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 (GalNAc-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 , His 259 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 His 259 residue.
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