Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Abstract 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 (SPR) assays to characterize interactions between endostatin, integrins and heparin/heparan sulfate (HP/HS). α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (KD~ 1.8 x10-8 M, 2-state model). Two arginine residues (Arg27 and Arg139) 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 αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to HP/HS. The ectodomain of the α5β1 integrin binds to HP with high affinity (KD=15.5 nM). The direct binding between integrins and HP/HS might explain why both HS and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.