Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain

Abstract

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.

Figure 1

A) Schematic representations of the human TSP-1 monomer and the recombinant fragments used in this study. Abbreviation for the structural domains are: N, amino-terminal domain; C, procollagen like domain; P, properdin like, type I repeat; E, EGF-like, type II repeat; Ca, calcium-binding, type III repeats; G, globular carboxy-terminal domain. Residues are related to the initiating methionine. B) Purified recombinant proteins were resolved by SDS-PAGE and stained with Coomassie blue. Molecular weight markers are indicated in kDa. The type III repeats-containing domains migrated more slowly than predicted by their molecular weight, as reported (Anilkumar, Annis, Mosher & Adams, 2002; Hannah, Misenheimer, Annis & Mosher, 2003).

Figure 2

FGF-binding ability of CP123-1, P3E123-1, and E3CaG-1. A) Binding of labeled FGF-2 to plastic coated with increasing concentrations of TSP-1 or TSP-1 fragment. B) Binding of labeled TSP-1 or fragments, at the indicated concentrations, to immobilized FGF-2. Data are the amount of bound FGF-2, as absorbance (Abs), mean and SD of triplicates. C) Formation of the complex between FGF-2 and the indicated TSP-1 fragment in solution. Samples were analyzed in Western blot. Arrow indicates the putative FGF-2/E3CaG-1 complex. Molecular weight markers (in kDa) are on the left. Experiments in A were done in calcium-free buffers, in B calcium concentration was < 0.1 mM.

Figure 3

Localization of the FGF-binding site in the type III repeats of TSP-1. A) Binding of labeled FGF-2 to plastic coated with increasing concentrations of TSP-1 fragments. Data are the amount of bound FGF-2, as absorbance, mean and SD of triplicates. B) Binding of labeled E3Ca-1 or Ca-1, at the indicated concentrations, to immobilized FGF-2. C) Competition assay: binding of labeled TSP-1, E3Ca-1 and Ca-1 to immobilized FGF-2 in the presence of 2.5 µM unlabeled E3Ca-1, Ca-1, or TSP-1 used as competitors (see legend). Data are the percentage of control binding, in the absence of competitors. D) Effect of the monoclonal anti-TSP-1 antibodies B5.2 and D4.6 (50 µg/ml) on the binding of TSP-1, E3Ca-1, or Ca-1 to immobilized FGF-2. Binding is the percentage of control, in the absence of antibodies. Experiments were done in calcium-free buffers.

Figure 4

Real time biomolecular studies of FGF-2 interaction with TSP-derived peptides. A) Overlay of blank-subtracted sensograms showing the binding of FGF-2 (1400, 1120, 840, 560, 280 and 140 nM from top to bottom) to immobilized E123CaG-1. B) Competition assay. FGF-2 was loaded onto the BIAcore CM5 sensorchip containing immobilized E123CaG-1 in the absence or in the presence of the indicated TSP-1-fragment (1.5 µM). Data are the percentage of FGF-2 bound to the sensorchip at the equilibrium in the absence of any competitor. Experiments were done in calcium-free buffers.

Figure 5

Effect of calcium on the interaction of TSP-1 and its fragments with FGF-2. Binding of 20 nM labeled TSP-1, E3Ca-1 or Ca-1 to immobilized FGF-2 was tested in the presence of the indicated concentration of CaCl2. Data are the amount of bound labeled protein, as the percentage of maximal binding (in calcium-free buffer), mean and SD of triplicates, from one experiment representative of 4–6. Inset: labeled FGF-2 and E3CaG-1 in solution were incubated in calcium-free buffer (control) or in the presence of CaCl₂ (1 mM) or EDTA (5 mM). The samples were then analyzed by Western blot.

Figure 6

Antiangiogenic activity of the TSP-1-type III repeats. A) Binding of FGF-2 to endothelial cells. BAEC were incubated with labeled FGF-2 in the absence (control, white column) or presence of the indicated recombinant fragment (7.5 µM grey columns, 15 µM striped columns, 30 µM black columns). The amount of cell-bound FGF-2 is expressed as absorbance. B) Endothelial cell proliferation. BAEC were exposed to the indicated concentration of E3Ca-1 (triangles), Ca-1 (squares), or TSP-1 (25 µg/ml, lines) with (black symbols, solid line) or without (white symbols, dashed line) 5 ng/ml FGF-2 and incubated for 3 days. Proliferation is expressed as absorbance. C) FGF-2-induced angiogenesis in the CAM assay. FGF-2 was administered in the absence or presence of E3Ca-1 on day 8 and pictures were taken 4 days later (n=10). Original magnification: 50 x.