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。

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.

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郭瑞庭 馬來蝮蛇蛇毒基因的選殖及蛇毒蛋白之功能及三度空間結構之研究。
國立成功大學生物化學研究所碩士論文，1998。

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。

謝堯勳 探討去組合蛋白之RGD功能區間及C端區域在辨識組合蛋白αvβ3及α5β1的角色。 國立成功大學生物化學研究所碩士論文，2006。

郭芷歆 探討ARGDMX motif在Rhodostomin中對於辨識整合蛋白αvβ3、αIIbβ3、α5β1所扮演的角色。 國立成功大學生物化學研究所碩士論文，2011。