College of Chemistry, Fuzhou University, Fuzhou, 350116, China.
College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian, 350116, China.
Biochem Biophys Res Commun. 2024 Nov 26;735:150484. doi: 10.1016/j.bbrc.2024.150484. Epub 2024 Jul 31.
Endothelial hyperpermeability-induced vascular dysfunction is a prevalent and significant characteristic in critical illnesses such as sepsis and other conditions marked by acute systemic inflammation. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and Tie2 serve as transmembrane receptors within endothelial cells (ECs), playing pivotal roles not only in maintaining EC-EC junctions but also in influencing vasculogenesis, vessel homeostasis, and vascular remodeling.
At present, the molecular basis of the PECAM-1-Tie2 interaction remains inadequately elucidated. In the study, recombinant soluble PECAM-1 (sPECAM-1) and Tie2 (sTie2) were expressed by Drosophila S2 and HEK293 expression systems, respectively. The interactions between sPECAM-1 and sTie2 were investigated using the Surface Plasmon Resonance (SPR) and size-exclusion chromatography methods. An immunofluorescence assay was used to detect the binding of sPECAM-1 and sTie2 on endothelial cells.
PECAM-1 was found to bind with sTie2 in a sodium and pH-dependent manner as confirmed by the ELISA, the D5-D6 domains of PECAM-1 might play a crucial role in binding with sTie2. Surface Plasmon Resonance (SPR) results showed that the full length of sPECAM-1 has the strongest binding affinity (K = 48.4 nM) with sTie2, compared to sPECAM-1-D1-D4 and sPECAM-1-D1-D2. This result is consistent with that in the ELISA. In addition, size-exclusion chromatography demonstrated that sPECAM-1, sTie2, and Ang1 can form a ternary complex.
In this study, we determined that sPECAM-1 binds to sTie2 in a pH and sodium-dependent manner. The full length of sPECAM-1 has the strongest binding affinity, and the D5-D6 domains in sPECAM-1 play a crucial role in the interaction between sPECAM-1 and sTie2.
内皮细胞通透性增加引起的血管功能障碍是败血症等危重病以及其他以急性全身炎症为特征的疾病中的一种普遍且重要的特征。血小板内皮细胞黏附分子-1（PECAM-1）和 Tie2 作为内皮细胞（ECs）中的跨膜受体，不仅在维持 EC-EC 连接中发挥关键作用，而且在影响血管生成、血管稳态和血管重塑中也发挥关键作用。
目前，PECAM-1-Tie2 相互作用的分子基础仍未得到充分阐明。在这项研究中，通过 Drosophila S2 和 HEK293 表达系统分别表达了重组可溶性 PECAM-1（sPECAM-1）和 Tie2（sTie2）。使用表面等离子体共振（SPR）和分子筛层析法研究了 sPECAM-1 和 sTie2 之间的相互作用。使用免疫荧光法检测 sPECAM-1 和 sTie2 在血管内皮细胞上的结合。
ELISA 证实 PECAM-1 以钠离子和 pH 值依赖的方式与 sTie2 结合，PECAM-1 的 D5-D6 结构域可能在与 sTie2 结合中发挥关键作用。表面等离子体共振（SPR）结果表明，全长 sPECAM-1 与 sTie2 具有最强的结合亲和力（K=48.4nM），而 sPECAM-1-D1-D4 和 sPECAM-1-D1-D2 则较弱。这一结果与 ELISA 一致。此外，分子筛层析显示 sPECAM-1、sTie2 和 Ang1 可以形成三元复合物。
在这项研究中，我们确定 sPECAM-1 以 pH 值和钠离子依赖的方式与 sTie2 结合。全长 sPECAM-1 具有最强的结合亲和力，sPECAM-1 中的 D5-D6 结构域在 sPECAM-1 和 sTie2 之间的相互作用中发挥关键作用。
