Design of Integrin α5β1-Specific Antagonist Using the Ninth and Tenth Module of Fibronectin Type III Domain

• Main Authors: Chun-YipPang, 彭俊業
• Other Authors: Woei-Jer Chuang
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/2wwh7a

碩士 === 國立成功大學 === 生物化學暨分子生物學研究所 === 103 === Integrin α5β1 is a fibronectin receptor that regulates complex biological cell behavior, such as differentiation, development, wound healing, and tumor progression. Many studies showed that the blockade of integrin α5β1 inhibited VEGF-induced angiogenesis, leading to the inhibition of tumor growth or tumor regression. In this study we used the ninth and tenth module of fibronectin type III domain (9,10Fn3) as the protein scaffold to study the effect of 1376Pro-His-Ser-Arg-Asn (PHSRN) motif, the synergy site, on its integrin α5β1 binding affinity. In our previous study we have successfully designed an integrin α5β1-specific antagonist, 910LPCRA, with the IC50 value of 65.0 nM. We now used site-directed mutagenesis to engineer the synergy site of 910LPCRA to improve its activity in inhibiting integrin α5β1. In this study I have successfully expressed and purified twenty-nine 9.10Fn3 mutants in E. coli to homogeneity. According to the results of cell adhesion assay, DHSRN and PHSEN mutants exhibited 9- and 〉781- fold decreases in the inhibition of integrin α5β1.The mutation of 1376PHSRN into 1376KHSRN and 1376KKKRT caused 2.9- and 1.8-fold decreases in their activity. This was consistent with the result of molecular docking experiment that a positively charged residue in the1376 position destabilized the salt bridge interaction between R1379 and D154 of integrin α5 subunit. We also found that S1378K mutants exhibited 2-3-fold increase in the inhibition of integrin α5β1. These results showed that the 1377 and 1378 positions with positively charge amino acid residues in the synergy site can increase its integrin α5β1 binding affinity. In particular, the 1376YKKRN mutant exhibited highest inhibitory activity with the IC50 value of 22.8 nM. We also found that the dimeric 910LPCRA (9,10Fn3-9,10Fn3) mutant exhibited 7-fold increase in inhibiting integrin α5β1 with the IC50 value of 9.8 nM. The results of this study will serve as the basis for the design of integrin α5β1-specific antagonist.