Specificities of Heparin-binding Sites from the Amino-Terminus and Type 1 Repeats of Thrombospondin-1

• Published in: Archives of biochemistry and biophysics 2000-02, Vol.374 (1), p.13-23
• Main Authors: Yu, Haini, Tyrrell, David, Cashel, JoAnne, Guo, Neng-hua, Vogel, Tikva, Sipes, John M., Lam, Lun, Fillit, Howard M., Hartman, Jacob, Mendelovitz, Simona, Panel, Amos, Roberts, David D.
• Format: Article
• Language: English
• Subjects: affinity chromatography; Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Cattle; Chromatography, Affinity; Chromatography, High Pressure Liquid - methods; Heparin - chemistry; Heparin - metabolism; heparin-binding proteins; Humans; Kinetics; Molecular Sequence Data; oligosaccharides; Oligosaccharides - chemistry; Oligosaccharides - metabolism; peptides; Radioligand Assay; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Repetitive Sequences, Amino Acid; Swine; Thrombospondin 1 - chemistry; Thrombospondin 1 - genetics; Thrombospondin 1 - metabolism; thrombospondins
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1999.1597

Interactions of heparin with intact human thrombospondin-1 (TSP1) and with two heparin-binding fragments of TSP1 were characterized using chemically modified heparins, a vascular heparan sulfate proteoglycan, and a series of heparin oligosaccharides prepared by partial deaminative cleavage. The avidity of TSP1 binding increased with oligosaccharide size, with plateaus at 4 to 6 and at 8 to 10 monosaccharide units. The dependence on oligosaccharide size for binding to the recombinant amino-terminal heparin-binding domain of TSP1 was the same as that of the intact TSP1 molecule but differed from that of a synthetic heparin-binding peptide from the type 1 repeats, suggesting that the interaction between intact TSP1 and heparin is primarily mediated by the amino-terminal domain. Based on activities of chemically modified heparins, binding to TSP1 depended primarily on 2-N- and 6-O-sulfation of glucosamine and to a lesser degree on 2,3-O-sulfation and the carboxyl residues of the uronic acids. In contrast, all of these modifications were required for binding of heparin to the type 1 repeat peptides. Affinity purification of heparin octasaccharides on immobilized TSP1 type 1 repeat peptides revealed a preference for oligosaccharides containing the disaccharide sequence IdoA(2-OSO3)α1-4-GlcNS(6-OSO3). Binding of these oligosaccharides to the peptide required the Trp residues. These data demonstrate that the heparin-binding specificities of intact TSP1 and peptides from the type 1 repeats overlap with that of basic fibroblast growth factor (FGF2) and are consistent with the ability of these TSP1-derived molecules to inhibit FGF2-stimulated angiogenesis.