CD44 enhances E-selectin-PSGL-1 interaction by binding to distinct sites on E-selectin

Precise roles of E-selectin in inflammatory cascades, particularly concerning its primary ligands PSGL-1 and CD44, remain incompletely understood. Existing observations such as their co-localization, shared E-selectin-triggered signaling pathways, and cooperative contributions to cellular adhesion hint at a potential interplay of the ligands. We combined molecular simulations and experimental measurements to investigate the cooperative relationship between PSGL-1 and CD44 and elucidate the underlying structural mechanisms. Simulations predicted that CD44 binds to the hinge region between Lectin and EGF domains of E-selectin, spatially distinct from the PSGL-1 binding epitope located to the head interface of E-selectin Lectin domain. This spatial arrangement allowed for simultaneous binding of both PSGL-1 and CD44 to the same E-selectin molecule, and CD44 binding was predicted to enhance E-selectin-PSGL-1 interaction. Experimental validation upon flow chamber assay exhibited that adhesion of both PSGL-1 and CD44-coated microbead to E-selectin-coated substrate was significantly higher than either ligand-coated alone, even at comparable coating densities. Moreover, both atomic force microscopy and acoustic force spectroscopy assays demonstrated that the binding strength of E-selectin-PSGL-1 interaction increased in the presence of soluble CD44. Co-localization of PSGL-1 and CD44, the precondition for binding cooperatively to the same E-selectin molecule, was also confirmed on both HL-60 and primary PMN cells using Immunofluorescence assay. These findings provided a more comprehensive understanding in differential roles and underling mechanisms of E-selectin-ligand interactions in immune responses.