Characterization of Endostatin Binding to Heparin and Heparan Sulfate by Surface Plasmon Resonance and Molecular Modeling

Abstract

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...

The First Draft of the Endostatin Interaction Network

“…Known interactions were analyzed to validate SPR arrays and the Biacore Flexchip as reliable tools for the investigation of interactions established by endostatin. We confirmed by this technique previously described interactions between heparin/heparan sulfate and several extracellular proteins including endostatin (8,9,26), collagens I and V (27,28), fibronectin, and transglutaminase-2. Protein-protein interactions between endostatin and laminin (29), endostatin and ␣5␤1 integrin (10,11), or between tissue transglutaminase and ␣5␤1 integrin (30) were also confirmed, as were interactions between proteins and proteoglycans such as the collagen VI-biglycan interaction (31) ( Table 1).…”

“…Source of Proteins and Glycosaminoglycans-Recombinant human endostatin, the trimeric C-terminal domain of collagen XVIII called NC1 and several mutants were produced by human embryonic kidney cells expressing Epstein-Barr virus nuclear antigen (293-EBNA cells) according to established protocols (8,9,11). Amino acid residues were numbered starting from the first amino acid residue of endostatin (His 1 , also referred to as His 132 when numbering starts from the first amino acid of the entire C-terminal domain NC1 of collagen XVIII).…”

“…Endostatin binds to several membrane proteins including ␣5␤1 and ␣v␤3 integrins (10,11), heparan sulfate proteoglycans (glypican-1 and -4) (12), and KDR/Flk1/VEGFR2 (13). We have previously characterized the binding of endostatin to heparan sulfate chains (9), and of endostatin to integrins (11). Furthermore, we have shown that ␣5␤1, ␣v␤3, and ␣v␤5 integrins bind to heparin/heparan sulfate (11).…”

“…It inhibits angiogenesis and tumor growth (1)(2)(3). The effect of endostatin depends on its concentration (4,5), on the length of exposure (6), on the type of endothelial cells (7), and on the growth factor inducing cell proliferation (fibroblast growth factor 2 or VEGF) 3 (8,9). Endostatin binds to several membrane proteins including ␣5␤1 and ␣v␤3 integrins (10,11), heparan sulfate proteoglycans (glypican-1 and -4) (12), and KDR/Flk1/VEGFR2 (13).…”

Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo

“…The immobilization of the biotinylated GAG onto a streptavidin-coated CM4 sensor chip was performed according to an established protocol, described recently (45). The actual binding interactions were recorded at 25°C in PBS, pH 7.4, containing 0.01% (v/v) P20 surfactant (BIAcore AB).…”