背景:内皮高通透性诱导的血管功能障碍是严重疾病如败血症和其他以急性全身性炎症为标志的疾病中普遍且重要的特征。血小板内皮细胞粘附分子-1(PECAM-1)和Tie2作为内皮细胞(ECs)的跨膜受体,不仅在维持EC-EC连接方面发挥关键作用,而且在影响血管生成方面发挥关键作用,血管稳态,和血管重塑。
目标:目前,PECAM-1-Tie2相互作用的分子基础仍未充分阐明。在研究中,通过果蝇S2和HEK293表达系统表达重组可溶性PECAM-1(sPECAM-1)和Tie2(sTie2),分别。使用表面等离子体共振(SPR)和尺寸排阻色谱法研究了sPECAM-1和sTie2之间的相互作用。免疫荧光法用于检测sPECAM-1和sTie2在内皮细胞上的结合。
结果:发现PECAM-1以钠和pH依赖性方式与sTie2结合,如ELISA所证实的,PECAM-1的D5-D6结构域可能在与sTie2的结合中起关键作用。表面等离子体共振(SPR)结果显示,与sPECAM-1-D1-D4和sPECAM-1-D1-D2相比,sPECAM-1的全长具有与sTie2最强的结合亲和力(KD=48.4nM)。该结果与ELISA中的结果一致。此外,大小排阻色谱显示sPECAM-1、sTie2和Ang1可以形成三元复合物。
结论:在这项研究中,我们确定sPECAM-1以pH和钠依赖性方式与sTie2结合。全长的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之间的相互作用中起着至关重要的作用。
BACKGROUND: 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.
OBJECTIVE: 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.
RESULTS: 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 (KD = 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.
CONCLUSIONS: 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.