The Role of Sialylated Glycans in Human Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1)-mediated Trans Homophilic Interactions and Endothelial Cell Barrier Function

• Published in: The Journal of biological chemistry Vol. 291; no. 50; pp. 26216 - 26225
• Main Authors: Lertkiatmongkol, Panida, Paddock, Cathy, Newman, Debra K., Zhu, Jieqing, Thomas, Michael J., Newman, Peter J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.12.2016; American Society for Biochemistry and Molecular Biology
• Subjects: adhesion; Amino Acid Substitution; Cell Communication - physiology; Cell Line; endothelial cell; Endothelial Cells - cytology; Endothelial Cells - metabolism; Glycobiology and Extracellular Matrices; glycosylation; Humans; Mutation, Missense; permeability; platelet endothelial cell adhesion molecule (PECAM); Platelet Endothelial Cell Adhesion Molecule-1 - chemistry; Platelet Endothelial Cell Adhesion Molecule-1 - genetics; Platelet Endothelial Cell Adhesion Molecule-1 - metabolism; Polysaccharides - chemistry; Polysaccharides - genetics; Polysaccharides - metabolism; sialic acid; Sialic Acids - chemistry; Sialic Acids - genetics; Sialic Acids - metabolism; Thrombin - chemistry; Thrombin - genetics; Thrombin - metabolism; vascular biology; platelet endothelial cell adhesion molecule (PECAM); sialic acid; endothelial cell; permeability; glycosylation; vascular biology; adhesion
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.756502

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.