Section of Experimental Oncology and Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, 25123, Italy.
Tumor Angiogenesis Unit, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Stezzano, 87, Bergamo, 24126, Italy.
Angiogenesis. 2019 Feb;22(1):133-144. doi: 10.1007/s10456-018-9644-3. Epub 2018 Aug 30.
Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.
血栓反应蛋白-1（TSP-1）和 TSP-2 具有相似的结构和功能，包括显著的抗血管生成活性。我们之前已经证明，TSP-1 抗血管生成活性的一种机制是其三型重复结构域与成纤维细胞生长因子-2（FGF2）的相互作用，影响生长因子的生物利用度和血管生成活性。由于 TSP-2 中的三型重复结构域是保守的，因此本研究旨在研究 TSP-2 是否也保留了与 FGF2 相互作用的能力。通过固相结合和表面等离子体共振分析，分析了 TSP-1 和 TSP-2 及其重组结构域与 FGF2 的结合特性。TSP-2 与 FGF2 具有高亲和力（Kd=1.3 nM）结合。TSP-2/FGF2 结合被钙和肝素抑制。TSP-2 的 FGF2 结合结构域位于三型重复结构域中，最小相互作用序列被鉴定为重复 3C 中的 GVTDEKD 肽，对应于 TSP-1 的活性序列 KIPDDRD。TSPs 的重复 11C 中也鉴定出第二个潜在的 FGF2 结合序列。计算对接分析预测，TSP-2 和 TSP-1 衍生的七肽与 FGF2 具有相似的结合特性相互作用。因此，基于 TSP-1 活性序列的小分子阻断了 TSP-2/FGF2 相互作用。TSP-2 与 FGF2 的结合削弱了生长因子与细胞受体相互作用的能力，因为 TSP-2 衍生片段阻止了 FGF2 与肝素（用作硫酸乙酰肝素蛋白聚糖的结构类似物）和 FGFR-1 的结合。这些发现确定 TSP-2 为一种新的 FGF2 配体，与 TSP-1 具有相同的分子要求与生长因子相互作用，并具有类似的能力来阻断 FGF2 与促血管生成受体的相互作用。这些特征可能有助于 TSP-2 的抗血管生成和抗肿瘤活性，为未来的治疗应用提供了依据。
