Abstract
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.
Highlights
In an effort to increase yields and generate novel crystal forms of carbohydraterecognition domain 4 (CRD4) that avoid formation of domain-swapped dimers seen in previous experiments, the expression construct was modified by changing the expression system and removing potentially flexible regions
Interdomain linkers on either side of CRD4 were retained in the CRD4 expression system employed previously, in which the ompA signal sequence was used to direct CRD4 to the periplasm of Escherichia coli to facilitate disulfide bond formation [24]
In these earlier experiments, folded CRD4 was extracted from the periplasm and purified by affinity chromatography
Summary
Sugar structures on microorganisms targeted by the receptor include mannans on the surface of yeasts, mannose-capped lipo-arabinomannans on mycobacteria, and high-mannose oligosaccharides on the surfaces of viruses [10,11,12] These diverse ligands are bound to three different types of protein domains in the extracellular portion of the mannose receptor (Fig. 1). These two domains are usually designated carbohydraterecognition domains (CRDs) 4 and 5 Interaction of these domains with mannose-containing oligosaccharides accounts for binding of the receptor to lysosomal enzymes, nonhelical fragments of collagen, and viral glycoproteins and can account for binding to mannose residues on the surfaces of other pathogens. In the present studies, binding competition experiments, glycan array analysis, and X-ray crystallography have been used to define the binding selectivity of CRD4 and the molecular mechanisms by which this selectivity is achieved
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