The minimal active domain of endostatin is a heparin-binding motif that mediates inhibition of tumor vascularization.
Endostatin constitutes the COOH-terminal 20,000 Da proteolytic fragment of collagen XVIII and has been shown to possess antiangiogenic and antitumorigenic properties. In the present study, we have investigated the role of the heparin-binding sites in the in vivo mechanism of action of endostatin. The majority of the heparin binding is mediated by arginines 155/158/184/270 in endostatin, but there is also a minor site constituted by arginines 193/194. Using endostatin mutants lacking either of these two sites, we show that inhibition of fibroblast growth factor-2-induced angiogenesis in the chicken chorioallantoic membrane requires both heparin-binding sites. In contrast, inhibition of vascular endothelial growth factor-A-induced chorioallantoic membrane angiogenesis by endostatin was only dependent on the minor heparin-binding site (R193/194). These arginines were also required for endostatin to inhibit fibroblast growth factor-2- and vascular endothelial growth factor-A-induced chemotaxis of primary endothelial cells. Moreover, we show that a synthetic peptide corresponding to amino acids 180-199 of human endostatin (which covers the minor heparin-binding site) inhibits endothelial cell chemotaxis and reduces tumor vascularization in vivo. Substitution of arginine residues 193/194 for alanine attenuates the antiangiogenic effects of the peptide. These data show an essential role for heparin binding in the antiangiogenic action of endostatin.
Olsson
Anna-Karin
AK
Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Uppsala, Sweden. Anna-Karin.Olsson@genpat.uu.se
Johansson
Irja
I
Akerud
Helena
H
Einarsson
Barbro
B
Christofferson
Rolf
R
Sasaki
Takako
T
Timpl
Rupert
R
Claesson-Welsh
Lena
L
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002633:Chemotaxis / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply; D043169:Endostatins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000494:pharmacology; D005354:Fibrosarcoma / Q000098:blood supply*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic / Q000188:drug therapy / Q000378:metabolism; D018919:Neovascularization, Physiologic; D010190:Pancreatic Neoplasms / Q000098:blood supply*; D010446:Peptide Fragments / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000494:pharmacology
D043169:Endostatins; D010446:Peptide Fragments; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D006493:Heparin