Section of Vector Biology, Laboratory of Malaria and Vector Research, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2010 Feb 26;5(2):e9356. doi: 10.1371/journal.pone.0009356.
Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV) family of extracellular-associated (matricellular) proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C (vWF), thrombospondin type 1 (TSP), and C-terminal growth factor cysteine knot (CT) domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.
In this report, surface plasmon resonance (SPR) experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC) with K(D) in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44)) of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44), and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44) in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44) at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules.
The finding that Cyr61 interacts with the SMTB(1-44) domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.
Cyr61 是细胞外基质相关（基质细胞）蛋白的 CCN（Cyr61、结缔组织生长因子、NOV）家族的成员，具有四个不同的功能模块，即胰岛素样生长因子结合蛋白（IGFBP）、血管性血友病因子 C 型（vWF）、血小板反应蛋白 1 型（TSP）和 C 端生长因子半胱氨酸结（CT）域。肝素硫酸蛋白聚糖据称介导 Cyr61 与基质和细胞表面的相互作用，但其他细胞外相关蛋白的作用尚未揭示。
在本报告中，表面等离子体共振（SPR）实验和固相结合实验表明，重组 Cyr61 与固定化单体或多聚体 vitronectin（VTNC）相互作用，K(D) 值在纳摩尔范围内。值得注意的是，Cyr61 的结合位点被确定为 VTNC 的 somatomedin B 结构域（SMTB(1-44)），该结构域介导其与 PAI-1、uPAR 和整合素 alphav beta3 的相互作用。因此，PAI-1 竞争 Cyr61 与固定化 SMTB(1-44)的结合，Cyr61 减弱 uPAR 介导的 U937 对 VTNC 的黏附。相反，等温滴定量热法显示 Cyr61 在溶液中对 SMTB(1-44)没有显示出高亲和力结合。然而，竞争性 ELISA 显示，多聚体 VTNC、热改性单体 VTNC 或高浓度的 SMTB(1-44) 减弱 Cyr61 与固定化 VTNC 的结合，而单体 VTNC 则无效。因此，VTNC 的固定化暴露了识别 Cyr61 的高亲和力隐匿表位，正如许多抗体、β-内啡肽和其他分子的报道。
Cyr61 与 SMTB(1-44)结构域相互作用的发现表明，VTNC 代表 CCN 家族成员与基质的锚定点。结果在 CCN 和 VTNC 在基质生物学和血管生成中的作用的背景下进行了讨论。
