探討ARGDMX motif在Rhodostomin中對於辨識整合蛋白αvβ3、αIIbβ3和α5β1所扮演的角色

Integrins are a family of heterodimeric cell-surface adhesion receptors, which mediate the outside-in and inside-out signaling and are involved in cell proliferation, adhesion, detachment, migration, and apoptosis. Eight out of twenty-four integrins recognize an Arg-Gly-Asp (RGD) motif within their ligands. Many studies have shown that alternations in the residues in or flanking the RGD motif of RGD-containing proteins affect their binding specificities and affinities for integrins. For example, disintegrins containing a RGDW motif exhibit higher inhibitory activity for integrin αIIbβ3, and disintegrins containing an RGDN motif selectively bind to integrins αvβ3 and α5β1. Rhodostomin (Rho) is the most potent integrin inhibitor, which is isolated from snake venom and contains a PRGDMP motif and 68 amino acid residues. In this study I used Rho as a protein scaffold to study the role of the ARGDMX motif of RGD-containing disintegrins in recognition of integrins. I have successfully expressed and purified eighteen Rho ARGDMX mutants in P. pastoris. There is no significant difference between Rho and these mutants in inhibiting platelet aggregation. The mutation on the ARGDMX motif of Rho caused 0.8-30.7-, 2.9-150.2-, and 0.2-65.4-folds decreases in inhibiting integrins αvβ3, αIIbβ3, and α5β1 in comparison with those of Rho. The relative sensitivity of this position were α5β1 (285-fold) > αIIbβ3 (52-fold) > αvβ3 (40-fold), showing that the X position of the ARGDMX motif in Rho is very sensitive to integrin α5β1 but not to integrins αvβ3 and αIIbβ3. The results also showed that Rho mutants with hydrophobic amino acid residues, such as W, F, Y, I, V, and A, exhibited higher affinity for integrin αvβ3, and Rho mutant with P, V, and T had higher binding affinity to integrin α5β1. In contrast, Rho mutants with charge amino acid residues, such as D, E, K, R, and H, exhibited lower binding affinity for integrin α5β1. We also found that the mutants ARGDMA, ARGDMI, and ARGRMW can selectively inhibit integrin αvβ3. The results of this study will serve as the basis for designing integrins αvβ3 and α5β1-specific disintegrins.