Proceedings of the National Academy of Sciences | 2021
Galectin-3 N-terminal tail prolines modulate cell activity and glycan-mediated oligomerization/phase separation
Abstract
Significance The functional role of the N-terminal tail (NT) of galectin-3 (Gal-3) with its numerous proline residues (27 out of 113 in human Gal-3) has remained enigmatic since its discovery. Our study enlightens the galectin field on the role of specific Gal-3 NT prolines and opens a window into understanding the mechanisms of Gal-3 proline polymorphisms (e.g., at P64) in various pathological disorders. In addition, we show that Gal-3 oligomerization, triggered by binding to glycoconjugates either in solution or on the plasma membrane and disrupted endomembranes, is a dynamic process analogous to liquid–liquid phase separation (LLPS). This dynamic behavior of Gal-3 illuminates a mechanism underlying the formation, regulation, and function of clusters of glycosylated cell surface receptors. Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3–mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3–mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid–liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding–triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT–NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT–CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.