Highly branched complex-type N-glycans on integrin β1 in extracellular vesicles enhance the binding of sEVs to laminin on recipient cells.

Small extracellular vesicles (sEVs) play a crucial role in intercellular communication. Recently, we discovered that sEVs bind to recipient cells via interactions between integrin heterodimers and laminins. In this study, we explored how sEV-associated glycans regulate this binding. We performed glycomic analyses of asparagine-linked (N-linked) glycans on integrin β1 in tumor-derived sEVs, revealing that N-glycan structures in sEVs differ from those in their cells of origin. Highly branched, complex-type N-glycans on integrin β1 were predominant in sEVs, whereas less branched complex-type and oligomannose-type N-glycans were more abundant in cells. Furthermore, the sialic acid content of sEV N-glycans was higher than that of cellular glycans. Simultaneous single-particle imaging and super-resolution movie observation revealed that knocking out the glycosyltransferase MGAT5, which synthesizes highly branched complex-type N-glycans, reduced the binding ability of tumor-derived sEVs to laminin on both glass and recipient cell membranes. Although α2,3-sialidase treatment of sEVs did not alter their laminin-binding ability, the α2-3,6,8 sialidase treatment and KO of ST6GAL1 significantly decreased it. Observations following the removal of the N-glycan from the I-like domain of integrin β1 via an NQ mutation revealed that the glycan at N269 enhances binding. Additionally, an antibody against the activated state of integrin β1 bound more frequently to WT integrin β1 in sEVs than to the N269Q mutant, indicating that the glycan at N269 is essential for maintaining the protein's activated conformation. Thus, using advanced imaging techniques, we elucidated the regulatory mechanisms by which N-glycans control the binding of sEVs to laminin on recipient cells.