同整合蛋白的辨識能力互相比較下,發現對整合蛋白αIIbβ3、αvβ5、αvβ3及α5β1分別有13倍、7倍、4倍及2倍的差異,在抑制血小板凝集則是有2倍的差異。在專一結合整合蛋白αvβ3的A48RLDDL序列搭配不同的C端序列中,和A48RLDDL-P65RYH比較下,發現對於整合蛋白αvβ3的抑制能力有提升1.0~3.8倍,而對於整合蛋白α5β1、αIIbβ3及血小板凝集則是有1.0-10.0, 2.3-6.3, 2.0-13.0倍下降的趨勢。在專一結合整合蛋白αIIbβ3的A48KGDWN序列搭配不同的C端序列中,和A48KGDWN-P65RYH比較下,發現對於整合蛋白αIIbβ3的抑制能力下降0.2-2.6倍,抑制血小板凝集則是下降0.5~2.0倍,對於整合蛋白α5β1、αvβ3則沒有很大的差別。另外,我們也發現C端序列會造成對整合蛋白LIBS的表現,並且大部份的突變株在對於整合蛋白LIBS的表現量和對整合蛋白所造成的抑制黏著能力是呈現正相關的,使我們可以利用LIBS的表現量來解釋去整合蛋白抑制整合蛋白黏著能力的情形。在本篇研究中,我們發現C端序列在辨識整合蛋白αIIbβ3上有較高的敏感度。並且,發現突變株重組蛋白A48RLDDL-P65GLYG對整合蛋白αvβ3具有更高的專一性,也發現其可以抑制黑色素細胞瘤A375的爬行。這些研究結果可以做為未來設計對整合蛋白αvβ3及αIIbβ3有專一性的去整合蛋白的基礎。
英文摘要 Integrins are α/β heterodimeric receptor that mediated cell to cell and cell to extracellular matrix interaction. They are involved in cell adhesion, detachment, apoptosis and signal transduction in mammals. Eight out of twenty-four integrins recognize an Arg-Gly-Asp (RGD) motif within their ligands. Disintegrins are a family of RGD/KGD containing proteins have high affinity to integrins and are the most potent integrin antagonists. It has been shown that C-terminal region of disintegrins are important for their activity and selectivity. For example, echistatin binds to αvβ3 with high affinity and αIIbβ3 and induces extensive conformational changes resulting in expression of ligand-induced binding site (LIBS) epitopes. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and failed to induce LIBS epitopes on cells transfected with αvβ3 and αIIbβ3 genes, but it retained its ability to bind purified αvβ3. In this study I used rhodostomin (Rho) as protein scaffold to study the effect of C-terminus of disintegrins on their activity of integrins. Rho is a snake venom protein isolated from Calloselasma Rhodostoma and contains 68 amino acid residues with a PRGDMP motif. Six C-terminal regions, including -P65RWNDL, -P65RNRFH, -P65RNPFHA, -P65RNPWNG, -P65RNGLYG, and -P65GLYG, fused with wild-type, integrin v3-specific A48RLDDL mutant, and αIIbβ-specific A48KGDWN mutant of Rho were constructed. I have successfully expressed eighteen C-terminal region mutants in P. pastoris and purified to homogeneity. Cell adhesion analysis showed that the relative sensitivity on mutation of C-terminal region with Rho was αIIbβ3(13-fold)>αvβ5(7-fold)>αvβ3(4-fold)>α5β1(2-fold) ≈ platelet aggregation(2-fold). The C-terminal mutants fused with Rho A48RLDDL motif caused 0.9-5.2-folds increase in inhibiting integrins αvβ3 and 1.0-10-, 2.3-6.3-, 2.0-13-folds decrease in inhibiting α5β1, αIIbβ3 and platelet aggregation. The C-terminal mutants fused with Rho A48KGDWN motif caused 0.2-2.6-folds decrease in inhibiting integrins αIIbβ3, and less effect on inhibiting integrins α5β1 and αvβ3. We also found that Rho C-terminal mutants can induce LIBS on integrin β3, and the levels of LIBS induced by the mutants were correlated with their activity in inhibiting cell adhesion. In conclusions, we found that C-terminal region of disintegrin are more sensitivity to integrin αIIbβ3. Based on the results of Rho mutant, we designed a potent and selective integrin αvβ3-specific mutant, an A48RLDDL-P65GLYG mutant. It can inhibit the migration of human melanoma cell A375 with the IC50 value of 118 nM. The study will serve as the basis for designing integrin αvβ3 and αIIbβ3-specific disintegrin.
論文目次 目錄
中文摘要 I
英文摘要 III
誌謝 IV
目錄 V
表目錄 VIII
圖目錄 IX
附錄表目錄 X
附錄圖目錄 XI
縮寫檢索表 XII
儀器 XIII

第一章 緒論 1
1-1 背景資料 1
1-1-1 整合蛋白(integrin)之介紹 1
1-1-2 整合蛋白與疾病 4
1-1-3 去整合蛋白(disintegrin)之介紹 4
1-1-4 蛇毒蛋白及馬來腹蛇去整合蛋白(Rhodostomin) 6
1-1-5 整合蛋白與去整合蛋白之受體辨識區域相關研究及應用 7

第二章 研究目標與策略 12

第三章 材料與方法 15
3-1 Rhodostomin與C端突變蛋白的製備 15
3-1-1 實驗菌株、質體和培養基配方 15
3-1-2 重組基因之建構 17
3-1-3 重組蛋白之表現與純化 20
3-1-4 重組蛋白之質譜鑑定 26
3-2 Rhodostomin與C端突變蛋白抑制血小板凝集功能之研究 27
3-2-1 血小板之製備 27
3-2-2 C端突變蛋白抑制血小板凝集功能之分析 28
3-3 細胞株及培養方法 29
3-3-1 細胞培養 29
3-4 Rhodostomin與C端突變蛋白抑制細胞黏著之研究 32
3-4-1 ECM之製備 32
3-4-2 突變株重組蛋白抑制細胞黏著之分析 37
3-5 Rho與C端突變蛋白導致整合蛋白LIBS表現之研究 39
3-5-1 C端突變蛋白導致整合蛋白LIBS表現之分析 40
3-6 C端突變蛋白抑制黑色素細胞瘤A375 cell細胞爬行之研究 41
3-6-1 C端突變蛋白抑制黑色素細胞瘤A375 cell細胞爬行之分
析 42

第四章 結果 43
4-1 Rhodostomin以及C端突變蛋白的製備和鑑定 43
4-2 C端突變蛋白對於抑制血小板凝集的結果 44
4-3 C端突變蛋白抑制細胞黏著的結果 45
4-3-1 細胞黏著實驗條件 45
4-3-2 RGD motif P48RGDMP搭配不同C端序列對整合蛋白黏著
的影響 47
4-3-3 RGD motif A48RLDDL、A48KGDWN搭配不同C端序列對整
合蛋白黏著的影響 47
4-3-4 C端突變蛋白之間之比較及對於整合蛋白所造成之影響 49
4-4 C端突變蛋白誘導LIBS表現的結果 49
4-4-1 誘導LIBS表現之實驗條件 50
4-4-2 C端突變蛋白對於整合蛋白αvβ3的LIBS表現量結果 50
4-4-3 C端突變蛋白對於整合蛋白αIIbβ3的LIBS表現量結果 51
4-5 Rho及針對整合蛋白αvβ3有特異性之C端突變蛋白對於
黑色素細胞瘤的影響 51
4-5-1 A375細胞表面整合蛋白含量之檢定 52
4-5-2 Rho及針對整合蛋白αvβ3有特異性之C端突變蛋白抑
制黑色素胞瘤的移動 53
第五章 討論 54
5-1 C端突變蛋白對於整合蛋白的影響 54
5-1-1 抑制血小板凝集實驗以及整合蛋白αIIbβ3黏著實驗
的探討 54
5-1-2 R66對於整合蛋白αIIbβ3之影響探討 55
5-2 C端突變蛋白對於整合蛋白LIBS表現量之探討 55

第六章 結論 58

參考文獻 60
表 64
圖 71
附錄 100

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