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

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.

SUBMITTER: Feinberg H

PROVIDER: S-EPMC7949135 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe innate pattern recognition C-type-lectin receptors (CLRs), including mannose receptor (MRC1; CD206), have been suggested to functionally interact with allergens and are critical in controlling immune response. Fibrocytes have been considered to play a role in allergic asthma. Here we sought to investigate the functional interaction of cockroach allergens with CD206 in fibrocytes.MethodsProfiling of N-linked glycans from natural purified cockroach allergen Bla g 2 was accomplished by MALDI-MS. The binding activity of cockroach allergens to CD206 was determined by solid-phase binding assays. Levels of CD206 expression on human fibrocytes and CD206 mediated signaling and cytokine production in Bla g 2 treated fibrocytes were determined.ResultsProfiling of N-linked glycans from Bla g 2 revealed a predominance of small, mannose-terminated glycans with and without fucose. Significant binding of Bla g 2 to CD206 was observed, which was inhibited by yeast mannan (a known CD206 ligand), free mannose, and a blocking antibody (anti-hMR). Flow cytometric analyses of human fibrocytes (CD45(+) and collagen-1(+)) showed selective expression of CD206 on fibrocytes. Functionally, a concentration-dependent uptake of FITC labeled Bla g 2 by fibrocytes was observed, but was significantly inhibited by anti-hMR. Bla g 2 can stimulate up-regulation of inflammatory cytokines including TNF-alpha and IL-6 and activation of nuclear factor kappa B (NF-kB/p65), p38 mitogen-activated protein kinase (p38), ERK, and JNK in cultured fibrocytes. This increased secretion of TNF-alpha and IL-6 and activation of NF-kB, ERK, and JNK was significantly inhibited by the addition of either mannan or mannose. Furthermore, Bla g 2 induced increase in TNF-alpha and IL-6 production was also inhibited by the use of NF-kB, ERK, and JNK inhibitors.ConclusionThese results provide evidence supporting the existence of a functional cockroach allergen-CD206 axis in human fibrocytes, suggesting a role for CD206 in regulating allergen induced allergic responses in asthma.

Project description:Computer modeling of complexation of mono- and oligosaccharide ligands with the main (fourth) carbohydrate-binding domain of the mannose receptor CD206 (CRD4), as well as with the model receptor concanavalin A (ConA), was carried out for the first time, using methods of molecular dynamics and neural network analysis. ConA was shown to be a relevant model of CD206 (CRD4) due to similarity of the structural organization of the binding sites and high correlation of the values of free energies of complexation between the literature data and computer modeling (r > 0.9). Role of the main factors affecting affinity of the ligand-receptor interactions is discussed: the number and nature of carbohydrate residues, presence of Me-group in the O1 position, type of the glycoside bond in dimannose. Complexation of ConA and CD206 with ligands is shown to be energetically caused by electrostatic interactions (E) of the charged residues (Asn, Asp, Arg) with oxygen and hydrogen atoms in carbohydrates; contributions of hydrophobic and van der Waals components is lower. Possibility of the additional stabilization of complexes due to the CH-π stacking interactions of Tyr with the Man plane is discussed. The role of calcium and manganese ions in binding ligands has been studied. The values of free energies of complexation calculated in the course of molecular dynamics simulation correlate with experimental data (published for the model ConA): correlation coefficient r = 0.68. The Pafnucy neural network was trained based on the set of PDBbind2020 ligand-receptor complexes, which significantly increased accuracy of the energy predictions to r = 0.8 and 0.82 for CD206 and ConA receptors, respectively. A model of normalization of the complexation energy values for calculating the relevant values of ΔGbind, Kd is proposed. Based on the developed technique, values of the dissociation constants of a series of CD206 complexes with nine carbohydrate ligands of different structures were determined, which were not previously known. The obtained data open up possibilities for using computer modeling for the development of optimal drug carriers capable of active macrophage targeting, and also determine the limits of applicability of using ConA as a relevant model for studying parameters of the CD206 binding to various carbohydrate ligands.

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

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