Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Reidunn Jetne,Naomi Fukai,Florence Ruggiero,Sylvie Ricard-Blum,Christophe Moreau,Bjorn R. Olsen,Emilie Chautard,Martin J. Humphries,Clément Faye

doi:10.1074/jbc.m109.002840

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 assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-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 alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

Highlights

Endostatin is an endogenous inhibitor of angiogenesis that inhibits proliferation and migration of endothelial cells [1,2,3]
The broad range of molecular targets of endostatin suggests that multiple signaling systems are involved in mediating its anti-angiogenic action [11], and several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanisms of action are not as fully elucidated as they are for other endogenous angiogenesis inhibitors [11]
Interactions between several integrins and endostatin have been studied previously in solid phase assays [12] and in cell models [12, 15, 16], no molecular data are available on the binding site of endostatin to the integrins

Summary

EXPERIMENTAL PROCEDURES

Source of Proteins and Glycosaminoglycans—Recombinant human endostatin, the trimeric C-terminal domain of collagen XVIII called NC1 (noncollagenous 1) and several mutants (D104N and the double mutant R27A/R139A) were produced by human embryonic kidney cells expressing Epstein-Barr virus nuclear antigen (293-EBNA cells) according to established protocols [9, 10]. Inhibition experiments were performed by preincubating a RGD-containing peptide (GRGDSPK; Sigma) diluted at 50 ␮g/ml in the running buffer with ␣v␤3 and ␣5␤1 integrins (30 ␮g/ml) at room temperature for 1 h before injection over immobilized endostatin. Full-length ␣5␤1, ␣v␤3, and ␣v␤5 integrins (30 ␮g/ml) were injected at 20 ␮l/min for 4 min over immobilized glycosaminoglycans with 25 mM Tris, pH 7.5, containing 150 mM NaCl, 0.1 mM CaCl2, 2 mM MgCl2, and 0.005% P20 as running buffer. The cells were harvested by treatment with 1% EDTA at ϳ80 –90% confluence and were suspended (3 ϫ 105 cells/ml) in 25 mM Tris, pH 7.4, containing 150 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, and P20 0.005% They were stored in a water bath at 37 °C without shaking until injection and were examined under an optical microscope to check cell viability before injection at 30 ␮l/min for 5 min over immobilized heparin or heparan sulfate in a Biacore 3000. As the circularity index approaches 0, it indicates an increasingly elongated shape, whereas a value of 1 indicates a perfect circle

RESULTS

SPR binding assays

Heterogeneous ligand Heterogeneous ligand

Integrin subunit

Heparan Sulfate at the Cellular

Findings

DISCUSSION