整合蛋白是由α和β兩個異質次單元所構成的穿膜蛋白，會調控細胞跟細胞以及細胞跟胞外間質之間的交互作用。其被發現參與在非常多生理反應中，像是血管新生、血栓、發炎以及骨質疏鬆等。因此，整合蛋白被視為具有潛力的治療標靶。去整合蛋白為含有RGD/KGD motif的蛇毒蛋白或生肽鏈的總稱，其對整合蛋白有高度的親和性而被視為有潛力的拮抗劑。而在過去的研究中發現，去整合蛋白的RGD motif附近區域以及C端區域對於辨識整合蛋白的活性及選擇性扮演著重要的角色。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。Rho為去整合蛋白，來源是馬來蝮蛇，含有六段雙硫鍵，且具有一個PRGDMP motif以及一個PRYH的C端區域。而在我的研究中，主要利用三種策略分別探討C端的重要性：(1)利用wild type的Rho對其C端區域PRYH分別進行alanine scanning的分析。(2)利用對αIIbβ3低親和力的突變株39KKARTICARGRGDNP，將其C端區域以-65PRWNDL, -65PRNRFH, -65PRNPFHA, -65PRNPWNG, -65PRNGLYG, -65PGLYG, -65PGLY and -65PGYH 這些C端保守序列進行置換。(3)利用 39KKARTICARGRGDNP-65PXLYG以不同性質的胺基酸P、Y、R、K、D和E進行置換。首先，我成功的利用P. pastoris系統表現並純化出18個不同的突變蛋白。在第一個策略的細胞黏著實驗中發現，P65A、R66A和Y67A分別對α5β1、αIIbβ3和αvβ6的抑制能力有2.4、5.5和3.3倍的下降。另外，在39KKARTICARGRGDNP-65PXLYG的細胞黏著實驗中發現，R66G、R66P、R66Y、R66D及R66E對αIIbβ3的細胞黏著實驗中，有2~16.4倍的抑制能力的下降，而R66K則保留了對於αIIbβ3的抑制能力。由此可知，在第66位點對於αIIbβ3辨識上，帶正電的胺基酸會有較大的親和力，而帶負電的胺基酸則會降低親和力。而由結果也指出，R66對於C端區域在辨識整合蛋白αIIbβ3上扮演關鍵的角色。而在我的研究中也成功地設計出對於αvβ3及α5β1具有專一性的突變株39KKARTICARGRGDNP-65PDLYG，其對於αvβ3, α5β1, αvβ5, αvβ6, and αIIbβ3的IC50值分別為6.6, 15.4, 833.7, 1996.2, and > 450958.0 nM (12%)。將可作為對αvβ3、αvβ5、αvβ6和α5β1設計拮抗劑，作為設計未來設計藥物的基礎。

Integrin are α/β heterodimeric transmembrane proteins that mediate cell to cell and cell to extracellular matrix interactions. They are involved in many biological processes and serve as important therapeutic targets. Disintegrins are a family of snake venom proteins containing an Arg- Gly-Asp (RGD) motif and have high affinity to integrins. It has been shown that the RGD loop and C-terminal region of disintegrins are important for their activity and selectivity. Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma and contains 68 amino acid residues with a 48PRGDMP motif and 65PRYH C-terminal tail. In this study I used Rho as the protein scaffold to study the effect of C-terminal region on their integrin binding affinities and selectivity. I have successfully expressed three types of Rho C-terminal mutants. The cell adhesion analysis and alanine scanning of 65PRYH region of Rho showed that the activities of P65A, R66A, and Y67A mutants had 2.4-, 5.5-, and 3.3-fold decreases in inhibiting integrins α5β1, αIIbβ3, and αvβ6. The analysis of the 39KKARTICARGRGDNP-65PXLYG mutants also showed that the R66G, R66P, R66Y, R66D and R66E mutants exhibited 2.0-16.4-fold decreases in inhibiting integrin αIIbβ3; however, the R66K mutant retained similar activity in inhibiting integrin αIIbβ3. I also found that 39KKARTICARGRGDNP-65PDLYG mutant was integrins αvβ3 and α5β1-specific disintegrin. These findings showed that C-terminal region of Rho is important for the recognition of integrins, and integrin αIIbβ3 preferred positively charged R and K residues but not negatively charged D and E residues at the 66 position of Rho.

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