Structural analysis of carbohydrate binding by the macrophage mannose receptor CD206.

The human mannose receptor expressed on macrophages and hepatic endothelial cells scavenges released lysosomal enzymes, glycopeptide fragments of collagen, and pathogenic microorganisms and thus reduces damage following tissue injury. The receptor binds mannose, fucose, or N-acetylglucosamine (GlcNAc) residues on these targets. C-type carbohydrate-recognition domain 4 (CRD4) of the receptor contains the site for Ca2+-dependent interaction with sugars. To investigate the details of CRD4 binding, glycan array screening was used to identify oligosaccharide ligands. The strongest signals were for glycans that contain either Manα1-2Man constituents or fucose in various linkages. The mechanisms of binding to monosaccharides and oligosaccharide substructures present in many of these ligands were examined in multiple crystal structures of CRD4. Binding of mannose residues to CRD4 results primarily from interaction of the equatorial 3- and 4-OH groups with a conserved principal Ca2+ common to almost all sugar-binding C-type CRDs. In the Manα1-2Man complex, supplementary interactions with the reducing mannose residue explain the enhanced affinity for this disaccharide. Bound GlcNAc also interacts with the principal Ca2+ through equatorial 3- and 4-OH groups, whereas fucose residues can bind in several orientations, through either the 2- and 3-OH groups or the 3- and 4-OH groups. Secondary contacts with additional sugars in fucose-containing oligosaccharides, such as the Lewis-a trisaccharide, provide enhanced affinity for these glycans. These results explain many of the biologically important interactions of the mannose receptor with both mammalian glycoproteins and microbes such as yeast and suggest additional classes of ligands that have not been previously identified.

Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle.

The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages.

Organization of the extracellular portion of the macrophage galactose receptor: a trimeric cluster of simple binding sites for N-acetylgalactosamine.

The properties of the human macrophage galactose receptor have been investigated. Specificity for N-acetylgalactosamine (GalNAc) residues with exposed 3- and 4-hydroxyl groups explains virtually all of the results obtained from a recently expanded array of synthetic glycans and is consistent with a model for the structure of the binding site. This simple interaction is sufficient to explain the ability of the receptor to bind to tumor-cell glycans bearing Tn and sialyl-Tn antigens, but not to more elaborate O-linked glycans that predominate on normal cells. This specificity also allows for binding of parasite glycans and screening of an array of bacterial outer membrane oligosaccharides confirms that the receptor binds to a subset of these structures with appropriately exposed GalNAc residues. A key feature of the receptor is the clustering of binding sites in the extracellular portion of the protein, which retains the trimeric structure observed in the cell membrane. Chemical crosslinking, gel filtration, circular dichroism analysis and differential scanning calorimetry demonstrate that this trimeric structure of the receptor is stabilized by an α-helical coiled coil that extends from the surface of the membrane to the globular carbohydrate-recognition domains. The helical neck domains form independent trimerization domains. Taken together, these results indicate that the macrophage galactose receptor shares many of the features of serum mannose-binding protein, in which clusters of monosaccharide-binding sites serve as detectors for a simple epitope that is not common on endogenous cell surface glycans but that is abundant on the surfaces of tumor cells and certain pathogens.