整合蛋白是異質雙體的細胞表面黏著受器的一員，媒介許多由「內至外」和「外至內」的訊息傳遞，並且參與許多細胞繁殖、細胞黏著、分離、移動和細胞凋亡現象。在24種整合蛋白裡，有8種整合蛋白是可以有效地辨識蛋白質中的RGD motif作為受質。Rhodostomin (Rho)是由蛇毒純化而來的整合蛋白抑制劑，擁有一個PRGDMP序列，分子由68個胺基酸組成。近年來有許多研究顯示含有RGD序列的蛋白中，改變RGD本身或周圍序列會影響和整合蛋白結合的專一性和親和力。舉例來說，去整合蛋白擁有一個RGDW motif對整合蛋白αIIbβ3有高度抑制能力；去整合蛋白有RGDN motif則選擇性地和整合蛋白αvβ3和α5β1結合。因此，本實驗中利用Rho當作蛋白質鷹架研究在去整合蛋白有個ARGDMX motif在辨識整合蛋白上扮演一個怎樣的角色。首先，先成功地利用P. pastoris表現系統表現以及純化18株Rho ARGDMX突變株。抑制血小板凝集實驗中，Rho突變株以及Rho對血小板的抑制能力並沒有明顯的差別。在細胞黏著的實驗中，Rho的ARGDMX突變株與Rho相較之下抑制整合蛋白αvβ3、αIIbβ3和α5β1的結合能力分別下降了0.8-30.7、2.9-150.2和0.2-65.4倍。有趣的是比較Rho的ARGDMX突變株對Rho的ARGDMP突變株發現針對不同的整合蛋白αvβ3、αIIbβ3和α5β1分別造成40、52和285倍抑制能力下降，這表示X位置的胺基酸改變對於整合蛋白α5β1有顯著的影響而非整合蛋白αvβ3和αIIbβ3。當X位置為疏水性胺基酸如W、F、Y、I、V和A時，突變蛋白對整合蛋白αvβ3有高度親和力；當X位置為P、V和T時則對整合蛋白α5β1有較高的親和力。相反地，帶電荷的胺基酸D、E、K、R和H取代X時，降低了對整合蛋白α5β1的辨識能力。本研究中同時也找到了三種可以選擇性地抑制整合蛋白αvβ3的Rho突變蛋白，分別是ARGDMA、ARGDMI和ARGDMW。而這些研究的結果可作為未來設計對整合蛋白αvβ3或α5β1有專一性的去整合蛋白的基礎。

Integrins are a family of heterodimeric cell-surface adhesion receptors, which mediate the outside-in and inside-out signaling and are involved in cell proliferation, adhesion, detachment, migration, and apoptosis. Eight out of twenty-four integrins recognize an Arg-Gly-Asp (RGD) motif within their ligands. Many studies have shown that alternations in the residues in or flanking the RGD motif of RGD-containing proteins affect their binding specificities and affinities for integrins. For example, disintegrins containing a RGDW motif exhibit higher inhibitory activity for integrin αIIbβ3, and disintegrins containing an RGDN motif selectively bind to integrins αvβ3 and α5β1. Rhodostomin (Rho) is the most potent integrin inhibitor, which is isolated from snake venom and contains a PRGDMP motif and 68 amino acid residues. In this study I used Rho as a protein scaffold to study the role of the ARGDMX motif of RGD-containing disintegrins in recognition of integrins. I have successfully expressed and purified eighteen Rho ARGDMX mutants in P. pastoris. There is no significant difference between Rho and these mutants in inhibiting platelet aggregation. The mutation on the ARGDMX motif of Rho caused 0.8-30.7-, 2.9-150.2-, and 0.2-65.4-folds decreases in inhibiting integrins αvβ3, αIIbβ3, and α5β1 in comparison with those of Rho. The relative sensitivity of this position were α5β1 (285-fold) > αIIbβ3 (52-fold) > αvβ3 (40-fold), showing that the X position of the ARGDMX motif in Rho is very sensitive to integrin α5β1 but not to integrins αvβ3 and αIIbβ3. The results also showed that Rho mutants with hydrophobic amino acid residues, such as W, F, Y, I, V, and A, exhibited higher affinity for integrin αvβ3, and Rho mutant with P, V, and T had higher binding affinity to integrin α5β1. In contrast, Rho mutants with charge amino acid residues, such as D, E, K, R, and H, exhibited lower binding affinity for integrin α5β1. We also found that the mutants ARGDMA, ARGDMI, and ARGRMW can selectively inhibit integrin αvβ3. The results of this study will serve as the basis for designing integrins αvβ3 and α5β1-specific disintegrins.

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陳秋月 利用馬來蝮蛇去整合蛋白以研究整合蛋白們所辨識的序列和製備胺基酸選擇性同位素標示的蛋白。國立成功大學基礎醫學研究所博士論文，2005。
杜威德 利用Rhodostomin當模板設計具整合蛋白a5b1專一性之拮抗劑。國立成功大學生物化學研究所碩士論文，2006。