Gal-3 N-terminal prolines within residues 69 to 117 regulate CRD glycan binding, phase separation and Gal-3-mediated cellular function.

Abstract

Galectin-3 (Gal-3), the only chimeric galectin, is a central driver and potential biomarker for cancer progression, fibrotic disorders, and heart failure. As a key proinflammatory factor, Gal-3 aggravates various disease states by promoting immune cell activation, cytokine storm amplification, and cell migration. Previously, we reported on the structural and functional role of prolines within Gal-3 NT residues 1-68. Here, we report on how the 14 NT prolines within the 69-117 segment affect Gal-3 structure and function. Circular dichroism (CD) and fluorescence spectroscopies showed that none of these proline-to-alanine substitutions altered overall Gal-3 conformation, whereas some of them did modulate glycoprotein-induced phase separation and ligand binding (lactose (Lac), LacNAc, (LacNAc)₃), as well as Gal-3-mediated cell function, i.e., cell activation, hemagglutination, and endocytosis. Three of these prolines were found to be the most crucial to Gal-3-mediated activities, with P86A and P113A attenuating glycan-binding, glycoprotein-induced phase separation, and cellular function and P117A enhancing these processes. Correlation analysis of all prolines within the Gal-3 NT revealed that prolines within the 1-68 segment primarily modulate cellular function via glycan binding, whereas prolines within the 69-117 segment modulate glycan-induced phase separation and cellular function. Overall, our findings reveal distinct functional roles for specific Gal-3 NT prolines, provide mechanistic insight into Gal-3 structure-function relationships, and identify potential NT prolines as targets for ameliorating various Gal-3-related pathological disorders.