Lertkiatmongkol Panida, Paddock Cathy, Newman Debra K, Zhu Jieqing, Thomas Michael J, Newman Peter J
From the Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin 53201, and.
the Departments of Pharmacology.
J Biol Chem. 2016 Dec 9;291(50):26216-26225. doi: 10.1074/jbc.M116.756502. Epub 2016 Oct 28.
Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) is a major component of the endothelial cell intercellular junction. Previous studies have shown that PECAM-1 homophilic interactions, mediated by amino-terminal immunoglobulin homology domain 1, contribute to maintenance of the vascular permeability barrier and to its re-establishment following inflammatory or thrombotic insult. PECAM-1 glycans account for ∼30% of its molecular mass, and the newly solved crystal structure of human PECAM-1 immunoglobulin homology domain 1 reveals that a glycan emanating from the asparagine residue at position 25 (Asn-25) is located within the trans homophilic-binding interface, suggesting a role for an Asn-25-associated glycan in PECAM-1 homophilic interactions. In support of this possibility, unbiased molecular docking studies revealed that negatively charged α2,3 sialic acid moieties bind tightly to a groove within the PECAM-1 homophilic interface in an orientation that favors the formation of an electrostatic bridge with positively charged Lys-89, mutation of which has been shown previously to disrupt PECAM-1-mediated homophilic binding. To verify the contribution of the Asn-25 glycan to endothelial barrier function, we generated an N25Q mutant form of PECAM-1 that is not glycosylated at this position and examined its ability to contribute to vascular integrity in endothelial cell-like REN cells. Confocal microscopy showed that although N25Q PECAM-1 concentrates normally at cell-cell junctions, the ability of this mutant form of PECAM-1 to support re-establishment of a permeability barrier following disruption with thrombin was significantly compromised. Taken together, these data suggest that a sialic acid-containing glycan emanating from Asn-25 reinforces dynamic endothelial cell-cell interactions by stabilizing the PECAM-1 homophilic binding interface.
血小板内皮细胞黏附分子1（PECAM-1）是内皮细胞间连接的主要成分。先前的研究表明，由氨基末端免疫球蛋白同源结构域1介导的PECAM-1同源相互作用有助于维持血管通透性屏障，并在炎症或血栓形成损伤后使其重新建立。PECAM-1聚糖约占其分子质量的30%，新解析的人PECAM-1免疫球蛋白同源结构域1的晶体结构表明，从第25位天冬酰胺残基（Asn-25）发出的聚糖位于反式同源结合界面内，提示Asn-25相关聚糖在PECAM-1同源相互作用中发挥作用。为支持这一可能性，无偏分子对接研究表明，带负电荷的α2,3唾液酸部分以有利于与带正电荷的Lys-89形成静电桥的方向紧密结合到PECAM-1同源界面内的一个凹槽中，先前已证明该位点的突变会破坏PECAM-1介导的同源结合。为验证Asn-25聚糖对内皮屏障功能的贡献，我们生成了在该位置不进行糖基化的PECAM-1的N25Q突变形式，并检测其在内皮样REN细胞中对血管完整性的贡献能力。共聚焦显微镜显示，尽管N25Q PECAM-1正常聚集在细胞间连接处，但这种PECAM-1突变形式在凝血酶破坏后支持通透性屏障重新建立的能力明显受损。综上所述，这些数据表明，从Asn-25发出的含唾液酸聚糖通过稳定PECAM-1同源结合界面来加强动态内皮细胞间相互作用。
