Specificities of heparin-binding sites from the amino-terminus and type 1 repeats of thrombospondin-1.
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-OSO(3))alpha1-4-GlcNS(6-OSO(3)). 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.
Yu
H
H
Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Tyrrell
D
D
Cashel
J
J
Guo
N H
NH
Vogel
T
T
Sipes
J M
JM
Lam
L
L
Fillit
H M
HM
Hartman
J
J
Mendelovitz
S
S
Panel
A
A
Roberts
D D
DD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013552:Swine; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D009844:Oligosaccharides; D011994:Recombinant Proteins; D019700:Thrombospondin 1; D006493:Heparin