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Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands
Department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands
Department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands
To whom correspondence should be addressed: Dept. of Molecular Cell Biology and Immunology, Amsterdam UMC, Location VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. Tel.: 31-204440355
Department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands
7 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. 1 Supported by the FCT-Portugal IF project (IF/00780/2015) and the UCIBIO funding UID/Multi/04378/2013, co-financed by the FEDER (POCI-01-0145-FEDER-007728). 2 Supported by Ministerio de Economía y Competitividad Projects CTQ2015-67727-R and UNLR13-4E-1931. 3 Present address: Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, The Netherlands. 4 Present address: Dept. of Pharmaceutical Sciences and Chemistry, Utrecht University, 3584 CG Utrecht, The Netherlands.
The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (αGalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGLshort H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGLshort, His259 mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGLshort H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His259 residue.
The C-type lectin family is a large family of soluble and transmembrane proteins that share the specific recognition of carbohydrate structures, which in most cases is Ca2+-dependent (
The primary binding site within this CRD is composed of a conserved three-amino acid motif, which largely determines the class of glycans the lectin is able to engage (e.g. the Gln-Pro-Asp (QPD) motif predicts binding to galactose or GalNAc) (
). In addition, secondary binding sites fine-tune the carbohydrate recognition, thereby creating enormous diversity in C-type lectin specificity and function.
The human QPD-containing macrophage galactose-type lectin (MGL; CLEC10A or CD301) is exclusively expressed by macrophages and dendritic cells within the immune system (
). Triggering of MGL on these human dendritic cells conveys immune inhibitory signals, leading to production of the anti-inflammatory cytokines and the ability to block unwanted inflammatory responses (
Human T cell activation results in extracellular signal-regulated kinase (ERK)-calcineurin-dependent exposure of Tn antigen on the cell surface and binding of the macrophage galactose-type lectin (MGL).
). Humans harbor only one MGL gene, which is subject to extensive splicing, generating different long and short MGL isoforms. Alternative splicing mainly occurs around exon 6, encoding the terminal stalk region (
The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.
The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.
). Another relatively abundant variant is the MGL 6B variant, which compared with the MGLshort contains an additional 27 amino acids in its stalk region (Fig. 1A, MGLlong).
Figure 1Differential GalNAc binding of MGL variants.A, amino acid sequence of the human MGL splice variants and the mouse MGL1. The stalk region and CRD are depicted, showing the 27-amino acid insertion in the stalk region of the MGLlong variant. Boxed in gray are the primary binding site (QPD) of MGL and the localization of the K237A and H259T mutations. B, binding of MGL variants determined by glycan array. Depicted are the binding to selected O-glycan core structures (left), Helminth-associated glycans (middle), and glycosphingolipids (right). Full glycan array results can be found in Table S1. Significance was calculated using two-way analysis of variance and Bonferroni’s multiple-comparison test. Data represent mean ± S.E. LacCer, lactosylceramide. C, binding of the MGL variants to PAA-coated glycoconjugates was determined using an ELISA-based assay. Human IgG1 was used as a negative control. One representative experiment of three is shown. Significance was calculated by nonlinear regression followed by one-way analysis of variance and Bonferroni’s multiple-comparison test. *, p < 0.05; **, p < 0.01; ***, p < 0.005.
The carbohydrate specificity of the MGLshort variant has been widely studied through surface plasmon resonance, glycan microarrays, and cellular binding assays, revealing its exclusive specificity for terminal GalNAc moieties, such as the Tn antigen (αGalNAc-Ser/Thr), the LacdiNAc structure (LDN; GalNAcβ1–4GlcNAc), and the GalNAc-Tyr moiety (
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
). In QPD-containing C-type lectins, the equatorial/axial configuration of the 3-OH and 4-OH groups in the GalNAc are crucial for glycan binding and chelation of the Ca2+ ion (
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
). It is noteworthy that MGL tolerates substitutions at the 6-OH position and therefore recognizes the sialylated Tn antigen (Neu5Acα2–6GalNAcα-Ser/Thr) (
). Despite their high homology and the shared QPD motif, MGL1 and MGL2 differ substantially in their carbohydrate specificities. MGL1 mainly interacts with Lewis X (Galβ1–4(Fucα1–3)GlcNAc) and Lewis A (Galβ1–3(Fucα1–4)GlcNAc) (
), whereas MGL2 recognizes terminal α-/β-GalNAc ((sialyl)Tn antigen, LDN), as well as the terminal galactose in the core 1/T antigen and core 2 O-glycan structures (
), 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 Ala89 and Thr111 (corresponding to Ala256 and Thr278 in the full-length MGL1; Fig. 1A). These amino acids are replaced by arginine and serine in MGL2, respectively. Although no crystal structure of human MGL is available, glycan accommodation by MGLlong has been studied by saturation transfer-difference NMR measurements and molecular modeling, revealing fine-structural insights into the interaction of Gal/GalNAc and MUC1-Tn-glycopeptides with MGL (
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
). These NMR binding studies have verified that galactose is a much weaker ligand than GalNAc. The NHAc group of GalNAc participates in additional H-bond and CH-π interactions, which are absent in the case of galactose. Moreover, an increased number of H bonds were formed when MGL engaged a Tn-glycopeptide; however, this was not reflected by an improved affinity, probably due to the subtle enthalpy–entropy equilibrium of any ligand–protein recognition event.
Our knowledge on MGL has substantially increased over the last decade; nevertheless, the carbohydrate fine specificities and ligand-binding properties of the different human MGL splice variants have never been directly compared and have been assumed to be identical. Here, we aimed to elucidate the molecular recognition features of human MGLshort and MGLlong isoforms by evaluating their carbohydrate specificities using glycan and Tn-peptide microarrays. In addition, we mutated the His259 in the CRD of human MGL, equivalent to Thr111 in MGL1, to Thr in the MGLshort variant (H259T) to elucidate whether this amino acid is also crucial for the exclusive GalNAc-binding properties of human MGL.
Results
Carbohydrate recognition profiles of MGL variants
The MGL 6B (MGLlong) and 6C (MGLshort) splice variants vary in a region of 27 amino acids present in the terminal stalk domain of MGLlong only (Fig. 1A). We constructed recombinant MGL-Fc proteins, consisting of the extracellular domains of MGLlong and MGLshort fused to the human IgG1-Fc domain for detection. Previously, Ala89 and Thr111 (Ala256 and Thr278 in the full-length MGL1) were shown to be crucial for the Lewis X preference of murine MGL1 (
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
), we mutated human MGLshort at the corresponding amino acids (H259T and K237A) to elucidate whether these amino acids also contribute to the fine specificity of human MGL (Fig. 1A). In our initial screens, the MGLshort K237A/H259T double mutant showed identical binding characteristics as the MGLshort single H259T mutant (data not shown); therefore, we omitted the K237A/H259T mutant from further analyses. All MGL-Fc proteins displayed equal binding to three independent anti-MGL antibodies (Fig. S1B), confirming the correct folding and indicating that the H259T mutation did not induce significant structural changes.
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
), MGLshort recognized the O-glycans αGalNAc, sialyl-Tn, and core 6 (GlcNAcβ1–6GalNAcα); the helminth glycans LDN and its fucosylated derivate (LDNF; GalNAcβ1–4(Fucα1–3)GlcNAc); and the glycosphingolipid structures GM2 (GalNAcβ1–4(Neu5Acα2–3)Galβ1–4Glc) and GD2 (GalNAcβ1–4(Neu5Acα2–8Neu5Acα2–3)Galβ1–4Glc) (Fig. 1B; full results in Table S1). Although differences were subtle, MGLlong displayed significantly higher binding to core 6, LDN, and LDNF than MGLshort and significantly lower binding to GM2 and GD2, as deduced by glycan microarrays. In contrast, the MGLshort H259T failed to interact with sialyl-Tn, LDNF, GM2, or GD2 on the array, whereas αGalNAc, the core 6 structure, and LDN were still bound (Fig. 1B). None of the MGL variants interacted with the core 1/T antigen, LacNAc, Lewis X, lactosylceramide, or GM3 structures on this array.
Next, we performed ELISA-based binding assays using polyacrylamide (PAA)-coupled glycoconjugates. All MGL variants, including the mutant, strongly bound the PAA-coupled GalNAc (Fig. 1C). Recognition of PAA-coupled sialyl-Tn and LDN by MGLshort and MGLlong was comparable, whereas MGLshort H259T showed diminished binding to both. Again, no binding could be detected to galactose, core 1/T antigen, or Lewis X. In contrast to the glycan microarray, no differential binding between MGLshort and MGLlong to the LDN moiety could be observed. The discrepancy with glycan microarrays might be explained by the multivalent nature of the PAA-glycoconjugate (20–25 glycans/molecule) versus the monovalent presentation on the array.
Differential glycan binding of the MGLshort H259T mutant
Overall, the carbohydrate recognition profiles of MGLshort and MGLlong appeared to be quite similar; therefore, we focused our further investigations on the differential recognition of MGLshort and the MGLshort H259T mutant. Although the Thr111 amino acid (corresponding to His259 in human MGL) has been implicated in the exclusive Lewis X specificity of mouse MGL1 (
), neither the MGLshort 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 His259 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.
To further extract carbohydrate determinants associated with MGL binding, we analyzed our array results using the publicly available Glycopattern web-based resource (https://glycopattern.emory.edu)7 (
). We first employed the GlycanMotifMiner to discover glycan binding motifs for MGLshort and MGLshort H259T. A binding motif was defined as a structural glycan element that contained the motif and was classified as an MGL-binding glycan. For both MGL variants, we identified the extended core 6 O-glycan Galβ1–3GlcNAcβ1–6GalNAc as the main glycan-binding motif (Table 1). In addition, MGLshort recognized the LDN motif and the globoside glycolipid GalNAcβ1–3Gal (Gb4Cer). The MGLshort H259T only seemed to favor core 6–derived structures (Table 1). Although not identified as a glycan-binding motif, some additional glycans emerged as high-affinity ligands for MGL on the glycan microarray. These included the single α- and β-GalNAc and 6-sulfated LDN (Table 2).
Table 1Glycan binding motifs of MGLshort and MGLshort H259T
To evaluate the differential glycan binding of MGLshort and MGLshort H259T, we used the side-by-side comparison tool in the Glycopattern software. Here, we identified 10 glycan structures that displayed high binding to MGLshort, but failed to interact with the MGLshort H259T (Table 3). Five of these contain the GalNAcα1–3(Fucα1–2)Galβ blood group A antigen. Furthermore, we observed diminished binding to the synthetic GalNAcβ1–3GalNAcα epitope, the ganglioside GM2, sialylated LDN, a synthetic ganglioside containing polysialic acid, and the Forssman antigen (GalNAcα1–3GalNAcβ) (Table 3).
Table 3Differential glycan binding of MGLshort H259T
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
). To address whether the His259 plays a role in the fine specificity of MGL toward Tn-containing glycopeptides, we employed a mucin glycopeptide array that included the known MGL ligands MUC1, MUC2, and CD45, which we interrogated using the MGLshort- and MGLshort H259T-Fc proteins (Fig. 2) (
Tumor-associated Tn-MUC1 glycoform is internalized through the macrophage galactose-type C-type lectin and delivered to the HLA class I and II compartments in dendritic cells.
Figure 2Binding profile of MGL variants to the Tn-glycopeptide array.A, glycopeptides and corresponding parental peptides were printed at 100 μm and probed using the MGLshort-Fc (left panels) and MGLshort H259T-Fc (right panels) at the indicated concentrations. Relevant protein carriers are indicated in the figure. Data represent mean ± S.D. (error bars). RFU, relative fluorescence units. B, list of peptide backbones and Tn attachment sites of glycopeptides present on the Tn-glycopeptide array. *, presence of a Tn antigen on Ser or Thr.
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
) displayed weaker binding to MGLshort (Fig. 2A). Curiously, MGLshort preferred the diglycosylated T*T* or T*XT* MUC2 peptides (where the asterisk denotes the presence of a Tn antigen on Ser or Thr) compared with the mono- and triglycosylated peptides, whereas the diglycopeptide S*T* MUC1 displayed much weaker binding to MGLshort (Fig. 2A). Additionally, MGLshort may favor a Thr-based Tn antigen compared with the Ser-linked Tn antigen on this array (compare peptides 29/30 with 31/32).
Interestingly, in the glycopeptide array, MGLshort H259T lost high-affinity binding toward MUC1 structures, even at high probing concentrations, while displaying a residual affinity toward MUC2- and CD45-derived Tn-glycopeptides (Fig. 2A), indicating that replacement of His259 by a threonine residue has an impact on the Tn-glycopeptide recognition by MGL. Indeed, the loss in affinity of the MGLshort H259T mutant toward Tn-glycopeptides could imply that the His259 residue interacts with the surrounding peptide backbone of Tn-glycopeptides.
MGLshort H259T has a diminished capacity to recognize cellular ligands
To confirm that the MGLshort H259T mutant also has a diminished capacity to interact with cellular MGL ligands, we performed flow cytometry–based MGL-Fc binding assays to colorectal carcinoma and Jurkat cell lines. The colorectal carcinoma cell lines HT29 and SW1398 were previously shown to contain large numbers of MGL-binding ligands (
). Whereas MGLshort displayed high binding to the colorectal carcinoma cell lines, the MGLshort H259T failed to recognize the HT29 cells and displayed a 10-fold decreased binding to the SW1398 cells (Fig. 3A). The Jurkat cell line has a deficiency in the COSMC chaperone leading to an absence of T-synthase activity and thus a complete lack of O-glycan elongation beyond the Tn stage (
). Again, we observed a reduced binding of MGLshort H259T to Jurkat cells (Fig. 3B), verifying that also on cells, MGLshort H259T has a reduced ability to engage Tn antigen. Together, our data indicate that although the H259T MGL mutant can still recognize Tn antigen, it does so with a strongly reduced affinity.
Figure 3MGLshort H259T displays poor recognition of GalNAc-containing glycan epitopes on tumor cells. Colorectal cancer cell lines HT29 and SW1398 (A) and the COSMC-deficient Jurkat cell line (B) were incubated with 5 μg/ml or the indicated concentrations of the MGL-Fc variants for 30 min at 4 °C. Binding was detected using an FITC-labeled goat anti-human Fc secondary antibody and analyzed by flow cytometry. One representative experiment of three is shown.
Molecular dynamics simulations of MGL and the H259T mutant
The involvement of His259 in the MGL-mediated recognition of Tn-glycopeptides seems to indicate that this residue is part of what we dubbed the secondary binding site in the MGL CRD. To check whether this secondary binding site establishes additional interactions with the surrounding peptide backbone of Tn-glycopeptides, we conducted molecular dynamics (MD) simulations on both MGL/Tn-glycopeptide and MGL H259T/Tn-glycopeptide complexes. The Tn-glycopeptides MUC2B Ac-PTT*TPLK-NH2 (peptide 15 in Fig. 2B) and MUC1 H2N-TRPAPGST*APPA-NH2 (peptide 6 in Fig. 2B) were selected based on the array results. A 3D model of the MGL CRD construct and the corresponding H259T mutant was built employing the homology model generated for MGL (residues Cys181–Leu308), as previously reported by us (
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
). Intensive 1-μs MD simulation was initially accomplished on both the WT MGLshort (MGL-His259) and the H259T mutant (MGL-Thr259) in explicit water and Ca2+ ions to evaluate the stability of both models. A structural ensemble of both proteins is depicted in Fig. S2. The calculated root mean square deviation shows that these molecules are equally stable and that the replacement of His259 by Thr does not induce major conformational perturbations (Fig. S2). Complexes of MGL-His259 and MGL-Thr259 with MUC2B and MUC1 glycopeptides were further generated as described by Marcelo et al. (
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
). Our previous MD and CORCEMA-ST analysis allowed us to conclude that the GalNAc monosaccharide interacts with MGL, employing two distinct binding modes (A and B), with alternative relative orientations of the galactosyl ring toward MGL (
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
), and in GalNAc-binding mode A, the NHAc fragment of GalNAc is close to the His259 to potentially establish a stable H bond. Thus, MD simulations of the generated complexes in binding mode A were further submitted to evaluate their stability and the existence of intermolecular interactions involving either His259 (WT MGL) or Thr259 (mutant) and the surrounding peptide structure in GalNAc-containing peptides. In the case of MUC2B, transient H bonds were detected with different peptide residues (Fig. 4, A–D). Despite the fact that these H bonds are not highly populated, the sum of these H bonds in the surrounding peptide sequence can help to stabilize the complex and thereby significantly increase the binding affinity of MGL toward Tn-glycopeptides. No H bonds were detected between Thr259 and peptide backbone in the MD simulation analysis of mutant MGL-Thr259/MUC2B complex. The replacement of His259 by Thr thus may abolish these additional interactions. Furthermore, as expected, a stable H bond was detected between the CO of the NHAc group of GalNAc and the polar hydrogen HΕ of His259.
Figure 4Selected frames of the MGL-His259/MUC2B complex extracted from 200-ns MD simulations.A–D, transient H bonds are established between the His259 and the peptide backbone (shown as yellow dashed lines). No H bonds were detected in the MGL-Thr259/MUC2B complex.
Interestingly, stable H bonds were not present between His259 and the peptide backbone of the MUC1-derived Tn-glycopeptide; however, a water pocket could be observed between the carbonyl CO of Thr linked to GalNAc and the polar hydrogen of the aromatic ring of His259 (Fig. 5). In contrast, no water oxygen density was detected involving the Thr259 amino acid for the complex generated using the MGLshort H259T mutant model. Overall, the MD results suggest that His259 plays a critical role in the MGL recognition of Tn-glycopeptides by establishing intermolecular interactions with the peptide backbone.
Figure 5Water-mediated binding between Tn-glycopeptide MUC1 (H2N-TRPAPGST*APPA-NH2) and His259 of MGLshort. Shown is the 2D-rdf function derived from 200-ns MD simulations between carbonyl (CO) of the glycosylated Thr and the polar nitrogen NΕ of the aromatic ring of His259 (top), together with the water density (shown in red) calculated for the MGL-His259/MUC1–derived Tn-glycopeptide complex. No water density was detected in the case of MGL-Thr259/MUC1-derived Tn-glycopeptide complex.
In this paper, we further defined the fine specificity of the human C-type lectin MGL through microarray analysis and MD simulations. Although MGL binding to the CFG glycan array has been analyzed before (
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
) (see also the CFG glycan array data website: http://www.functionalglycomics.org),7 the carbohydrate recognition profiles of the different human MGL splice variants and the H259T mutant have never been directly compared. In our glycan array analyses, all identified MGL ligands contained a nonreducing terminal GalNAc with the equatorial/axial 3-OH and 4-OH groups exposed, allowing the interaction with Ca2+ at the primary binding site. None of the 3- or 4-extended GalNAc structures were recognized by MGL in the glycan microarray, confirming its unique preference for terminal GalNAc residues (
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
). It is noteworthy that extension at C-6 was allowed. Elongation or sulfation of the 3-OH or 4-OH abrogated MGL binding, thus explaining why some structures that do contain an identified MGL-binding motif were still categorized as nonbinders by the Glycopattern software (Table 1).
Strikingly, the identified MGL-binding elements are highly restricted in their expression pattern, suggesting that MGL may have some unique and distinctive functions within the human body. The LDN motif is mainly found on intestinal helminths (
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
in: Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. 2nd Ed. Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY2009
) (http://www.functionalglycomics.org),7 whereas the Gb4Cer glycolipid is most abundant on human embryonic stem cells and up-regulated on colon cancer (
). Together, these studies indicate that MGL may recognize cancer cells through their glycolipid framework and/or LDN expression and may be involved in hormone action or turnover. Both potential roles have so far not been studied in relation to MGL biology, although the mouse MGL1 was recently shown to clear WT and hyposialylated von Willebrand factor (
), indicating that nonimmune-related functions might exist for human MGL as well.
Intriguingly, we could detect some subtle nuances in glycan specificity between the MGLshort and MGLlong splice variants, even though their CRDs are completely identical. The additional 27 amino acids in MGLlong stalk region could potentially affect multimerization of the receptor. The neck region of MGLshort has been shown to form trimers, generating a cluster of binding sites for glycans abundant on pathogens and tumor cells (
). So far, there are no indications that multimerization might be different for MGLlong. The MGL CRDs have some flexibility to engage differentially spaced glycans on the counteracting interface (
). It is therefore tempting to speculate that the 27-amino acid region alters the orientation and spacing of the individual MGL CRDs, thereby modulating the range of ligands that are able to interact. This phenomenon has already been demonstrated for DC-SIGNR, where the variable number of neck repeats in DC-SIGNR affects the orientation of the individual CRDs and subsequently the relative binding affinities for surface glycoproteins (
). Structural studies of MGLshort and MGLlong trimers are required to formally prove the effect of the extended neck region in MGLlong, as well as a more quantitative analysis of binding affinities of each interacting glycan with surface plasmon resonance.
Engagement of Tn antigens by MGL on human dendritic cells is thought to lead to immune tolerance via the MGL-induced secretion of the anti-inflammatory cytokine IL-10 and the ability to instruct the differentiation of suppressive CD4+ T cells (
Human T cell activation results in extracellular signal-regulated kinase (ERK)-calcineurin-dependent exposure of Tn antigen on the cell surface and binding of the macrophage galactose-type lectin (MGL).
). Moreover, through direct engagement of CD45 on activated T cells, MGL is able to inhibit T cell proliferation and cytokine release, while at the same time promoting T cell apoptosis (
Tumor-associated Tn-MUC1 glycoform is internalized through the macrophage galactose-type C-type lectin and delivered to the HLA class I and II compartments in dendritic cells.
). Therefore, we constructed a Tn-glycopeptide array harboring these immune-relevant MGL ligands. On our Tn-glycopeptide array, WT MGLshort seemed to prefer MUC2- and CD45-derived glycopeptides over the MUC1 glycopeptides. However, the MUC1 glycopeptides in this particular array represent only a subset of possible glycosylation patterns of the MUC1 repeats; thus, we cannot exclude the possibility that other MUC1 glycopeptides may be dominant for MGL binding. MGLshort also appeared to favor diglycosylated peptides, as well as GalNAc-Thr over GalNAc-Ser. In general, lectin affinity increases with the increased valency of glycan epitopes in a molecule (
). From the four CD45 glycopeptides tested, MGLshort preferred the tri- and tetraglycosylated peptides over the mono- and diglycosylated peptides. The preference for Tn clustering by MGL might be caused by the use of dimeric MGL-Fc constructs, which contain two MGL molecules coupled to one Fc-tail. However, further experiments are required to determine the minimal distance required in a multi-Tn-peptide to accommodate two MGL proteins. Differential recognition of GalNAc-Ser and GalNAc-Thr has been observed before and might be explained by the relatively fixed position of the GalNAc-Thr compared with the more flexible GalNAc-Ser (
Serine versus threonine glycosylation: the methyl group causes a drastic alteration on the carbohydrate orientation and on the surrounding water shell.
We did, however, find major differences in the glycan-binding profiles of the MGLshort and its H259T mutant, whereby the MGLshort H259T was still able to recognize a single GalNAc monosaccharide (Fig. 1C) and core 6 structures (Table 2). Nevertheless, the H259T mutant displayed a diminished capacity to bind elongated structures, such as the LDN epitope and sialyl-Tn (Fig. 1), as well as the blood group A determinant, the ganglioside GM2, sialylated LDN, and the Forsmann antigen (Table 3). Blood group antigens are highly expressed on erythrocytes and in a variety of other tissues, including the vascular endothelium, mucus secretions, and epithelial surfaces. Intriguingly, in a rat model for colon cancer, expression of blood group A antigen increased apoptosis resistance and facilitated immune escape (
Also on the Tn-glycopeptide array, a reduced binding capacity of the MGLshort H259T mutant could be observed. The local sequence context around the GalNAc-modified Ser or Thr residue thus emerges as an important factor for MGL binding specificity, which is further corroborated by the loss of affinity of MGLshort H259T to particular glycan structures as well as Tn-containing glycopeptides and Tn-expressing cells. This loss of affinity directly implies that MGL recognizes not only the GalNAc monosaccharide, but also the underlying glycan or protein backbone, through a secondary binding site, of which the His259 amino acid is a key element. Through MD simulations, we could confirm the MGL-mediated recognition of the peptide backbone in MUC1 (
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
) as well as in MUC2 through the His259 residue. Strikingly, the type of interactions, either through H bonds or water bridges, depended on the peptide sequence of the glycopeptide. We assume that also in the recognition of GalNAc-Tyr (
), MGL is able to engage the underlying tyrosine residue. Actually, the MGL binding mode shows clear parallels to the first structurally characterized Tn-glycopeptide–specific 237mAb antibody (
). Similar to MGL, the primary binding site of 237mAb is anchored by the Tn antigen, whereas the peptide backbone of podoplanin provides the additional specificity, thus conveying a dual recognition mode toward both Tn antigen and the podoplanin peptide backbone (
). The additional interactions involving the His259 residue, conveying fine specificity toward Tn-containing proteins or peptides, clearly illustrate the importance of exploring molecular recognition events for the optimal design of MGL-targeting structures for anti-tumor vaccines in light of the proposed role of MGL in impacting immunological responses through its highly exclusive recognition of tumor-associated glycan structures (
Glycosidic Tn-based vaccines targeting dermal dendritic cells favor germinal center B-cell development and potent antibody response in the absence of adjuvant.
The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.
)) was bought from Baseclear and cloned into the Sig-pIgG1-Fc vector. The MGLshort H259T mutant was generated by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Stratagene) and confirmed by sequencing. All MGL-Fc proteins were produced in CHO-K1 cells and purified from supernatants using HiTrap Protein A HP columns (GE Healthcare). MGL-Fc concentrations were determined by ELISA using human IgG1 as a standard, and purity was verified by Western blot analysis (Fig. S1A).
Antibody- and lectin-binding ELISAs
Anti-MGL antibodies 18E4 and 1G6.6 were generated as described before (
). Anti-MGL clone 125A10 was obtained from Dendritics. Isotype-matched control antibodies were purchased from eBioscience. To test the reactivity of these anti-MGL antibodies toward the different MGL-Fc proteins, NUNC MaxiSorp plates were coated overnight at room temperature with 5 μg/ml of the purified MGL-Fc proteins in 0.2 m NaHCO3 (pH 9.2). After washing with TSM (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm MgCl2, 1 mm CaCl2), plates were blocked for 30 min at 37 °C with TSM containing 1% BSA. Anti-MGL antibodies and isotype controls were added to the wells at 5 μg/ml in TSM plus 0.5% BSA and incubated for 1 h at room temperature. Plates were washed with TSM plus 0.05% Tween 20, and bound antibodies were detected using peroxidase-labeled goat anti-mouse IgG Fc-specific secondary antibodies (0.3 μg/ml in TSM plus 0.05% Tween 20). TMB (100 μg/ml TMB and 0.003% H2O2 in 0.1 m NaAc, pH 4) was used as a substrate to visualize the presence of bound peroxidase, and optical densities were measured at 450 nm.
To investigate MGL-Fc binding properties, NUNC MaxiSorp plates were coated overnight at room temperature with the indicated concentrations of polyacrylamide-coupled glycoconjugates (Lectinity) in 0.2 m NaHCO3 (pH 9.2). After washing with TSM, plates were blocked with 1% BSA plus TSM. MGL-Fc proteins (0.5 μg/ml) were added and incubated for 2 h at room temperature. Plates were washed with TSM plus 0.05% Tween 20, and bound MGL-Fc was detected using a peroxidase-labeled goat anti-human IgG-Fc (Jackson Immunoresearch Laboratories; 0.3 μg/ml in TSM plus 0.05% Tween 20). The reaction was developed using TMB (100 μg/ml TMB and 0.003% H2O2 in 0.1 m NaAc, pH 4), and optical densities were measured at 450 nm.
Glycan microarray analysis
The mammalian glycan array (version 5.1, containing 610 glycan structures) was developed by the Consortium of Functional Glycomics (www.functionalglycomics.org) from a library of natural and synthetic mammalian glycans with amino-linkers and printed in replicates of six onto N-hydroxysuccinimide–activated glass slides, forming covalent amide linkages. 70 μl of the MGL-Fc proteins (2 and 200 μg/ml in TSM containing 1% BSA and 0.05% Tween 20) was applied to the array and incubated at room temperature in a humidified chamber for 1 h. After extensive washing in TSM, bound MGL-Fc was detected using an Alexa 488–labeled anti-human IgG secondary antibody (1 μg/ml in TSM containing 1% BSA and 0.05% Tween 20). Fluorescence intensities were detected using a PerkinElmer Life Sciences ScanArray 5000 scanner, followed by image analyses using IMAGENE image analysis software (for representative images, see Fig. S3). The glycan array data (2 and 200 μg/ml) was analyzed using the Data mining feature in the GlycoPattern (https://glycopattern.emory.edu)7 software (
) to identify glycan binding motifs and glycans that differentially bound the MGL variants. Glycan structures were drawn using the Glycoworkbench software (
). All glycan microarray analyses were carried out in accordance with the MIRAGE (minimum information required for a glycomics experiment) guidelines for reporting glycan microarray-based data (
). Those incorporating lysine were acetylated at the N terminus with the lysine side chain providing the amino group for the reaction with the N-hydroxysuccinimide–functionalized slide. The others had a free N terminus through which they were immobilized on the slide (
). The glycopeptides were adjusted to the same concentration (100 μm) in printing buffer (300 mm sodium phosphate, pH 8.5) and spotted in replicates of 6. The Tn-glycopeptide was probed using MGL-Fc at a concentration of 10 and 1 μg/ml. Bound MGL-Fc was detected using an Alexa 488–labeled anti-human IgG secondary antibody at 5 μg/ml. Other assay conditions, buffers, and methods for imaging and processing of the data were identical to the screening of the glycan microarray and as described previously (Fig. S3) (
The human colon cancer cell lines SW1398 and HT29 were maintained in Dulbecco’s modified Eagle’s medium (Thermo Fischer Scientific) supplemented with 10% fetal calf serum and antibiotics. The Jurkat cell line was cultured in RPMI (Thermo Fisher Scientific) supplemented with 10% fetal calf serum and antibiotics. Colon cancer and Jurkat cell lines were incubated for 30 min at 4 °C with 5 μg/ml or the indicated concentrations of the different MGL-Fc variants. After washing with Hanks’ buffered saline solution containing 0.5% BSA, the bound MGL-Fc was counterstained for 30 min at 4 °C using an FITC-labeled goat anti-human IgG Fc (Jackson Immunoresearch). Cells were analyzed by flow cytometry on a CyAn flow cytometer (Beckman Coulter). Dead cells were excluded by 7-aminoactinomycin D discrimination (Molecular Probes).
MD simulations
MD simulations were performed using Amber16 implemented with the force field ff14SB (
) to properly reproduce the conformational behavior of the glycopeptides. The 3D models of the MGLshort and the corresponding mutant H259T were generated according to Marcelo et al. (
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
). 1-μs MD simulations were performed to compare the stability of both models for MGLshort and the MGLshort H259T. The MUC2 glycopeptide Ac-PTT*TPLK-NH2 (peptide 15; Fig. 2B) and the MUC1 glycopeptide H2N-TRPAPGST*APPA-NH2 (peptide 6; Fig. 2B) were selected to generate the corresponding MGLshort/Tn-glycopeptide and MGLshort H259T/Tn-glycopeptide complexes.
The 3D models of MGLshort and corresponding H259T mutant, as well as the MGL/Tn-glycopeptide complexes, were immersed in a box with a 10-Å buffer of TIP3P (
) water molecules plus Ca2+ and neutralized by adding explicit counterions (Na+). A two-stage geometry optimization approach was used. The first stage minimizes only the positions of solvent molecules and ions, and the second stage is an unrestrained minimization of all of the atoms in the simulation cell. The systems were then gently heated by incrementing the temperature from 0 to 300 K under a constant pressure of 1 atm and periodic boundary conditions. Harmonic restraints of 30 kcal/mol were applied to the solute, and the Andersen temperature coupling scheme (
) was used to control and equalize the temperature. The time step was kept at 1 fs during the heating stages. Water molecules are treated with the SHAKE algorithm, such that the angle between the hydrogen atoms is kept fixed. Long-range electrostatic effects are modeled using the particle mesh–Ewald method (
). An 8-Å cut-off was applied to Lennard-Jones interactions. Each system was equilibrated for 2 ns with a 2-fs time step at a constant volume and temperature of 300 K. Production trajectories, under the same simulation conditions, were then run for an additional 1 μs in the case of MGLshort and MGLshort H259T and 200 ns in the case of Tn-glycopeptide–containing complexes.
Statistics
Significant differences were evaluated using the GraphPad Prism software. A p value < 0.05 was considered to be significant.
Author contributions
F. M., F. C., D. L., and S. J. v. V. conceptualization; F. M., F. C., D. L., and S. J. v. V. formal analysis; F. M., N. S., F. C., J. C. v. d. H., I. M. V., D. L., D. F. S., and S. J. v. V. investigation; F. M., N. S., F. C., J. C. v. d. H., I. M. V., D. L., G.-J. P. B., D. F. S., and S. J. v. V. methodology; F. M., F. C., D. L., and S. J. v. V. writing-original draft; N. S., J. C. v. d. H., I. M. V., G.-J. P. B., and D. F. S. writing-review and editing; D. L. and S. J. v. V. funding acquisition; G.-J. P. B. supervision.
Human T cell activation results in extracellular signal-regulated kinase (ERK)-calcineurin-dependent exposure of Tn antigen on the cell surface and binding of the macrophage galactose-type lectin (MGL).
The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.
Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells.
Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.
Tumor-associated Tn-MUC1 glycoform is internalized through the macrophage galactose-type C-type lectin and delivered to the HLA class I and II compartments in dendritic cells.
in: Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. 2nd Ed. Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY2009 (Chapter 9, 20301232)
Serine versus threonine glycosylation: the methyl group causes a drastic alteration on the carbohydrate orientation and on the surrounding water shell.
Glycosidic Tn-based vaccines targeting dermal dendritic cells favor germinal center B-cell development and potent antibody response in the absence of adjuvant.
This work was supported by a bridging grant under National Institutes of Health Grant U54GM62116 (to the Consortium for Functional Glycomics) and P41GM103390 (to the Research Resource for Integrated Glycotechnology). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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