Irma van Die

Cutting Edge: Carbohydrate Profiling Identifies New Pathogens That Interact with Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin on Dendritic Cells

Dendritic cells (DC) are instrumental in handling pathogens for processing and presentation to T cells, thus eliciting an appropriate immune response. C-type lectins expressed by DC function as pathogen-recognition receptors; yet their specificity for carbohydrate structures on pathogens is not fully understood. In this study, we analyzed the carbohydrate specificity of DC-specific ICAM-3-grabbing nonintegrin (SIGN)/CD209, the recently documented HIV-1 receptor on DC. Our studies show that DC-SIGN binds with high affinity to both synthetic mannose- and fucose-containing glycoconjugates. These carbohydrate structures are abundantly expressed by pathogens as demonstrated by the affinity of DC-SIGN for natural surface glycans of the human pathogens Mycobacterium tuberculosis, Helicobacter pylori, Leishmania mexicana, and Schistosoma mansoni. This analysis expands our knowledge on the carbohydrate and pathogen-specificity of DC-SIGN and identifies this lectin to be central in pathogen-DC interactions.

A Novel Glycosulfopeptide Binds to P-selectin and Inhibits Leukocyte Adhesion to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO3) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLex) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO3 or C2-O-sLex do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6′, which contains sLex on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with aK d of ∼350 nm. GSP-6 (<5 μm) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLex (5 mm) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.

Specificity of DC-SIGN for mannose- and fucose-containing glycans.

The dendritic cell specific C‐type lectin dendritic cell specific ICAM‐3 grabbing non‐integrin (DC‐SIGN) binds to “self” glycan ligands found on human cells and to “foreign” glycans of bacterial or parasitic pathogens. Here, we investigated the binding properties of DC‐SIGN to a large array of potential ligands in a glycan array format. Our data indicate that DC‐SIGN binds with K d < 2 μM to a neoglycoconjugate in which Galβ1‐4(Fucα1‐3)GlcNAc (Lex) trisaccharides are expressed multivalently. A lower selective binding was observed to oligomannose‐type N‐glycans, diantennary N‐glycans expressing Lex and GalNAcβ1‐4(Fucα1‐3)GlcNAc (LacdiNAc‐fucose), whereas no binding was observed to N‐glycans expressing core‐fucose linked either α1‐6 or α1‐3 to the Asn‐linked GlcNAc of N‐glycans. These results demonstrate that DC‐SIGN is selective in its recognition of specific types of fucosylated glycans and subsets of oligomannose‐ and complex‐type N‐glycans.

LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediated immune recognition

Although Galβ1–4GlcNAc (LacNAc) moieties are the most common constituents of N-linked glycans on vertebrate proteins, GalNAcβ1–4GlcNAc (LacdiNAc, LDN)-containing glycans are widespread in invertebrates, such as helminths. We postulated that LDN might be a molecular pattern for recognition of helminth parasites by the immune system. Using LDN-based affinity chromatography and mass spectrometry, we have identified galectin-3 as the major LDN-binding protein in macrophages. By contrast, LDN binding was not observed with galectin-1. Surface plasmon resonance (SPR) analysis and a solid phase binding assay demonstrated that galectin-3 binds directly to neoglycoconjugates carrying LDN glycans. In addition, galectin-3 bound to Schistosoma mansoni soluble egg Ags and a mAb against the LDN glycan inhibited this binding, suggesting that LDN glycans within S. mansoni soluble egg Ags contribute to galectin-3 binding. Immunocytochemistry demonstrated high levels of galectin-3 in liver granulomas of S. mansoni-infected hamsters, and a colocalization of galectin-3 and LDN glycans was observed on the parasite eggshells. Finally, we demonstrate that galectin-3 can mediate recognition and phagocytosis of LDN-coated particles by macrophages. These findings provide evidence that LDN-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.

An antibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human β-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.

Core α1→3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex‐type N‐glycans from Arabidopsis thaliana, which contain R‐GlcNAcβ1→4(Fucα1→3)GlcNAcβ1‐Asn (core α1→3‐Fuc) and Xylβ1→2Manβ1→4GlcNAcβ1‐R (core β1→2‐Xyl) modifications, and honeybee phospholipase A2, which carries N‐glycans that contain the core α1→3‐Fuc epitope. Evidence is presented that core α1→3‐fucosylated N‐glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core α1→3‐Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N‐glycans carrying core α1→3‐Fuc and β1→2‐Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N‐glycans containing the core α1→3‐Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

DC-SIGN Mediates Binding of Dendritic Cells to Authentic Pseudo-LewisY Glycolipids of Schistosoma mansoni Cercariae, the First Parasite-specific Ligand of DC-SIGN

During schistosomiasis, parasite-derived glycoconjugates play a key role in manipulation of the host immune response, associated with persistence of the parasite. Among the candidate host receptors that are triggered by glycoconjugates are C-type lectins (CLRs) on dendritic cells (DCs), which in concerted action with Toll-like receptors determine the balance in DCs between induction of immunity versus tolerance. Here we report that the CLR DC-SIGN mediates adhesion of DCs to authentic glycolipids derived from Schistosoma mansoni cercariae and their excretory/secretory products. Structural characterization of the glycolipids, in combination with solid phase and cellular binding studies revealed that DC-SIGN binds to the carbohydrate moieties of both glycosphingolipid species with Galβ1–4(Fucα1–3)GlcNAc (LewisX) and Fucα1–3Galβ1–4(Fucα1–3)GlcNAc (pseudo-LewisY) determinants. Importantly, these data indicate that surveying DCs in the skin may encounter schistosome-derived glycolipids immediately after infection. Recent analysis of crystals of the carbohydrate binding domain of DC-SIGN bound to LewisX provided insight into the ability of DC-SIGN to bind fucosylated ligands. Using molecular modeling we showed that the observed binding of the schistosome-specific pseudo-LewisY to DC-SIGN is not directly compatible with the model described. To fit pseudo-LewisY into the model, the orientation of the side chain of Phe313 in the secondary binding site of DC-SIGN was slightly changed, which results in a perfect stacking of Phe313 with the hydrophobic side of the galactose-linked fucose of pseudo-LewisY. We propose that pathogens such as S. mansoni may use the observed flexibility in the secondary binding site of DC-SIGN to target DCs, which may contribute to immune escape.

Demonstration of glycosaminoglycans in Caenorhabditis elegans.

A considerable amount (approximately 1.6 μg from 1 mg of dried nematode) of non‐sulfated chondroitin, two orders of magnitude less yet an appreciable amount of heparan sulfate, and no hyaluronate were found in Caenorhabditis elegans nematodes. The chondroitin chains were heterogeneous in size, being shorter than that of whale cartilage chondroitin sulfate. The disaccharide composition analysis of heparan sulfate revealed diverse sulfation including glucosamine 2‐N‐sulfation, glucosamine 6‐O‐sulfation and uronate 2‐O‐sulfation. These results imply that chondroitin and heparan sulfate are involved in fundamental biological processes.

Receptor Structure for F1C Fimbriae of Uropathogenic Escherichia coli

ABSTRACT F1C fimbriae are correlated with uropathogenic Escherichia coli strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing E. coli. The binding of the F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), β1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc4Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM2 [GgO3Cer]) and gangliotetraosylceramide (asialo-GM1[GgO4Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant E. coli strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM2 (GgO3Cer). The bacterial interaction with asialo-GM1 (GgO4Cer) and asialo-GM2 (GgO3Cer) was strongly inhibited only by disaccharide GalNAcβ1-4Galβ linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb5Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAcβ1-4Galβ is linked to glucosylceramide as in asialo-GM2 (GgO3Cer). Thus, it is suggested that the disaccharide sequence GalNAcβ1-4Galβ of asialo-GM2 (GgO3Cer) which is positioned internally in asialo-GM1 (GgO4Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenicE. coli.

Galectin-3 Modulates Immune and Inflammatory Responses during Helminthic Infection: Impact of Galectin-3 Deficiency on the Functions of Dendritic Cells

ABSTRACT Galectin-3 (Gal-3) is a multifunctional β-galactoside-binding lectin that senses self-derived and microbial glycoconjugates. Although Gal-3 is important in immune reactions and host defense in some experimental models, the function of Gal-3 during helminthic diseases (e.g., schistosomiasis) is still elusive. We show that, compared to wild-type Schistosoma mansoni-infected mice, infected Gal-3−/− mice have a reduced number of T and B lymphocytes in the spleen, develop reduced liver granulomas at 7 weeks (acute phase) and 14 weeks (chronic phase) postinfection, and mount a biased cellular and humoral Th1 response. In an attempt to understand this latter phenomenon, we studied the role of endogenous Gal-3 in dendritic cells (DCs), the most potent antigen-presenting cells, both in vitro and in vivo. Although Gal-3 deficiency in DCs does not impact their differentiation and maturation processes, it greatly influences the strength (but not the nature) of the adaptive immune response that they trigger, suggesting that Gal-3 deficiency in some other cell types may be important during murine schistosomiasis. As a whole, this study implies that Gal-3 is a modulator of the immune/inflammatory responses during helminthic infection and reveals for the first time that Gal-3 expression in DCs is pivotal to control the magnitude of T-lymphocyte priming.