Binding Interactions of Rhodostomin with Integrins: Analysis by Site-Directed Mutagenesis and Cell Adhesion Assay

 

Chiu-Yueh Chen (陳秋月), Yen-Chin Chen, Szecheng J. Lo, and Woei-Jer Chaung.

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. Based on our preliminary results, the residues adjacent to RGD of Rho also play an important role in recognizing various integrins.

 

 

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