Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha 5 beta 1 and alpha v beta 3 integrins form stable complexes with immobilized endostatin (K-D = similar to 1.8 x 10(-8) M, two-state model). Two arginine residues (Arg(27) and Arg(139)) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alpha v beta 3 integrin at the interface between the beta-propeller domain of the alpha v subunit and the beta A domain of the beta 3 subunit. In addition, we report that alpha 5 beta 1 and alpha v beta 3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha 5 beta 1 integrin binds to haparin with high affinity (K-D = 15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha 5 beta 1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.