SPR interaction analysis of GM3 glycan with the integrin α5β1 head domain.

Integrins are heterodimeric receptors involved in cell adhesion and bidirectional signaling. Ganglioside GM3 in the outer leaflet of cell membranes possibly regulates integrin activity via direct interactions; however, its specific binding mode with integrins remains unclear. Therefore, in this study, we focused on the GM3 glycan moiety, which encounters the integrin ectodomain on the cell surface, and synthesized a soluble GM3 probe without hydrocarbon chains using a chemoenzymatic approach. Binding analysis using surface plasmon resonance (SPR) indicated that the synthetic GM3 probe interacts with the integrin α5β1 ectodomain with a significantly higher affinity than the lactosylceramide probe. Saturation transfer difference (STD) NMR spectra suggested that the integrin α5β1 ectodomain interacts with N-acetylneuraminic acid (Neu5Ac) at the GM3 terminal and α2-3Gal linkage. Notably, RGD peptide, an integrin ligand interacting with the heterodimer interface, competed with GM3 to bind to integrin α5β1 on the SPR sensor chip. Consistent with these results, the GM3-binding site predicted by Chai-1 partially overlapped with the RGD peptide-binding groove on integrin α5β1; however, the binding mode was different. RGD peptide bridged the α and β subunits of the integrin head moiety, whereas the GM3 probe found at the cleft between the subunits. The Neu5Ac-Gal moiety primarily interacts with the metal ion-dependent adhesion site and nearby residues in the β1 subunit. Overall, our findings suggest that gangliosides directly interact with integrin α5β1 at a previously unknown binding site, revealing a novel regulatory mechanism for the integrin activity.