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

https://doi.org/10.1006/abbi.1999.1597Get rights and content

Abstract

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.

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    1

    Present address: Department of Ophthalmology, Tulane University School of Medicine, New Orleans, LA 70112.

    2

    Present address: Kimberly-Clark Corporation, WRE, Neenah, WI 54956.

    3

    To whom correspondence should be addressed at National Institutes of Health, Building 10 Room 2A33, 10 Center Drive, MSC 1500, Bethesda, MD 20892-1500. Fax: (301) 402-0043. E-mail: [email protected].

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