Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin

Recombinant mouse endostatin produced by mammalian cells was shown to bind to heparin with a Kd of 0.3 mu M, suggesting that this interaction may play a role in its anti-angiogenic activity. Alanine mutagenesis demonstrated t hat a major site of four clustered arginines (positions 155, 158, 184 and 2 70) and a second site (R193,R194) are essential for binding. The same epito pes also participate in endostatin binding to heparan sulfate and sulfatide s but not in its binding to the extracellular protein ligands fibulin-1 and fibulin-2, Analyses with various heparin fragments demonstrated a minimum size (12mer) for efficient binding to endostatin and a crucial role of 2-O- and 6-O-sulfation, Furthermore, a substantial proportion (10-50%) of hepar an sulfate chains obtained from various tissues showed a distinct binding t o endostatin, indicating its potential to interact with extracellular and/o r membrane-bound proteoglycans, Angiogenesis induced by basic fibroblast gr owth factor-2 (FGF-2), but not by vascular endothelial growth factor (VEGF) , in a chick chorioallantoic membrane assay could be inhibited by endostati n in a dose-dependent manner. The mutational block of heparin binding decre ased endostatin inhibition to low levels but elimination of zinc binding ha d no effect.