Structure and Function Relationships of Integrins and Rhodostomin, an RGD-Containing Disintegrin

 

Woei-Jer Chuang (莊偉哲), Chiu-Yueh Chen, Yen-Chin Chen, Szecheng J. Lo, and Ming-Jer Tang

Department of Biochemistry, National Cheng Kung University Medical College, Tainan, Taiwan 701.

 

                    Integrins are a superfamily of ab heterodimeric cell surface receptors that function in cellular adhesion, migration and signal transduction. Nearly half of the over 25 known integrins recognize the RGD sequence in their adhesive proteins. Disintegrins are the RGD/KGD-containing proteins and have high potency in inhibiting integrin function. To date, more than 50 disintegrins have been isolated. They possess both a remarkable sequence homology and an equally notable variability in potency and selectivity in their interactions with integrins. The selectivity of integrins appears to derive from different conformations of RGD determinant and additional binding sites. Rhodostomin (Rho) is a potent inhibitor of platelet aggregation and consists of 68 amino acids including six disulfide bonds and an RGDMP sequence. In order to identify the motifs required for selective recognition of various integrins, we expressed Rho in Pichia pastoris system. Our previous report showed that Rho expressed in P. pastoris possesses the same function and structure as native protein. In this study, we mutated the RGD loop and C-terminal region of Rho and used cell adhesion and platelet aggregation assays to identify the sequences of disintegrins recognized by various integrins. In addition, we determined the solution structures of Rho and its integrin-specific mutants by heteronuclear multidimensional NMR spectroscopy and the hybrid distance geometry-dynamical simulated annealing method. This study may serve as the basis for uncovering the species and tissues in which evolution has utilized this adhesive domain.

 

 

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