Rusnati M, Tanghetti E, Urbinati C, Tulipano G, Marchesini S, Ziche M, Presta M
Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, 25123 Brescia, Italy.
Mol Biol Cell. 1999 Feb;10(2):313-27. doi: 10.1091/mbc.10.2.313.
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, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM. 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), FGF-2(86-96), 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 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 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, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, 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.
外源性神经节苷脂可影响成纤维细胞生长因子-2（FGF-2）的血管生成活性，但其作用机制尚未阐明。在此，研究了唾液酸糖脂与FGF-2之间可能的直接相互作用。尺寸排阻色谱法表明，天然的而非热变性的125I-FGF-2可与由神经节苷脂GT1b、GD1b或GM1形成的胶束结合。此外，神经节苷脂在微摩尔浓度下可保护天然FGF-2不被胰蛋白酶消化，相对效力顺序为GT1b > GD1b > GM1 = GM2 = 硫脂 > GM3 = 半乳糖神经酰胺，而脱唾液酸GM1、神经氨酸和N-乙酰神经氨酸乳糖则无效。对荧光染料标记的GM1与固定化FGF-2的结合数据进行Scatchard作图分析表明，FGF-2/GM1相互作用的解离常数（Kd）等于6微摩尔。这种相互作用受到唾液酸结合肽蜂毒素以及合成片段FGF-2(112 - 129)的抑制，在较小程度上也受到FGF-2(130 - 155)的抑制，而肽FGF-2(10 - 33)、FGF-2(39 - 59)、FGF-2(86 - 96)以及碱性肽HIV-1 Tat(41 - 60)则无效。这些数据确定FGF-2的COOH末端为假定的神经节苷脂结合区域。外源性神经节苷脂在微摩尔浓度下可抑制125I-FGF-2与内皮GM 7373细胞的高亲和力酪氨酸激酶FGF受体（FGFRs）结合。相对效力顺序为GT1b > GD1b > GM1 > 硫脂a = 唾液酸GM1。因此，GT1b、GD1b、GM1和GM2可阻止125I-FGF-2与可溶性重组细胞外FGFR-1结合，而GM3和脱唾液酸GM1则不能。相反，可溶性受体和游离肝素可抑制荧光染料标记的GM1与固定化FGF-2的相互作用。与它们的FGFR拮抗活性一致，游离神经节苷脂在相同浓度范围内可抑制FGF-2对内皮细胞的促有丝分裂活性。同样在这种情况下，GT1b在所测试的神经节苷脂中最为有效，而脱唾液酸GM1、神经氨酸、N-乙酰神经氨酸乳糖、半乳糖神经酰胺和硫脂则无效。总之，数据表明外源性神经节苷脂具有与FGF-2相互作用的能力。这种相互作用涉及FGF-2分子的COOH末端，并且取决于神经节苷脂分子中寡糖链的结构以及唾液酸残基的存在。当添加到内皮细胞培养物中时，外源性神经节苷脂可作为FGF-2拮抗剂。由于神经节苷脂由肿瘤细胞大量释放并在荷瘤患者血清中达到较高水平，我们的数据表明外源性神经节苷脂可能通过与FGF-2的直接相互作用影响内皮细胞功能，从而调节肿瘤新生血管形成。
