Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K D ϳ 2 M). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg 47 and Arg 66 , participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl 2 to endostatin enhanced its binding to heparan sulfate by ϳ40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.Endostatin (20 kDa), first identified by O'Reilly et al. (1), is a proteolytic fragment from the C-terminal domain of collagen XVIII. It inhibits tumor angiogenesis by interfering at several levels with growth factor signaling (2). Two heparin-binding sites have been identified in endostatin involving two clusters of arginine residues, and a zinc binding site is located in the N-terminal part of the molecule (see Refs. 3-5 for reviews). The possibility that its anti-angiogenic effect might be related to displacement of angiogenic factors from the surface of endothelial cells through binding of heparan sulfate has prompted several investigations of its interaction with heparin and heparan sulfate (6 -10). The role of zinc in the biological activity of endostatin remains controversial. Zinc binding has been reported to be essential for the anti-angiogenic activity of endostatin (11). Indeed, mutations of amino acids involved in zinc coordination to alanines significantly reduce its anti-tumoral activity, the mutants being unable to cause regression of Lewis lung carcinoma (11). On the other hand, zinc binding does not appear to be critical for the inhibitory effects of endostatin on VEGF 1 -induced migration and tumor growth in mice (12). However, it is of crucial importance to determine the co-factors, if any, that participate in the bind...

[reactions: see text] The glycosylation with trichloroacetimidates derived from different glycopyranoses bearing a nonparticipating group at C-2 was explored in different ionic liquids as solvents. The stereoselectivity of the reaction was significantly affected by the reaction media and by the anomeric configuration of the donor.

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