Interaction of Fibroblast Growth Factor-2 (FGF-2) with Free Gangliosides: Biochemical Characterization and Biological Consequences in Endothelial Cell Cultures

Abstract

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125 I-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 being GT 1b Ͼ GD 1b Ͼ GM 1 ϭ GM 2 ϭ sulfatide Ͼ GM 3 ϭ galactosyl-ceramide, whereas asialo-GM 1 , neuraminic acid, and Nacetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM 1 to immobilized FGF-2 indicates that FGF-2/GM 1 interaction occurs with a K d equal to 6 M. This interaction is inhibited by the sialic acid-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), FGF-2(39 -59), , and the basic peptide HIV-1 Tat(41-60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125 I-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 Ͼ GD 1b Ͼ GM 1 Ͼ sulfatide a ϭ sialo-GM 1 . Accordingly, GT 1b ,GD 1b , GM 1 , and GM 2 , but not GM 3 and asialo-GM 1 , prevent the binding of 125 I-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 tested while asialo-GM 1 , neuraminic acid, N-acetylneuramin-lactose, galactosylceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-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 may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization. § Corresponding author. E-mail address: presta@med.unibs.it.© 1999 by The American Society for Cell Biology 313 INTRODUCTIONGangliosides are neuraminic acid (NeuAc) 1 -containing glycosphingolipids. Under physiological conditions, gangliosides are mainly associated to the ce...

Pretreatment with PTD-Calbindin D 28k Alleviates Rat Brain Injury Induced by Ischemia and Reperfusion

“…The mechanism of PTD-mediated protein transduction, independent of energy and receptors binding, has not yet been fully elucidated. A nonspecific electrostatic interaction of basic amino acids PTD with cellular membrane might be the first crucial step (Bellet-Amalric et al, 2000); heparan sulfate in the cytoplasm (Rusnati et al, 1999;Ziegler and Seelig, 2004) and amphiphilic a-helices of PTD might be required during the transduction (Scheller et al, 1999). For the hippocampal slices, we obtained a similar result; after incubation with FITC-labeled PTD-CaBD, most of the cells showed FITC-positive staining.…”

Ganglioside GD1a inhibits HGF-induced motility and scattering of cancer cells through suppression of tyrosine phosphorylation of c-Met

“…When gangliosides are continuously presented in the culture medium with growth factors, the free gangliosides have been reported to inhibit the binding of growth factors to their receptors by direct interaction with growth factors. 25,26,28 However, in our study, we consider that GD1a, which is incorporated on the cell surface, acted as a negative regulator, because HGF-induced motility and tyrosine phosphorylation of c-Met were inhibited by expression of GD1a on the FBJ-LL cell surface induced by transfection of the cells with GM2/GD2 synthase cDNA or incorporation of GD1a on the cell surface by pretreating the cells with GD1a. We recently confirmed that HGF binds on the surface of cells expressing high levels of GD1a (data not shown).…”

“…It has been reported that gangliosides modify the biologic responses of cells to various growth factors, including epidermal growth factor (EGF), 24 fibroblast growth factor, 25 platelet-derived growth factor 26 and nerve growth factor. 27 However, there have been no reports regarding the effect of gangliosides on HGF and its receptor, c-Met.…”

Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis

“…Gangliosides bind FGF1, FGF2, and FGF4 via negatively charged Neu-Ac residues [147,148]. In the extracellular environment, gangliosides compete with free heparin for the binding to the growth factor.…”

“…In the extracellular environment, gangliosides compete with free heparin for the binding to the growth factor. On endothelial cells, free gangliosides prevent the binding of FGF2 to FGFRs and HSPGs, thus inhibiting FGF2-mediated cell proliferation [147].…”