Mapping Galectin-3 Ligand Networks Driving Renal Fibrosis Using Proximity-Labeling Proteomics 2253572

Abstract Introduction Galectin-3 (Gal-3) is a β-galactoside—binding lectin implicated in inflammation, epithelial injury, and fibrosis. Although Gal-3 is strongly upregulated in inflamed kidneys, its role in promoting renal fibrosis remains poorly defined. We hypothesized that Gal-3 interacts with cell-surface receptors that trigger profibrotic signaling during tubular injury. Methods To identify Gal-3—associated ligands, we generated a Gal-3—APEX2 fusion construct that enables proximity-dependent biotinylation of neighboring proteins in live BUMPT kidney proximal tubule epithelial cells. Cells were stimulated with TGF-β (0—10 ng/mL, 24 h) and treated with biotin-phenol/H₂O₂ to induce APEX2-mediated labeling. Results Immunofluorescence (IF) staining and western blot (WB) confirmed robust Gal-3-dependent biotinylation localized to the plasma membrane and perinuclear regions. Quantitative IF revealed dose-dependent increases in Gal-3 proximity activity at concentrations of 25 nM (FC = 16.6, p < 0.01), 50 nM (FC = 40.5, p < 0.0001), and 100 nM (FC = 88.7, p < 0.0001) with TGF-β incubation (10ng/ml). WB analysis showed a 2.7-fold increase (t = 36.5, p = 0.0007) in labeled proteins in TGF-β—treated cells (10ng/ml). Tandem-mass-tag proteomics identified 41 upregulated proteins (FC > 1.2 — 7.5) following TGF-β stimulation, including extracellular matrix components (TNC, TSP1, TIMP3), adhesion molecules (EPCAM, CLDN7, JAM3), and receptors (EPHA2, TLR2, SLC3A2). Mitochondrial and metabolic enzymes (SLC2A1, SHMT2, SDHB, IDH2) were also enriched, suggesting Gal-3-driven metabolic activation. Conclusion These findings demonstrate that Gal-3 proximity labeling enables proteome-wide mapping of Gal-3 interactors in renal epithelial cells. TGF-β—induced Gal-3 activation promotes recruitment of adhesion, metabolic, and extracellular matrix proteins, defining a molecular framework for Gal-3—mediated epithelial activation and fibrosis. Funding Source n/a Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)