Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen

Abstract

The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of N-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired 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 LewisX and GalNAc, respectively.

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope, LacdiNAc by Immune‐Related Lectins

“…To transfer these experimental NMR findings into a 3D structure, 500 ns molecular dynamics (MD) simulations of the hMGL/LDN and hMGL/LN complexes were carried out (see supporting information) using the coordinates of the X-ray crystal structure of the complex hMGL/ GalNAc (PDB ID: 6PY1). [36] The relative orientation between the GalNAc/Gal and GlcNAc units in LDN and LN bound structures defined for MD are compatible with the NMR data obtained for LDN in solution (Figure S13A). The saccharide conformation complies with the exo-anomeric effect.…”

“…In fact, the involvement of Tyr236 and His286 in the engagement of the NHAc group has been recently shown in the X-ray crystallographic analysis of hMGL bound to GalNAc monosaccharide (PDB ID: 6PY1). [36] The significant CSP difference found for Asp294 in the hMGL/LDN and hMGL/LN complexes (Figure 3C) fits with its proximity to the NHAc or OH functional groups depending on the ligand context, LDN or LN, respectively.…”

“…The 3D models of the hMGL complexed with LDN and LN were generated from X-ray structure of carbohydrate recognition domain of hMGL complexed with GalNAc monosaccharide (PDB code 6PY1). [36] The setup for the molecular dynamics was like that described previously in Diniz et al, [35] except for the time of the production step that was set to 500 ns instead of 100 ns. A detailed analysis of each MD trajectory was performed.…”

The Shape of Nanostructures Encodes Immunomodulation of Carbohydrate Antigen and Vaccine Development

“…To address the mechanism underlining the immunomodulation of the nanostructures, we first investigated the cellular internalization of AuNPs using mDCs because MGL (human macrophage galactose and N-acetylgalactosamine-specific Ctype lectin) receptors interact with Tn-antigen on mDCs engaged in receptor-mediated endocytosis. 61 Thus, it is hypothesized that disparity in the aspect ratio, nanostructure contact area, and antigen/adjuvant conjugation modulate the uptake mechanism and sequestration.…”

Nanoparticle Shape Governs Immunomodulation of MUC1 Antigen to Develop Anti-cancer Vaccine

“…To address the mechanism underlining the immunomodulation of the nanostructures, we first investigated the cellular internalization of AuNPs using mDCs because MGL (human macrophage galactose and Nacetylgalactosamine-specific C-type lectin) receptors interact with Tn-antigen on mDCs engaged receptor mediated endocytosis. 55 Thus, it is hypothesized that disparity in the aspect ratio, nanostructure contact area, and antigen/adjuvant conjugation modulate the uptake mechanism and sequestration.…”