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系統識別號 U0026-0812200915273635
論文名稱(中文) 利用馬來蝮蛇蛇毒蛋白作為模板設計對組合蛋白α5β1具有專一性的去組合蛋白
論文名稱(英文) Design of Integrin α5β1-specific Disintegrin Using Rhodostomin as the Scaffold
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) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 江佩儒
研究生(英文) Pei-ju Chiang
電子信箱 s1696106@mail.ncku.edu.tw
學號 s1696106
學位類別 碩士
語文別 中文
論文頁數 85頁
口試委員 指導教授-莊偉哲
口試委員-周三和
口試委員-鄭宏祺
中文關鍵字 連接區域  去整合蛋白  整合蛋白 
英文關鍵字 linker region  disintegrin  integrin a5b1 
學科別分類
中文摘要 整合蛋白(Integrins)存在於細胞表面的穿膜受體,由α、β次單元所組合而成的異質雙體,媒介著細胞間及與細胞外間質的作用,其所活化的訊息傳遞路徑調控著細胞的生理功能,包括: 細胞黏著、遷移、凋亡以及增生。在許多的細胞外間質中,有整合蛋白結合的序列RGD (Arg-Gly-Asp) motif,而此序列也廣泛地存在於自然界中的蛇毒去整合蛋白,使得這些去整合蛋白可以作為整合蛋白的拮抗劑。在過去的研究文獻中,發現整合蛋白α5β1不正常地過量表現在腫瘤的血管中,顯示出整合蛋白α5β1可作為一個很好的抗血管新生的腫瘤治療標的。Rhodostomin (Rho)是去整合蛋白,可抑制血小板的凝集,具有48PRGDMP53序列,含有68個胺基酸以及六對的雙硫鍵。目前,我們實驗室已經成功的使用Rho作為模板去設計分別針對整合蛋白αIIbβ3及αvβ3有特異性的去整合蛋白。在這個研究中,我們想要利用Rho的模板去設計對整合蛋白α5β1具有特異性的去整合蛋白。在之前的研究以及實驗室研究的結果,均指出連接區域KKKRT序列可以促進去整合蛋白與整合蛋白α5β1的結合;而在P48R的突變則是可以促使Rho與整合蛋白α5β1作專一性的辨認;根據噬菌體呈現(phage-display)的實驗結果,含有RGDGW、RGDGF、RGDGY及RGDGL序列的噬菌體與整合蛋白α5β1較好的親和性;依據整合蛋白α5β1之3D同源模擬結構的結果,發現配體與整合蛋白α5次單元的結合位置較小,和β1次單元則較大。將這些得到的研究資料訊息應用到我的研究設計中,表現了13株連接區域序列為39KKKRT43之Rho突變株,其中一株連接區域序列則是39SRAGK43 (39SRAGK43-48ARGDGF53),並將其純化之。經由質譜儀的分析後,可得到實驗值的分子量與實際值相差小於1Da的蛋白,表示這些Rho突變株的六對雙硫鍵皆有形成。我也利用抑制血小板凝集和抑制細胞黏著的方法,去評估這些蛋白對各個整合蛋白的特異性及其活性。在我的結果中,發現39KKKRT43連接區域序列對於與整合蛋白α5β1的結合能力增加了3.3~3.6倍,此結果也與實驗室先前的研究相符合,39KKKRT43連接區域序列的確可以增加與整合蛋白α5β1的結合能力。在48XRGDXW53的模板中,A48R的突變株對於與整合蛋白αIIbβ3及α5β1的結合能力則是分別變差了2.5-13.1倍和3.5-4.8倍。相較於與F52、 L52、G52及 D52突變株作比較,N52的突變與整合蛋白α5β1有較好的親和性,另外,在第53個位置的突變中,相較於芳香族胺基酸的突變,L53的突變與整合蛋白α5β1的結合能力增加了7.6倍。因此,我們發現了39KKKRT43連接區域序列和去整合蛋白上的R48及L53殘基,對於與各個整合蛋白的結合能力皆有不同的影響,而在未來,這些發現可以提供我們去設計對整合蛋白α5β1有特異性的去整合蛋白。
英文摘要 Integrins are a large family of heterodimeric cell-surface receptors that consist of α and β subunit. Integrins mediate cell-cell and cell-extracellular matrix (ECM) interactions and regulate cellular processes including adhesion, migration, apoptosis and proliferation through modulating downstream signaling pathways. In previous studies, integrin α5β1 was found uniform overexpressed on tumor blood vessels, indicating that integrin α5β1 plays an important role on angiogenesis. Therefore, integrin α5β1 is a potential drug target for antiangiogenetic cancer therapy. Rhodostomin (Rho) is a disintegrin, which can specifically inhibit platelet aggregation. It is composed of 68 amino acids and contains a 48PRGDMP53 sequence with a six-disulfide bond structure. In our previous study we found that Rho with a 39KKKRT43 linker sequence can increase its affinity to integrin α5β1, and P48R mutation can increase its selectivity to integrin α5β1. Phage-display experiments found that the phages containing RGDGW, RGDGF, RGDGY, and RGDGL sequences can selectively bind to integrin α5β1. According to 3D homology model structure of integrin α5β1, integrin α5 and β1 subunits have small and large binding pocket, respectively. Incorporating these information into the design, I expressed thirteen Rho mutants with 39KKKRT43 linker region and one with 39SRAGK43 linker sequence (39SRAGK43- 48ARGDGF53) and purified them to homogeneity. The deviations of the experimental molecular weights to the theoretical values of these mutants were less than 1 Da, indicating the formation of six disulfide bonds. I also used platelet aggregation and cell adhesion assays to examine their specificity and activity to integrinsαIIbβ3, αvβ3, and α5β1. I found that disintegrins with 39KKKRT43 linker sequence exhibited a 3.3-3.6-fold increase in inhibitory activity to integrins α5β1. It is consistent with our previous studies that the incorporation of 39KKKRT43 linker sequence into Rho can increase its binding to integrins α5β1. The A48R mutation on the 48XRGDXW53 motif exhibited 2.5-13.1- and 3.5-4.8-folds decrease in inhibitory activity to integrins αIIbβ3 and α5β1, respectively. In comparison of F52, L52, G52, and D52 mutations, N52 mutation exhibited better affinity to integrin α5β1. In contrast to the mutations on 53 position with aromatic amino acids, we found that L53 mutation exhibit 7.6-fold increase in inhibitory activity to integrin α5β1. In summary, we showed that the mutation on the 39KKKRT43 linker region and on the R48 and L53 residues have some effect on their binding to integrins α5β1, αIIbβ3 and αvβ3. These findings will provide molecular basis to design potent integrin α5β1-specific disintegrins.
論文目次 中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
表目錄 IX
圖目錄 X
縮寫檢索表 XI
儀器 XII

第1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 去整合蛋白(disintegrin)之介紹 4
1-1-3 馬來蝮蛇去整合蛋白(Rhodostomin;簡稱Rho)之介紹 6
1-1-4 酵母菌Pichia pastoris (P. pastoris) 表現系統之介
紹 7
1-1-5 對整合蛋白α5β1與去整合蛋白或與其配體結合的相關
研究 9
1-2 研究動機與目標 10

第2章 材料與方法 12
2-1 Rho與突變株重組蛋白之製備 12
2-1-1 實驗菌株、質體與培養基配方 12
2-1-2 重組基因之構築 14
2-1-3 重組蛋白之表現與純化 17
2-1-4 重組蛋白之質譜鑑定 21
2-2 Rho與突變株重組蛋白抑制血小板凝集功能之研究 22
2-2-1 血小板之製備 22
2-2-2 重組蛋白抑制血小板凝集功能之分析 22
2-3 Rho與突變株重組蛋白抑制細胞黏著之研究 23
2-3-1 細胞培養 24
2-3-2 Fibronectin之製備 25
2-3-4 重組蛋白抑制細胞黏著之分析 27

第3章 結果 29
3-1 Rho突變株重組蛋白之製備與鑑定 29
3-2 Rho與突變株重組蛋白之抑制血小板凝集及對整合蛋白
αIIbβ3、αvβ3及α5β1細胞黏著抑制活性的結果 30
3-2-1 測試抑制細胞黏著條件的結果 31
3-2-2 比較連接區域序列39KKKRT43與39SRAGK43對於抑制血小
板凝集以及整合蛋白αIIbβ3、αvβ3及α5β1黏著抑
制的影響 32
3-2-3 比較Rho突變株中A48及R48對抑制血小板凝集以及整合蛋
白αIIbβ3、αvβ3及α5β1細胞黏著抑制活性的影響 33
3-2-4 在Rho突變株中比較X52分別相對於G52及D52的突變株對
抑制血小板凝集以及整合蛋白αIIbβ3、αvβ3及α5β
1細胞黏著抑制活性的影響 34
3-2-5 探討Rho突變株中X53對抑制血小板凝集以及整合蛋白
αIIbβ3、αvβ3及α5β1細胞黏著抑制活性的影響 35

4章 討論 36
4-1 Rho與其突變株重組蛋白對於與整合蛋白的影響 36
4-2 連接區域序列39KKKRT43對於去整合蛋白Rho與整合蛋白
辨識裡所扮演的角色 37
4-3 R48突變株對於Rho去整合蛋白與整合蛋白α5β1辨認的
影響 39
4-4 設計對整合蛋白α5β1具有特異性序列的Rho突變株之討
論 40
4-5 去整合蛋白的研究對未來的醫藥貢獻 41

第5章 結論 42
參考文獻 44
表 51
圖 56
附錄 71
自述 85
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