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10 p.-5 fig.-1 graph. abst., The human macrophage galactose lectin (MGL) is anendocytic type II transmembrane receptor expressed on immaturemonocyte-derived dendritic cells and activated macrophages and playsa role in modulating the immune system in response to infections andcancer. MGL contains an extracellular calcium-dependent (C-type)carbohydrate recognition domain (CRD) that specifically bindsterminalN-acetylgalactosamine glycan residues such as the Tn andsialyl-Tn antigens found on tumor cells, as well as otherN-andO-glycans displayed on certain viruses and parasites. Even though theglycan specificity of MGL is known and several binding glycoproteinshave been identified, the molecular basis for substrate recognition hasremained elusive due to the lack of high-resolution structures. Here wepresent crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactionswith the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like theasialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. Thestructure provides the molecular basis for preferential binding ofN-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance dataacquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution.Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisXand GalNAc,respectively, This work was supported by funding from Gabrielle’s Angel Foundation for Cancer Research to G.B., grants 12/IA/1398and 16/IA/4419 from Science Foundation Ireland to P.V.M.and GOIPG/2016/858 from the Irish Research Council toA.G. F.M., H.C., and A.D. acknowledge Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding ProjectsIF/00780/2015 and PTDC/BIA-MIB/31028/2017 and UCI-BIO Project UIDB/04378/2020, as well as the Ph.D. grantattributed to A.D. (PD/BD/142847/2018). The NMRspectrometers are part of the National NMR Network(PTNMR) and are partially supported by InfrastructureProject 22161 (co-financed by FEDER through COMPETE2020, POCI, and PORL and FCT through PIDDAC). F.J.C.acknowledges funding from Agencia Estatal de Investigación(Spain) for Grant RTI2018-094751-B-C22 and CIBERES, aninitiative from the Spanish Institute of Health Carlos III. F.C.thanks Agencia Estatal de Investigación (Spain) for Grant RTI2018-099592-B-C2.