Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, NIH, Bethesda, MD, USA.
FEBS J. 2010 May;277(9):2192-205. doi: 10.1111/j.1742-4658.2010.07641.x.
Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a approximately 60 kDa PEDF-binding protein in bovine retina with Bos taurus F(1)-ATP synthase beta-subunit, and that F(1)F(o)-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F(1). Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F(1). Real-time binding as determined by surface plasmon resonance demonstrated that yeast F(1) interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F(1) and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F(1). Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of approximately 60 kDa. Antibodies to F(1)beta-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F(1), these results show that PEDF is a ligand for endothelial cell-surface F(1)F(o)-ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity.
色素上皮衍生因子（PEDF）是一种有效的血管生成抑制剂，能抑制内皮细胞的迁移和小管形成，它与内皮细胞表面的一种未知蛋白高亲和力结合。鉴于蛋白质指纹图谱显示牛视网膜中大约 60 kDa 的 PEDF 结合蛋白与牛 Bos taurus F（1）-ATP 合酶β亚基相匹配，并且最近已经鉴定出 F（1）F（o）-ATP 合酶的组成部分是细胞表面受体，我们研究了 PEDF 与 F（1）的直接结合。大小排阻超滤分析表明，重组人 PEDF 与重组酵母 F（1）形成复合物。表面等离子体共振实时结合实验表明，酵母 F（1）特异性且可逆地与人类 PEDF 相互作用。动力学评估表明 PEDF 具有高结合亲和力，这与 PEDF 与内皮细胞表面的亲和力一致。PEDF 阻断了 F（1）与另一种抗血管生成因子血管抑素之间的相互作用，这表明 F（1）上存在重叠的 PEDF 结合和血管抑素结合位点。内皮细胞表面表现出对大约 60 kDa 的 PEDF 结合蛋白的亲和力。针对 F（1）β亚基的抗体特异性捕获了内皮质膜中的 PEDF 结合成分。在存在 PEDF 的情况下，检测内皮细胞的细胞外 ATP 合成活性。PEDF 显著减少了内皮细胞产生的细胞外 ATP 量，这与细胞表面 ATP 合酶与 PEDF 之间的直接相互作用一致。这些结果不仅证明了 PEDF 与细胞表面 F（1）结合，还表明 PEDF 是内皮细胞表面 F（1）F（o）-ATP 合酶的配体。它们表明 PEDF 介导的 ATP 合酶抑制可能是 PEDF 发挥其抗血管生成活性的生化机制的一部分。
