Carbohydrate Binding Mechanism of the Macrophage Galactose-type C-type Lectin 1 Revealed by Saturation Transfer Experiments

Abstract

Macrophage galactose-type C-type lectins 1 and 2 (MGL1/2) are expressed on the surfaces of macrophages and immature dendritic cells. Despite the high similarity between the primary sequences of MGL1 and MGL2, they display different ligand specificities. MGL1 shows high affinity for the Lewis X trisaccharide, whereas MGL2 shows affinity for N-acetylgalactosamine. To elucidate the structural basis for the ligand specificities of the MGLs, we performed NMR analyses of the MGL1-Lewis X complex. To identify the Lewis X binding site on MGL1, a saturation transfer experiment for the MGL1-Lewis X complex where sugar-CH/CH 2 -selective saturation was applied was carried out. To obtain sugar moiety-specific information on the interface between MGL1 and the Lewis X trisaccharide, saturation transfer experiments where each of galactose-H5-, fucose-CH 3 -, and N-acetylglucosamine-CH 3 -selective saturations was applied to the MGL1-Lewis X complex were performed. Based on these results, we present a Lewis X binding mode on MGL1 where the galactose moiety is bound to the primary sugar binding site, including Asp-94, Trp-96, and Asp-118, and the fucose moiety interacts with the secondary sugar binding site, including Ala-89 and Thr-111. Ala-89 and Thr-111 in MGL1 are replaced with arginine and serine in MGL2, respectively. The hydrophobic environment formed by a small side chain of Ala-89 and a methyl group of Thr-111 is a requisite for the accommodation of the fucose moiety of the Lewis X trisaccharide within the sugar binding site of MGL1. Macrophage galactose (Gal)2 -type calcium-type (C-type) lectins (MGLs; CD301) are carbohydrate recognition proteins expressed on the surfaces of macrophages and immature dendritic cells in humans and rodents (1-4). In mice, two closely related MGLs, MGL1 and MGL2 (CD301a and CD301b), have been found (3), whereas humans have a single MGL (CD301) gene (2). MGLs have unique carbohydrate specificities toward galactose and N-acetylgalactosamine as monosaccharides, whereas many other C-type lectins on macrophages and dendritic cells have the mannose (Man)-type specificity, such as DC-SIGN and the macrophage mannose receptor (5). The cells expressing MGL1 are widely distributed in connective tissues (6). A variety of biological functions of MGL1 have been reported so far, such as the recognition and endocytosis of glycoproteins with terminal Gal/GalNAc moieties (7, 8), contribution to defense against tumor cell metastasis (9, 10), and clearance of apoptotic cells in embryos (11,12). MGL2 is also expressed on the cells that display the same distribution pattern as MGL1-positive cells (3). For human MGL, additional functions, involvements in the regulation of effector T cells (13) and the promotion of filovirus entry (14), have been reported.The MGL molecules are ϳ42-kDa type II transmembrane glycoproteins, which possess a cytoplasmic domain, a transmembrane domain, a neck domain, and a carbohydrate recognition domain (CRD) within each molecule (1, 3). Two potential N-glycosylation sites are present...

Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands

“…To elucidate the identity of other basic amino acids involved in binding CS444, we used perdeuterated CCL5 and unlabeled CS444 to carry out a cross saturation experiment, a technique that has been successfully used to identify carbohydrate binding residues in a previous study (Sakakura et al, 2008). The data revealed that the backbone amide proton of S1, Y3 and R17 are significantly perturbed by saturation of CS444 protons (Figure 6).…”

Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands

“…We constructed recombinant MGL-Fc proteins, consisting of the extracellular domains of MGL long and MGL short fused to the human IgG1-Fc domain for detection. Previously, Ala 89 and Thr 111 (Ala 256 and Thr 278 in the full-length MGL1) were shown to be crucial for the Lewis X preference of murine MGL1 (16). Based on these findings and our previous work using the MGL short splice variant (8), we mutated human MGL short at the corresponding amino acids (H259T and K237A) to elucidate whether these amino acids also contribute to the fine specificity of human MGL (Fig.…”

“…MGL1 mainly interacts with Lewis X (Gal␤1-4(Fuc␣1-3)GlcNAc) and Lewis A (Gal␤1-3(Fuc␣1-4)GlcNAc) (15), whereas MGL2 recognizes terminal ␣-/␤-GalNAc ((sialyl)Tn antigen, LDN), as well as the terminal galactose in the core 1/T antigen and core 2 O-glycan structures (15). These disparities in ligand recognition have been further elucidated by Sakakura et al (16), showing that whereas the galactose moiety of Lewis X is engaged by the QPD motif in the MGL1 CRD, the fucose residue is bound by a secondary binding site composed of Ala 89 and Thr 111 (corresponding to Ala 256 and Thr 278 in the full-length MGL1; Fig. 1A).…”

“…MGL) has been implicated in the exclusive Lewis X specificity of mouse MGL1 (16), neither the MGL short H259T nor the K237A/H259T double mutant recognized Lewis X on the glycan array or in the ELISA-based assay, indicating that these two amino acids are not sufficient to convey Lewis X recognition. Nevertheless, the His 259 seems essential for binding extended GalNAc structures, such as the sialyl-Tn and glycosphingolipids, and might therefore be a critical component of a secondary binding site in the MGL CRD.…”

Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease

“…TDPC protons were selectively irradiated in continuous wave mode for 2 sec and the amide proton signals of the protein were detected by the TROSY pulse scheme following the irradiation (Sakakura et al, 2008). The delay for relaxation between scans was set to 2 sec.…”