Species-Specific Roles for a2,3- and a2,6-Linked Sialic Acid Residues in PECAM-1-Mediated Homophilic Interactions

PECAM-1 is a 130 kDa type 1 transmembrane glycoprotein belonging to the immunoglobulin gene (Ig) superfamily, and is constitutively expressed on the surface of circulating blood cells and endothelial cells. In platelets, PECAM-1 serves as an inhibitory receptor to limit thrombus growth, while on endothelial cells, PECAM-1 is a major junctional protein that functions to sense flow, mediate leukocyte transendothelial migration, and regulate vascular permeability. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions, mediated by amino-terminal Ig-homology domain 1 (IgD1), are required for its functions on endothelial cells. Interestingly, PECAM-1 is a heavily glycosylated receptor, with two of its nine N -linked glycans present in IgD1. Murine PECAM-1 is modified by addition of α2,6-linked sialic acid residues, and studies performed using recombinant murine PECAM-1 protein have shown that PECAM-1/PECAM-1 homophilic interactions are abrogated upon removal of sialic acid, and inhibited by α2,6- sialylated oligosaccharides, suggesting that these glycans may be important in PECAM-1 homophilic binding. Murine PECAM-1, however, does not bind human PECAM-1. A chimeric protein comprised of the human PECAM-1 extracellular domain fused with human Fc (PECAM-1/IgG) produced by Chinese Hamster Ovary (CHO) cells, which are able to add only α2,3-linked sialic acid residues, has been successfully used to study PECAM-1-mediated adhesive interactions for many years. Taken together, these data suggest that the mechanisms by which PECAM-1 interacts with itself may differ between species. The purpose of the present investigation, therefore, was to examine the role of glycans, specifically terminal sialic acid residues, in human PECAM-1 homophilic interactions. To determine whether the specific linkage of sialic acid affects human PECAM-1 homophilic interactions, we examined the relative ability of α2,3-sialylated PECAM-1/IgG - produced in wild-type CHO cells which lack α2,6-sialyltransferase activity - and α2,6-sialylated PECAM-1/IgG - produced in CHO cells that had been stably transfected with ST6Gal-1, to bind to PECAM-1-transfected REN cells. We found that α2,3-sialylated PECAM-1/IgG bound more than twice as well as α2,6-sialylated PECAM-1/IgG (mean fluorescence intensity (MFI) of 457,250 ± 33,049 versus 280,333 ± 78,589; normal human IgG bound with an MFI of 158,550 ± 62,133). These findings were further supported by molecular docking studies, which showed a preferential orientation for interactions between α2,3-linked sialic acid and amino acid K89 within PECAM-1 Ig homology domain 1 - a residue known to be important in PECAM-1 homophilic interactions. Taken together, these data provide important new conceptual information that improves our understanding of the role of glycosylation in PECAM-1 homophilic adhesive interactions.