Grosso, Ana SofiaDiniz, AnaSoares, Cátia O.Goerdeler, FelixGimeno, AnaCoelho, PedroCoelho, HelenaLima, Carlos D.L.Pinheiro, BeneditaLete, Marta G.Garcia-Martin, FaynaJaroentomeechai, ThapakornGomes, JoanaReis, Celso A.Westerlind, UlrikaCorzana, FranciscoPalma, Angelina S.Clausen, HenrikJiménez-Barbero, Jesúsvan Vliet, Sandra J.Narimatsu, YoshikiMarcelo, Filipa2026-03-232026-03-232025-12-292691-3704PURE: 158207876PURE UUID: d6fc55ff-5310-48ff-b40b-51327e3686f5Scopus: 105028306402ORCID: /0000-0001-5797-6555/work/209454296ORCID: /0000-0001-5049-8511/work/209454532WOS: 001650824600001PubMed: 41614153PubMedCentral: PMC12848722http://hdl.handle.net/10362/201731Publisher Copyright: © 2025 The Authors. Published by American Chemical SocietyThe human macrophage galactose-type lectin (MGL) recognizes exposed GalNAc residues abundantly found in tumor O-glycans. Herein, we have used an integrative chemical, structural, and functional approach to unravel the intricate specificity and molecular determinants that underlie the recognition of Thomsen-nouveau (Tn), the sialylated variant (STn), and Thomsen-Friedenreich (TF) O-glycans by the carbohydrate recognition domain of the MGL (MGL-CRD) at the molecular and cellular levels. The MGL-CRD prefers binding to Tn > STn ≫ TF O-glycans. In this molecular context, NMR, isothermal titration calorimetry, and molecular dynamics simulations revealed quantitative key structural and dynamic differences in binding, depending on the O-glycan. Interestingly, the density of Tn epitopes was critical for engaging multiple MGL-CRDs to MUC1 Tn-glycopeptides; however, the enthalpy–entropy balance strongly influenced the affinity, and a higher Tn density did not improve the binding. Cell-based mucin arrays recapitulated the MGL-CRD binding preference (Tn > STn ≫TF), but no preference for a specific O-glycan pattern in mucins was observed. The MGL-CRD also selectively recognizes glycoengineered gastric cancer cells expressing Tn/STn. Conversely, in the cellular context, employing CHO cells expressing the full-length MGL (CHO+MGL) allowed analysis of the MGL binding properties in its native presentation toward tagged isolated mucin reporters. Specificity for short tumor-associated O-glycans without any preference for a specific mucin was confirmed. Stunningly, the CHO+MGL cells revealed that the MGL shows similar binding to the STn and TF mucin reporters, suggesting that its natural oligomeric state displays promiscuous binding to simple O-glycans. Conceptually, the key role of glycan and lectin presentations for binding is thus highlighted. Moreover, this suggests the compelling scenario that the MGL serves as a universal receptor for truncated cancer-associated O-glycans.138825096engGlycan and receptor presentationMacrophage galactose-type lectinMolecular recognitionTumor-associatedO-glycansAnalytical ChemistryChemistry (miscellaneous)Physical and Theoretical ChemistryOrganic ChemistrySDG 3 - Good Health and Well-beingjournal article10.1021/jacsau.5c00905https://www.scopus.com/pages/publications/105028306402https://www.webofscience.com/wos/woscc/full-record/WOS:001650824600001