整合蛋白 (integrins) 為穿越細胞膜的接受器，可調節細胞與其周遭組織如其他細胞以及細胞外間質的黏著，整合蛋白也參與細胞的訊息傳遞，因而影響細胞的形狀、移動以及細胞週期的調控，纖維連結蛋白 (fibronectin) 是一個會與整合蛋白結合的細胞外間質糖蛋白，纖維連結蛋白的組成包含有第一型 (Fn1) 、第二型 (Fn2) 及第三型 (Fn3) 三種不同類別的重複性單元 (homologous repeating domains)，其中第一型 (Fn1) 有12個，第二型 (Fn2) 有2個，第三型 (Fn3) 有15-17個，已知，第三型重複性單元的第九個與第十個模組 (module) 片段 (9,10Fn3) 或第十個模組 (module) 片段 (10Fn3) 可以分別與整合蛋白 α5β1 或 αvβ3 有辨識作用，但運用9,10Fn3 和10Fn3設計對整合蛋白的拮抗劑，必須使其具有較高的溶解度、穩定度、選擇性及活性，因此，本研究我們欲使用 9,10Fn3 和 10Fn3 作為鷹架蛋白 (scaffold) 來分別設計出對整合蛋白 α5β1 和/或 αvβ3 有專一性的拮抗劑 (integrin-specific antagonists)，期望: (1) 藉由結合去整合蛋白 (disintegrin) 的序列以及改變 RGD loop 上雙硫鍵的鍵結模式 (CXnC)，來設計出對整合蛋白 α5β1 和/或 αvβ3 有專一性的突變株; 並且，(2) 對專一性的突變株進行特性分析; 同時，(3) 探討專一性突變株之結構與功能間的關係; 最後，(4) 評價專一性突變株其應用上的意義。目前，已成功地用大腸桿菌表現系統表現出多於20種 9,10Fn3 和 10Fn3 及其突變株，且可以得到純的蛋白產物; 根據核磁共振 (NMR) 分析的結果顯示出，9,10Fn3 和10Fn3及其突變株有正確的摺疊 (fold); 而在加入一對雙硫鍵於 9,10Fn3 和 10Fn3 的RGD loop後，突變株會分別對 α5β1 或 αvβ3 有較高的親合力 (affinity) 與選擇性 (selectivity)，從血小板凝集實驗的結果顯示，Fn3系列突變株較不會影響血小板凝集，表示其較不會引起血小板缺乏症; 利用蛋白抑制細胞黏著性試驗的結果顯示，含有CX8C雙硫鍵的鍵結模式之9,10Fn3突變株對整合蛋白 α5β1 有較高專一性 (specificity)，而含有CX7C雙硫鍵的鍵結模式之10Fn3突變株則對整合蛋白 αvβ3 有較高專一性，並且我們篩選到 9,10Fn3L1408P(CRARGDNPDC) 為能專一性地抑制整合蛋白 α5β1 的突變株，其半抑制濃度為67 nM，而 10Fn3(CPRGDMPDC) 為能專一地抑制整合蛋白 αvβ3 的突變株，其半抑制濃度為93 nM; 此外，含有一對雙硫鍵的突變株，不僅有較高活性，更具有較高的溶解度 (solubility) 與熱穩定度 (thermostability); 經由大小排阻層析 (SEC) 搭配多角雷射光散射 (MALLS) 方法分析顯示，具專一性的突變株皆以單體 (monomer) 存在於溶液中;由熱分析儀 (DSC) 的結果呈現出，對整合蛋白 α5β1 或 αvβ3 有較高專一性的突變株其半解構溫度 (melting temperatures) 各為62.4 ℃和85.0 ℃; 再由硫酸銨沉澱法 (ammonium sulfate precipitation) 求得其溶解度分別為24.4 mg/mL和27.8 mg/mL。我們也利用 X-ray 結晶繞射 (diffraction) 方法解得其結構 (structure)，發現專一性突變株與野生株的結構差異，在 RGD loop 的構形和其中的 R 與 D 之呈現有所不同;嵌合模型的建構 (Haddock docking models) 闡明了，對整合蛋白 α5β1有較高專一性的突變株，其與 α5β1 的嵌合模型具有最低的嵌合能量 (docking energy)。從體內與體外的血管生成實驗顯示出，對整合蛋白 α5β1 或 αvβ3 有較高專一性的突變株，可各別地抑制 VEGF 或 bFGF 所誘導的血管新生 (angiogenesis);將突變株應用在將人類癌細胞植入老鼠的模式中發現，對整合蛋白 α5β1 或 αvβ3 有較高專一性的突變株，可分別地抑制人類黑色素瘤細胞 A375 或肺癌細胞 A549 的生長。此研究，我們設計出對整合蛋白 α5β1 的專一性突變株9,10Fn3L1408P(CRARGDNPDC) 以及對整合蛋白 αvβ3 的專一性突變株10Fn3(CPRGDMPDC)，並且證實其具有抑制血管新生的能力，顯示將其應用在癌症治療 (cancer therapy) 上的潛力。

Fibronectin (Fn) is an extracellular matrix glycoprotein that binds to integrins. Integrins are membrane-spanning receptors that mediate the attachment between a cell and the tissues surrounding it, which may be other cells or the extracellular matrix (ECM). They also play a role in cell signaling and thereby define cellular shape, mobility, and regulate the cell cycle. Fn is composed of three different types of homologous repeating domains, including Fn1, Fn2, and Fn3. Fn contains 12 of Fn1, 2 of Fn2, and 15-17 of Fn3 repeats. In particular, 9,10Fn3 and 10Fn3 can interact with integrins α5β1 and αvβ3, respectively. To use 9,10Fn3 and 10Fn3 as integrin drugs, it is essential to engineer them to have high solubility, stability, selectivity, and potency. Therefore, we propose to use 9,10Fn3 and 10Fn3 as the scaffolds to design integrins α5β1 and/or αvβ3-specific antagonists. In this study I have successfully (1) designed integrin-specific variants by the incorporation of disintegrin sequences and the modification of disulfide bond pattern (CXnC) within RGD loop; (2) characterized the properties of integrins-specific 9,10Fn3 and 10Fn3 variants; (3) determined structure and function relationships of integrins-specific 9,10Fn3 and 10Fn3 variants; and (4) evaluated functional significance of integrins-specific 9,10Fn3 and 10Fn3 variants. I have expressed >20 9,10Fn3 and 10Fn3 variants in E. coli and purified them to homogeneity. NMR analysis showed that recombinant 9,10Fn3 and 10Fn3 variants had the correct folds. After the incorporation of a disulfide bond into the RGD loop of 9,10Fn3 and 10Fn3, they exhibited higher affinity and selectivity to integrins α5β1 and αvβ3. Functional analysis showed that Fn3 variants had lower activity in inhibiting platelet aggregation, suggesting that they had low thrombocytopenia effect. Cell adhesion inhibition study showed that integrin α5β1 preferred the CX8C pattern of 9,10Fn3 variants; and integrin αvβ3 preferred the CX7C pattern of 10Fn3 variants. 9,10Fn3L1408P(CRARGDNPDC) variant selectively inhibited integrin α5β1 with an IC50 value of 67 nM, and 10Fn3 (CPRGDMPDC) variant selectively inhibited integrin αvβ3 with an IC50 value of 93 nM. The incorporation of disulfide bond can increase not only their affinity to integrins but also their solubility and thermostability. Size exclusion chromatography with multi-angles laser light scattering (SEC-MALLS) results showed that integrin α5β1-specific 9,10Fn3 variant and αvβ3-specific 10Fn3 variant were monomer in solution predominately. Differential scanning calorimetry (DSC) data displayed that their melting temperatures were 62.4 ℃ and 85.0 ℃ respectively. Solubility measurements by ammonium sulfate precipitation showed that their solubilities were 24.4 mg/mL and 27.8 mg/mL. X-ray structures of integrin α5β1-specific 9,10Fn3 variant and αvβ3-specific 10Fn3 variant were determined by protein crystallography. Structural comparison between wild-type and these variants showed that the differences were found from the orientation of the RGD-containing loop, as well as the R and D residues. The docking of integrin α5β1-specific 9,10Fn3 variant into integrins showed that its integrin α5β1 had the lowest docking energy. In vitro HUVEC tube formation, migration and in vivo matrigel plug assay revealed that integrin α5β1-specific 9,10Fn3 variant and αvβ3-specific 10Fn3 variant can reduce VEGF-induced and bFGF-induced angiogenesis respectively. The study of mouse xenograft model showed that they significantly suppressed tumor growth with human melanoma cancer A375 cells and human lung cancer A549 cells. We here demonstrate that 9,10Fn3L1408P(CRARGDNPDC) and 10Fn3(CPRGDMPDC) variants with anti-angiogenic property can selectively inhibit integrins α5β1 and αvβ3, indicating their potential use in cancer therapy.

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