Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies:...

The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (αGalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGL^short) and the long MGL 6B (MGL^long) splice variants, as well as of a histidine-to-threonine mutant (MGL^short H259T). Although the MGL^short and MGL^long variants displayed similar binding properties on the glycan array, the MGL^short H259T mutant failed to interact with the sialyl-Tn epitope. As the MGL^short H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGL^short H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGL^short H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGL^short, His²⁵⁹ mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGL^short H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His²⁵⁹ residue.