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。在過去關於去整合蛋白研究中，發現了RGD motif 周圍的序列會影響去整合蛋白對整合蛋白辨識，例如：當我們將位於RGD motif N-terminal 的胺基酸由Proline 改變成Alanine 後，會使得去整合蛋白對於整合蛋白α5β1 的親和力增加。因此，在我的研究中，我們想要利用Rhodostomin (Rho)這個去整合蛋白當作一個模板來研究XRGD motif 的X對於去整合蛋白辨識整合蛋白的影響。Rhodostomin (Rho)是一個從馬來腹蛇蛇毒純化出來的去整合蛋白，總共有68 個胺基酸，在結構上帶有6 對雙硫鍵，而且在第48-53 的位置上帶有一個PRGDMP motif。在成功的利用P. pastoris 的蛋白表現系統表現了17 株Rho蛋白的突變株後，根據抑制血小板凝集實驗和細胞黏著抑制實驗所得到的數據，發現位於RGD motif N-terminal 的胺基酸確實在去整合蛋白辨認整合蛋白上扮演著重要的角色。根據17 個Rho 蛋白突變株在抑制整合蛋白αvβ3、α5β1、和αIIbβ3 所得到的IC50數值倍數的改變分別是0.6-3.2 倍、0.2-18 倍、和0.7-30 倍，由這樣的結果可以發現，當我們改變位於RGD motif N-terminal 的胺基酸時，對去整合蛋白和整合蛋白的影響是αIIbβ3>α5β1>αvβ3。我們也發現，當位於RGD motif N-terminal 的胺基酸為酸性胺基酸(Glu 和Asp)時，對於整合蛋白αvβ3 的抑制是有選擇性的。當序列為GRGDMP 時，對於整合蛋白α5β1 和αvβ3 的抑制是有選擇性的。這樣的結果能夠提供做為日後設計對整合蛋白αvβ3 和α5β1 具有特異性的去整合蛋白藥物的參考資料。

Integrins are a large family of α β heterodimeric receptors on cell surface. There are 24 integrins have been identified in mammals, and they mediate cell to cell and extraceullar matrix (ECM) interactions and regulate cell processes, including adhesion, migration, proliferation, and apoptosis. Many members of the integrin family recognize an Arg-Gly-Asp (RGD) motif within their ligands. Disintegrins are a family of RGD-containing proteins isolated from snake venoms and are the most potent integrin antagonists. It is reported that the residues flanking the RGD motif of integrin ligands are important for regulating integrin recognition. For example, it has been demonstrated that the mutation of the residue N-terminal to the RGD motif from proline to alanine on disintegrins can enhance their recognition of integrin α5β1. In this study I used rhodostomin (Rho) as the scaffold to study the role of the residue in the XRGD motif in recognizing integrins. Rho is a disintegrin that contain 68 amino acid including 6 disulfide bonds and a PRGDMP sequence at the positions of 48-53. In this study I have successfully expressed and purified seventeen XRGD mutants in the P. pastoris expression system. According to the results of cell adhesion and platelet aggregation assays, I found that the residue N-terminal to the RGD motif of Rho plays an important role for their integrin recognition. The mutations on the residue N-terminal to the RGD motif in inhibiting integrins αvβ3, α5β1, and αIIbβ3 have affinity changed of 0.6-3.2 folds, 0.2-18 folds, and 0.7-30 folds, respectively. These findings showed that the residue N-terminal to the RGD motif of Rho was more sensitive to integrin α5β1. We also found that Rho mutants containing acidic residues (D and E) N-terminal to the RGD motif selectively inhibited integrin αvβ3, and Rho mutant containing the GRGD motif selectively inhibited integrins αvβ3 and α5β1. The results of this study will serve as the basis for designing integrins αvβ3 and α5β1-specific disintegrins.

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