Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Germany.
J Biotechnol. 2010 Jan 1;145(1):54-9. doi: 10.1016/j.jbiotec.2009.10.005.
Fibroblast growth factor 2 (FGF2) plays a pivotal role in cell proliferation, angiogenesis and neuroprotection. Several clinical trials using this growth factor in bone regeneration, wound healing and cardioprotection are initiated but the inadequate stability of FGF2 after application is one major problem. Binding of ATP to FGF2 and other growth factors has been demonstrated recently. Here we report that ATP, other nucleoside triphosphates and sodium triphosphate protect FGF2 from trypsin, plasmin and neutrophile elastase digestion in vitro. A molar ratio of 2:1 (ligand/FGF2) is sufficient for these protective effects. ADP shows only little, AMP no stabilizing effect on FGF2 indicating that the number of phosphate residues is important. Protection of FGF2 by ATP can be abolished by the addition of alkaline phosphatase hydrolyzing free and FGF2-bound ATP. The mutant FGF2 (K128A/R129A/K134A/K144A) with strongly reduced ATP-binding capacity revealed no detectable protease resistance after incubation with ATP. Furthermore, a stabilizing effect of ATP on FGF2 could also be demonstrated in cell culture experiments. ATP bound to FGF2 increased FGF2-dependent human umbilical vein endothelial cells proliferation when the growth factor was treated with neutrophile elastase or heat. For the first time these data demonstrate protection of FGF2 by bound ATP, other nucleoside triphosphates or sodium triphosphate from rapid protease digestion. Our data provide new evidence that nucleoside triphosphates are capable of protecting FGF2 and favours such stabilization for various, especially medical applications.
成纤维细胞生长因子 2（FGF2）在细胞增殖、血管生成和神经保护中发挥关键作用。目前已经启动了几项使用该生长因子进行骨再生、伤口愈合和心脏保护的临床试验，但 FGF2 应用后的稳定性不足是一个主要问题。最近已经证明了 FGF2 和其他生长因子与 ATP 的结合。在这里，我们报告 ATP、其他核苷三磷酸和三磷酸钠可以在体外保护 FGF2 免受胰蛋白酶、纤溶酶和中性粒细胞弹性蛋白酶的消化。配体/FGF2 的摩尔比为 2:1 就足以产生这些保护作用。ADP 对 FGF2 只有很小的稳定作用，AMP 则没有，这表明磷酸盐残基的数量很重要。碱性磷酸酶可以水解游离和与 FGF2 结合的 ATP，从而消除 ATP 对 FGF2 的保护作用。具有强烈降低 ATP 结合能力的突变体 FGF2（K128A/R129A/K134A/K144A）在与 ATP 孵育后没有检测到明显的蛋白酶抗性。此外，还可以在细胞培养实验中证明 ATP 对 FGF2 的稳定作用。当用中性粒细胞弹性蛋白酶或热处理 FGF2 时，与 FGF2 结合的 ATP 增加了 FGF2 依赖性人脐静脉内皮细胞的增殖。这些数据首次证明了结合的 ATP、其他核苷三磷酸或三磷酸钠可以保护 FGF2 免受快速蛋白酶消化。我们的数据提供了新的证据，表明核苷三磷酸能够保护 FGF2，并支持其在各种应用中的稳定性，特别是在医学应用中。
