CHARACTERIZATION OF THE INTERACTION BETWEEN HUMAN GALECTIN-3 AND SULFATED GLYCOSAMINOGLYCANS

Galectin-3 (Gal-3) is a β-galactoside-binding protein widely expressed in various human tissues. Intracellularly, it regulates apoptosis, cell cycle, and signaling; extracellularly, it forms networks that promote cell–cell and cell–matrix interactions. Gal-3 is associated with cancer by modulating tumor cell adhesion, migration, and invasion, thereby favoring growth and metastatic dissemination. Its overexpression in several cancers makes it a promising biomarker and therapeutic target. Gal-3 is also known to interact with sulfated glycosaminoglycans, components of the extracellular matrix that regulate signaling and tissue organization. This interaction may influence matrix structure, cell signaling, and disease development, but details on specificity and conformational effects remain poorly understood. The aim of this work was to characterize, through biophysical and structural techniques, the interaction of galectin-3 with different sulfated glycosaminoglycans, in order to understand the effects of this binding on the protein. Recombinant human Gal-3 was expressed and purified, and the glycosaminoglycans chondroitin-4-sulfate (CS4), chondroitin-6-sulfate (CS6), and heparin were used. Analyses were performed in buffers mimicking physiological and tumor microenvironments. Intrinsic tryptophan fluorescence was used to monitor changes in the aromatic environment indicative of interaction. Protein secondary structure and thermal stability were assessed by circular dichroism and thermal denaturation. Complex formation was evaluated by saturation transfer difference nuclear magnetic resonance (STD-NMR). Fluorescence showed that Gal-3 binds to GAGs, with variations depending on the ligand and pH. Circular dichroism revealed changes in the protein’s secondary structure, while thermal stability was unaffected, indicating that the overall fold remains stable. STD-NMR spectroscopy confirmed direct and specific binding between Gal-3 and the GAGs. Together, these findings expand the understanding of Gal-3/GAG interactions, with implications for physiological functions and cancer-related processes.