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

Abstract

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.

Keywords: Carbohydrate-recognition domain; Galactose receptor; Glycan array; Glycan-binding receptor; Lectin.

Fig. 1.

Organization of the MGL polypeptide. (A) A summary of two potential views of MGL organization based on paradigms from mannose-binding protein (left), in which CRDs interact extensively and DC-SIGN (right), in which the neck regions mediate oligomer formation independently of the CRDs. (B) Linear diagrams of the MGL polypeptide based on the most abundant isoform, which is designated isoform 3. Additional sequences found in some cDNAs from alternatively spliced mRNA molecules are present in isoforms 1 and 2. Regions expressed are shown, along with the sequence of the neck region divided into four sub-regions, which are color-coded to correspond to the sequence above. Two of the sub-regions contain heptad repeat patterns of nonpolar aliphatic amino acid side chains highlighted with green shading. Glycosylation sites are noted with arrows. The disulfide bonding pattern in the CRD is also indicated. (C) SDS–PAGE (17.5% gel) of purified fragments of MGL. Left panel, samples without reducing agent. Right panel, samples reduced with 2-mercaptoethanol. Gel was stained with Coomassie blue. (D) SDS–PAGE (10% gel) of the extracellular fragment of MGL subjected to chemical crosslinking with bis-sulfosuccinimidylsuberate. Concentrations of cross-linking reagent are indicated at the top of each well. Gel was stained with Coomassie blue. (E) Gel-filtration analysis of fragments of MGL on a Superdex S200 column. (F) Gel-filtration analysis of CRD and extended CRD of MGL on a Superdex S75 column. In (E and F), elution positions of standards are indicated in kDa at the bottom.

Fig. 2.

Glycan array and pathogen array analysis of MGL-binding specificity. (A) Synthetic glycan array. Results are arranged in rank order based on decreasing signal observed on the glycan array probed with fluorescently labeled extracellular fragment of MGL at 10 μg/mL. Similar results were obtained at 1 and 90 μg/mL. The complete glycan array results are provided in Supplementary data, Table S1. All signals shown in color result from binding of glycans containing GalNAc with free 3- and 4-hydroxyl groups. In the top 65 glycans, the single exception to this rule is a strong signal for a simple α-linked fucose residue (Position 7). No studies with MGL binding to earlier versions of the glycan array have revealed binding to this sugar and none of the 170 other glycans on the array that contain α-linked fucose but lack GalNAc give signals above background, suggesting that this signal does not reflect an alternative binding specificity for glycans. (B) A model of the binding site of MGL based on the structure of the CRD of mannose-binding protein modified with portions of the MGL sequence so that it binds GalNAc (Feinberg et al. 2000). This figure was prepared with PyMol based on Protein Data Bank entry 1fih. The sequences of the model protein and human MGL are compared in Supplementary data, Figure S1. (C) Bacterial O-specific polysaccharide array. Each oligosaccharide is represented five times printed directly and five times printed after derivatization with a disubstituted oxamine linker, each at 0.03, 0.06, 0.125, 0.25 and 0.5 mg/mL. Red, GalNAc residues in terminal positions on branches; blue, 6-substituted GalNAc in the backbone sequence; green, 3- or 4-substituted GalNAc in the backbone sequence. A complete list of the bacterial sources of the lipopolysaccharides and their O-specific polysaccharide core structures is provided in Supplementary data, Table S2.

Fig. 3.

Conformations of MGL fragments analyzed by circular dichroism. Circular dichroism spectra for the entire extracellular fragment of MGL and for the CRD and extended CRD fragments were normalized on a molar basis so that difference spectra corresponding to the neck could be obtained by subtraction. (A) Calculation of difference spectrum corresponding to the entire neck domain. (B) Calculation of difference spectrum corresponding to portion of the neck adjacent to the CRD. (C) Circular dichroism spectrum of the CRD as a function of Ca²⁺ concentration. (D) Ca²⁺-dependence of circular dichroism of the CRD at 229 nm. Values for the molar ellipticity at 229 nm were fitted to a third-order binding equation using SigmaPlot.

Fig. 4.

Limited proteolysis of the extracellular fragment of MGL. SDS–PAGE of digestions conducted for 30 min at 37°C at increasing concentrations of protease. Gel was stained with Coomassie blue. The bacterially expressed CRD fragment of MGL was run in parallel for comparison in the final lane of the left-hand panel.

Fig. 5.

Stability of MGL fragments analyzed by differential scanning calorimetry. Proteins were extensively dialyzed against 100 mM NaCl, 10 mM HEPES, pH 7.4 and 2.5 mM CaCl₂ for calorimetry. Concentrations of proteins were (A) extracellular fragment of MGL 2.7 mg/mL; (B) extended CRD, 0.7 mg/mL; (C) CRD, 10.0 mg/mL.

Fig. 6.

Stability of MGL fragments analyzed by circular dichroism. (A and B) Folded conformation of the CRD (0.07 mg/mL) was assessed by comparing the molar ellipticity at 229 nm, which reflects the folded conformation of the tryptophan residue adjacent to the Ca²⁺ that forms the sugar-binding site (see Figure 3C and D). (C and D) Folded conformation of the neck domain (0.27 mg/mL) was assessed based on the ellipticity at 222 nm of the extracellular fragment of MGL, which is sensitive to the α-helical conformation that arises dominantly from the neck domain. Proteins were dialyzed extensively against 10 mM Tris-Cl, pH 7.4 and 2.5 mM CaCl₂. Spectra represent averages of 10 scans each, taken after a 2-min stabilization period at each temperature. Data were fitted to first order transitions in order to determine the midpoint of the unfolding transitions.

Fig. 7.

Purification and analysis of the neck domain of MGL. (A) SDS–PAGE (17.5% gel) of neck domain purified on immobilized Ni²⁺ in the presence of urea (lane 1, Coomassie blue; lane 2, detection with antibody to the His₆ tag) and following further purification on immobilized Ni²⁺ in the absence of urea (lane 3, Coomassie blue). (B) Gel-filtration analysis of the neck domain of MGL on a Superdex S75 column. Concentrations of starting samples (100 μL) are indicated. (C) Conformation of the neck domain analyzed by circular dichroism. (D) Stability of the neck domain determined by differential scanning calorimetry. Protein concentration was 0.65 mg/mL.