Identification of Galectin-3 As a High-Affinity Binding Protein for Advanced Glycation End Products (AGE): A New Member of the AGE-Receptor Complex

Abstract

Background: Advanced glycation end products (AGE), the reactive derivatives of nonenzymatic glucose-protein condensation reactions, are implicated in the multiorgan complications of diabetes and aging. An AGE-specific cellular receptor complex (AGE-R) mediating AGE removal as well as multiple biological responses has been identified. By screening an expression library using antibody against a previously identified component of the AGE-R complex p90, a known partial cDNA clone was isolated with homology to galectin-3, a protein of diverse identity, and member of the galectin family. Materials and Methods: To explore this unexpected finding, the nature of the interactions between galectin-3 and AGE was studied using intact macrophage-like RAW 264.7 cells, membrane-associated and recombinant galectin-1 through -4, and model AGE-ligands (AGE-BSA, FFI-BSA). Results: Among the members of this family (galectin-1 through 4), recombinant rat galectin-3 was found to exhibit high-affinity 125I-AGE-BSA binding with saturable kinetics (kD 3.5 × 107 M−1) that was fully blocked by excess unlabeled naturally formed AGE-BSA or synthetic FFI-BSA, but only weakly inhibited by several known galectin-3 ligands, such as lactose. In addition to the p90, immunoprecipitation with anti-galectin-3, followed by 125I-AGE-BSA ligand blot analysis of RAW 264.7 cell extracts, revealed galectin-3 (28 and 32 kD), as well as galectin-3-associated proteins (40 and 50 kD) with AGE-binding activity. Interaction of galectin-3 with AGE-BSA or FFI-BSA resulted in formation of SDS-, and β-mercaptoethanol-insoluble, but hydroxylamine-sensitive high-molecular weight complexes between AGE-ligand, galectin-3, and other membrane components. Conclusions: The findings point toward a mechanism by which galectin-3 may serve in the assembly of AGE-R components and in the efficient cell surface attachment and endocytosis by macrophages of a heterogenous pool of AGE moieties with diverse affinities, thus contributing to the elimination of these pathogenic substances.