The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal N-acetylgalactosamine 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 (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort 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 MGLshort H259T variant lost high-affinity binding towards Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in wild-type MGLshort, His259 mediates H-bonds directly or engages the Tn glycopeptide backbone through water molecules. These bonds were lost in MGLshort 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 comprising the His259 residue.
|Number of pages||1311|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 2019|
Marcelo, F., Supekar, N., Corzana, F., van der Horst, J. C., Vuist, I. M., Live, D., ... van Vliet, SJ. (2019). Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands. Journal of Biological Chemistry, 1300.