Glycan binding proteins (GBPs) belong to two distinct domains, namely, glycosaminoglycan (GAG)-binding proteins (GAGBPs) and lectins. While GAGBPs recognize proteoglycans and heir constituent GAGs, lectins interact with N- and O-linked glycans of glycoproteins. The multi-functional human protein Galectin-3 (Gal-3) is a bona fide lectin that plays significant roles in many cellular and pathological processes including cancer and SARS Covid. Gal-3 binds to neutral galactosyl moieties, primarily N-acetyllactosamine (LacNAc) residues, on glycoproteins. We found that despite being a lectin, Gal-3 possesses the characteristics of a GAGBP. Gal-3 binds to heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC) as well as chondroitin sulfate proteoglycans (CSPGs). Heparin and CSB are monovalent ligands for Gal-3 but CSA, CSC, and a bovine CSPG engage in multivalent binding with Gal-3 and form noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs by Gal-3 was completely abolished when Gal-3 was preincubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was reversed by β-lactose. Both observations strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. We have mapped the GAG binding site of Gal-3 and identified the contact amino acid residues by mutational studies. Further investigation of GAG recognition mechanism by Gal-3 was done by computational modeling with the help of the GlycoMIP Modeling Facility. Modeling studies revealed how the structures of the GAGs, particularly the orientation of the sulfate groups, regulated their binding interactions. In addition, GlycoMIP Modeling Facility helped understand the intriguing cross-linking properties of CSA, CSC and a CSPG. Elucidation of the GAG binding site of Gal-3 and understanding the GAG binding mechanism will help unravel the mystery behind the muti-tasking abilities of Gal-3. Knowledge acquired from this study might be useful for designing function-specific inhibitors of Gal-3.

Interactions of human Galectin-3 with glycosaminoglycans

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