Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: Biochemical characterization and biological consequences in endothelial cell cultures

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. He re, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but n ot heat-denatured, I-125-FGF-2 binds to micelles formed by gangliosides GT( 1b), GD(1b), or GM(1). Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency bein g GT(1b) > GD(1b) > GM(1) = GM(2) sulfatide > GM(3) = galactosyl-ceramide, whereas asialo-GM(1), neuraminic acid, and N-acetylneuramin-lactose were in effective. Scatchard plot analysis of the binding data of fluorochrome-labe led GM(1) to immobilized FGF-2 indicates that FGF-2/GM(1) interaction occur s with a K-d equal to 6 mu M. This interaction is inhibited by the sialic a cid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129 ) and, to a lesser extent, FGF-2(130-155), whereas peptides FGF-2(10-33), F GF-2(39-59), FGF-2(86-96), and the basic peptide HIV-1 Tat(41-60) were inef fective. These data identify the COOH terminus of FGF-2 as a putative gangl ioside-binding region. Exogenous gangliosides inhibit the binding of I-125- FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT(1b) > GD(1b) > GM(1) > sulfatide a = sialo-GM(1). Accordingly, GT(1b ),GD(1b), GM(1), and GM(2), but not GM(3) and asialo-GM(1), prevent the bin ding of I-125-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM(1) to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT(1b) was the most effective among the gangliosides te sted while asialo-GM(1), neuraminic acid, N-acetylneuramin-lactose, galacto sylceramide, and sulfatide were ineffective. Ln conclusion, the data demons trate the capacity of exogenous gangliosides to interact with FGF-2. This i nteraction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic ac id residue(s) in the ganglioside molecule. Exogenous gangliosides act as FG F-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides ma y affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.