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 K(d) of 0.3 microM, suggesting that this interaction may play a role in its anti-angiogenic activity. Alanine mutagenesis demonstrated that a major site of four clustered arginines (positions 155, 158, 184 and 270) and a second site (R193, R194) are essential for binding. The same epitopes also participate in endostatin binding to heparan sulfate and sulfatides 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 heparan sulfate chains obtained from various tissues showed a distinct binding to endostatin, indicating its potential to interact with extracellular and/or membrane-bound proteoglycans. Angiogenesis induced by basic fibroblast growth factor-2 (FGF-2), but not by vascular endothelial growth factor (VEGF), in a chick chorioallantoic membrane assay could be inhibited by endostatin in a dose-dependent manner. The mutational block of heparin binding decreased endostatin inhibition to low levels but elimination of zinc binding had no effect.
Sasaki
T
T
Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany.
Larsson
H
H
Kreuger
J
J
Salmivirta
M
M
Claesson-Welsh
L
L
Lindahl
U
U
Hohenester
E
E
Timpl
R
R
D000482:Allantois / Q000098:blood supply; D019943:Amino Acid Substitution; D020533:Angiogenesis Inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002642:Chick Embryo; D002823:Chorion / Q000098:blood supply; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D043169:Endostatins; D016228:Endothelial Growth Factors / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008222:Lymphokines / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020533:Angiogenesis Inhibitors; D002135:Calcium-Binding Proteins; D003094:Collagen; D043169:Endostatins; D016228:Endothelial Growth Factors; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D006497:Heparan Sulfate; D008222:Lymphokines; D010446:Peptide Fragments; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C060820:fibulin; C084612:fibulin 2