Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands

• Type of Material: Article
• Authors: Filipa Marcelo, REQUIMTE; Nitin Supekar, University of Georgia; Francisco Corzana, Universidad de La Rioja; Joost C. van der Horst, Vrije Universiteit Amsterdam; Iiona Vuist, Vrije Universiteit Amsterdam; David Live, University of Georgia; Geert-Jan P.H. Boons, University of Georgia; David Smith, Emory University; Sandra J. van Vliet, Vrije Universiteit Amsterdam
• Language: English
• Date: 2019-01-25
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2019 Marcelo et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.RA118.004957
• Title of Journal or Parent Work: Journal of Biological Chemistry
• Volume: 294
• Issue: 4
• Start Page: 1300
• End Page: 1311
• Grant/Funding Information: This work was supported by a bridging grant under National Institutes of Health Grant U54GM62116 (to the Consortium for Functional Glycomics) and P41GM103390 (to the Research Resource for Integrated Glycotechnology)
• Keywords: ANTIGEN; C-type lectin; carbohydrate recognition domain; glycan; Biochemistry & Molecular Biology; Science & Technology; alternative splicing; molecular modeling; carbohydrate specificity; C-TYPE LECTIN; MGL; peptide array; mutant; RECEPTOR; splice variant; Life Sciences & Biomedicine; THREONINE GLYCOSYLATION; STRUCTURAL ELEMENTS; DENDRITIC CELLS; Tn antigen; ANTIBODY; glycopeptide; microarray; EXPRESSION; GLYCOPEPTIDES
• Research Categories: Biology, Molecular

Abstract

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 (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 toward 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 WT 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 containing the His259 residue.