| Summary: | 碩士 === 國立成功大學 === 生物化學暨分子生物學研究所 === 100 === Integrins are cell-surface adhesion receptors that affect cell adhesion, migration and proliferation. Fibronectin (FN) is an extracellular matrix glycoprotein that binds to integrins. FN is composed of three different types of homologous repeating domains, including FN-I, FN-II, and FN-III. FN contains 12 of FN-I, 2 of FN-II, and 15-17 of FN-III repeats. In particular, FN-III10 can bind to integrin αvβ3 with low affinity. To use FN-III10 as integrin drugs, it is essential to engineer them to have high stability, selectivity, and potency. Therefore, we propose to use FN-III10 as the scaffold to design integrin αvβ3-specific antagonists. In this study I: (1) incorporate the integrin-specific recognition sequences into FN-III based on the results of our study on disintegrin or reported sequences; (2) modify disulfide bond pattern (CXnC) within RGD loop; (3) characterize integrin αvβ3-specific FN-III10 variants; and (4) study anti-tumor activity of integrin αvβ3-specific FN-III10 variants. Now I have expressed 〉 20 FN-III10 variants in E. coli and purified them to homogeneity. Functional analysis showed that all FN-III10 variants had lower activity in inhibiting platelet aggregation, suggesting that they exhibited low thrombocytopenia effect. Cell adhesion inhibition study demonstrated that FN-III10(PRGDMPD) mutant incorporating disintegrin sequence had higher affinity to integrin αvβ3. The incorporation of disulfide linkage into the RGD loop showed that integrin v3-specific FN-III10 variants preferred the CX7C but not CX4C and CX8C patterns, and FN-III10(CPRGDMPDC) selectively inhibited integrin αvβ3 with an IC50 value of 93 nM. Differential scanning calorimetry analysis and solubility measurements by ammonium sulfate precipitation showed that their Tm values and solubility FN-III10(CPRGDMPDC) (85 oC; 27.8 mg/ml) 〉 FN-III10 (81oC; 7.3 mg/ml) 〉 FN-III10(PRGDMPD) (74 oC; 5.3 mg/ml). These results indicate that the incorporation of a disulfide bond into the RGD loop of FN-III10 can increase its thermostability and solubility. NMR and dynamics analyses demonstrated that FN-III10(CPRGDMPDC) has the correct folding and the RGD loop of FN-III10(CPRGDMPDC) exhibited higher rigidity with preference for integrin v3 binding. Integrin αvβ3-specific FN-III10 (CPRGDMPDC) protein can inhibit A375 cell migration and HUVEC tube formation in vitro. The xenograft mouse model showed that it only slightly reduced the growth of Lewis lung cancer cells in C57/B6 mice; however, it can prolong their survival in vivo. In conclusions, we have successfully engineered FN-III10 scaffold to produce an integrin αvβ3-specific variant with high potency, selectivity, thermostability, and solubility. Our results also showed that this integrin αvβ3-specific FN-III10 variant is superior than the reported variant, FN-III10(PRGDWNEG). The further optimization of integrin v3-specific FN-III10 variant for the treatment of integrin αvβ3-related diseases is ongoing.
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