Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

Computational analysis of carbohydrate recognition based on hybrid QM/MM modeling: a case study of norovirus capsid protein in complex with Lewis antigen.

Norovirus is a major pathogen of nonbacterial acute gastroenteritis in humans and animals. Carbohydrate recognition between norovirus capsid proteins and Lewis antigens is considered to play a critical role in initiating infection of eukaryotic cells. In this article, we first report a detailed atomistic simulation study of the norovirus capsid protein in complex with the Lewis antigen based on ab initio QM/MM combined with MD-FEP simulations. To understand the mechanistic details of ligand binding, we analyzed and compared the carbohydrate recognition mechanism of the wild-type P domain protein with a mutant protein. Small structural differences between two capsid proteins are observed on the weak interaction site of residue 389, which is located on the solvent exposed surface of the P domain. To further clarify affinity differences in ligand binding, we directly evaluated free energy changes of the ligand binding process. Although the mutant protein loses its interaction energy with the Lewis antigen, this small amount of energy penalty is compensated for by an increase in the solvation stability, which is induced by structural reorganization at the ligand binding site on the protein surface. As a sum of these opposite energy components, the mutant P domain obtains a slightly enhanced binding affinity for the Lewis antigen. The present computational study clearly demonstrated that a detailed free energy balance of the interaction energy between the capsid protein and the surrounding aqueous solvent is the mechanistic basis of carbohydrate recognition in the norovirus capsid protein.

Embryoglycan: a highly branched poly-N-acetyllactosamine in pluripotent stem cells and early embryonic cells.

Embryonal carcinoma cells, stem cells of teratocarcinomas, are pluripotent stem cells and also prototypes of embryonic stem cells. Embryonal carcinoma cells contain large amounts of a highly branched poly-N-acetyllactosamine called embryoglycan, which has a molecular weight of approximately 10,000 or greater, and is asparagine-linked. This glycan was found by analyses of fucose-labeled glycopeptides, and its characteristics were established by biochemical analyses. The content of embryoglycan progressively decreases during the in vitro differentiation of embryonal carcinoma cells. Embryoglycan is also abundant in mouse embryonic stem cells and preimplantation mouse embryos, and decreases during embryogenesis. Embryoglycan carries a number of carbohydrate markers of murine pluripotent stem cells. Lewis x markers, such as SSEA-1, 4C9 antigen, and binding sites for Lotus tetragonolobus agglutinin are of particular importance. 4C9 antigenicity requires clustering of Lewis x, best accomplished by poly-N-acetyllactosamine branching, whereas SSEA-1 does not. Although in vivo evidence is lacking, these epitopes have been suggested to participate in cell-to-cell and cell-to-substratum adhesion. Other markers on embryoglycan include α-galactosyl antigens such as ECMA-2, and binding sites for Dolichos biflorus agglutinin, the epitope of which is considered to be identical to Sda antigen, namely, GalNAcß1-4(NeuAcα2-3)Galß1-4GlcNAc. While embryoglycan is also present in human teratocarcinoma cells, the carbohydrate markers characterized in human pluripotent stem cells to date are largely carried by glycolipids and keratan sulfate. Information on embryoglycan and markers carried by it may assist in the development of new markers of human pluripotent stem cells and their progenies.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Interactions of antitumour Sialyl Lewis X liposomes with vascular endothelial cells.

Recently, we showed that tetrasaccharide selectin ligand SiaLeX provided targeted delivery of liposomes loaded in the bilayer with melphalan lipophilic prodrug to tumour endothelium followed by severe injury of tumour vessels in a Lewis lung carcinoma model. Here, we study the impact of SiaLeX ligand on the interactions of liposomes with human umbilical vein endothelial cells (HUVEC) using flow cytometry, spectrofluorimetry and confocal microscopy. Liposomes composed of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol ester of melphalan, 8:1:1, by mol, and varying percentages of lipophilic SiaLeX conjugate were labelled with BODIPY-phosphatidylcholine. The increase in SiaLeX content in liposomes led to a proportional increase in their uptake by cytokine-activated cells as opposed to non-activated HUVEC: for 10% SiaLeX liposomes, binding avidity and overall accumulation increased 14- and 6-fold, respectively. The early stages of intracellular traffic of targeted liposomes in the activated cells were monitored by co-localisation with the trackers of organelles. Endocytosis of SiaLeX liposomes occurred mostly via clathrin-independent pathways, which does not contradict the available literature data on E-selectin localisation in the plasma membrane. Using dual fluorescence labelling, with rhodamine-labelled phospholipid and calcein encapsulated at self-quenching concentrations, we found that SiaLeX liposomes undergo rapid (within minutes) internalisation by activated HUVEC accompanied by the disruption of liposomes; non-activated cells consumed a negligible dose of liposomes during at least 1.5h. Our data evidence the selective effect of SiaLeX formulations on activated endothelial cells and indicate their potential for intracellular delivery of melphalan lipophilic prodrug.

Schistosoma mansoni α1,3-fucosyltransferase-F generates the Lewis X antigen.

Genetic evidence suggests that the Schistosoma mansoni genome contains six genes that encode α1,3-fucosyltransferases (smFuTs). To date, the activities and specificities of these putative fucosyltransferases are unknown. As Schistosoma express a variety of fucosylated glycans, including the Lewis X antigen Galß1-4(Fucα1-3)GlcNAcß-R, it is likely that this family of genes encode enzymes that are partly responsible for the generation of those structures. Here, we report the molecular cloning of fucosyltransferase-F (smFuT-F) from S. mansoni, as a soluble, green fluorescent protein fusion protein and its acceptor specificity. The gene smFuT-F was expressed in HEK freestyle cells, purified by affinity chromatography, and analyzed toward a broad panel of glycan acceptors. The enzyme product of smFuT-F effectively utilizes a type II chain acceptor Galß1-4GlcNAc-R, but notably not the LDN sequence GalNAcß1-4GlcNAc-R, to generate Lewis X type-glycans, and smFuT-F transcripts are present in all intramammalian life stages.

The three-dimensional structure of the extracellular adhesion domain of the sialic acid-binding adhesin SabA from Helicobacter pylori.

The gastric pathogen Helicobacter pylori is a major cause of acute chronic gastritis and the development of stomach and duodenal ulcers. Chronic infection furthermore predisposes to the development of gastric cancer. Crucial to H. pylori survival within the hostile environment of the digestive system are the adhesins SabA and BabA; these molecules belong to the same protein family and permit the bacteria to bind tightly to sugar moieties Lewis(B) and sialyl-Lewis(X), respectively, on the surface of epithelial cells lining the stomach and duodenum. To date, no representative SabA/BabA structure has been determined, hampering the development of strategies to eliminate persistent H. pylori infections that fail to respond to conventional therapy. Here, using x-ray crystallography, we show that the soluble extracellular adhesin domain of SabA shares distant similarity to the tetratricopeptide repeat fold family. The molecule broadly resembles a golf putter in shape, with the head region featuring a large cavity surrounded by loops that vary in sequence between different H. pylori strains. The N-terminal and C-terminal helices protrude at right angles from the head domain and together form a shaft that connects to a predicted outer membrane protein-like ß-barrel trans-membrane domain. Using surface plasmon resonance, we were able to detect binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X) but not to Lewis(A), Lewis(B), or Lewis(Y). Substitution of the highly conserved glutamine residue 159 in the predicted ligand-binding pocket abrogates the binding of the SabA adhesin domain to sialyl-Lewis(X) and Lewis(X). Taken together, these data suggest that the adhesin domain of SabA is sufficient in isolation for specific ligand binding.

Inflammatory cytokines regulate the expression of glycosyltransferases involved in the biosynthesis of tumor-associated sialylated glycans in pancreatic cancer cell lines.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma containing several pro-inflammatory cytokines, which are described to modulate the expression of important genes related to tumor promotion and progression. In the present work we have investigated the potential role of these cytokines in the biosynthesis of tumor-associated carbohydrate antigens such as sialyl-Lewis(x) (SLe(x)) through the regulation of specific glycosyltransferase genes. METHODS: Two human PDAC cell lines MDAPanc-3 and MDAPanc-28 were treated with pro-inflammatory cytokines IL-1ß, TNFα, IL-6 or IL-8, and the content of tumor-associated carbohydrate antigens at the cell membrane was analyzed by flow cytometry. In addition, variation in the mRNA expression of sialyltransferase (ST) and fucosyltransferase (FUT) genes, which codify for the ST and FucT enzymes involved in the carbohydrate antigens' biosynthesis, was determined. The inflammatory microenvironment of PDAC tissues and the expression of Lewis-type antigens were analyzed by immunohistochemistry to find a possible correlation between inflammation status and the presence of tumor-associated carbohydrate antigens. RESULTS: IL-1ß stimuli increased SLe(x) and α2,6-sialic acid levels in MDAPanc-28 cells and enhanced the mRNA levels of ST3GAL3-4 and FUT5-7, which codify for ST and FucT enzymes related to SLe(x) biosynthesis, and of ST6GAL1. IL-6 and TNFα treatments increased the levels of SLe(x) and Le(y) antigens in MDPanc-3 cells and, similarly, the mRNA expression of ST3GAL3-4, FUT1-2 and FUT6, related to these Lewis-type antigens' biosynthesis, were increased. Most PDAC tissues stained for SLe(x) and SLe(a) and tended to be expressed in the tumor samples with a higher presence of inflammatory immune cells. CONCLUSIONS: The inflammatory microenvironment can modulate the glycosylation pattern of PDAC cells, increasing the expression of tumor-associated sialylated antigens such as SLe(x), which contributes to pancreatic tumor malignancy.

Structural characterization of the DC-SIGN-Lewis(X) complex.

Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a C-type lectin highly expressed on the surface of antigen-presenting dendritic cells. DC-SIGN mediates interactions among dendritic cells, pathogens, and a variety of epithelia, myeloid cells, and endothelia by binding to high mannose residues on pathogenic invaders or fucosylated residues on the membranes of other immune cells. Although these interactions are normally beneficial, they can also contribute to disease. The structural characterization of binding geometries is therefore of interest as a basis for the construction of mimetics that can mediate the effects of abnormal immune response. Here, we report the structural characteristics of the interaction of the DC-SIGN carbohydrate recognition domain (CRD) with a common fucosylated entity, the Lewis(X) trisaccharide (Le(X)), using NMR methods. Titration of the monomeric DC-SIGN CRD with Le(X) monitored by 2D NMR revealed significant perturbations of DC-SIGN cross-peak positions in (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra and identified residues near the binding site. Additionally, saturation transfer difference (STD) and transferred nuclear Overhauser effect (trNOE) NMR experiments, using a tetrameric form of DC-SIGN, identified binding epitopes and bound conformations of the Le(X) ligand. The restraints derived from these multiple experiments were used to generate models for the binding of Le(X) to the DC-SIGN CRD. Ranking of the models based on the fit of model-based simulations of the trNOE data and STD buildup curves suggested conformations distinct from those seen in previous crystal structures. The new conformations offer insight into how differences between binding of Lewis(X) and mannose-terminated saccharides may be propagated.

Competition between core-2 GlcNAc-transferase and ST6GalNAc-transferase regulates the synthesis of the leukocyte selectin ligand on human P-selectin glycoprotein ligand-1.

The binding of selectins to carbohydrate ligands expressed on leukocytes regulates immunity and inflammation. Among the human selectin ligands, the O-linked glycans at the N-terminus of the leukocyte cell-surface molecule P-selectin glycoprotein ligand-1 (PSGL-1, CD162) are important because they bind all selectins (L-, E-, and P-selectin) with high affinity under hydrodynamic shear conditions. Analysis of glycan microheterogeneity at this site is complicated by the presence of 72 additional potential O-linked glycosylation sites on this mucinous protein. To overcome this limitation, truncated forms of PSGL-1, called "PSGL-1 peptide probes," were developed. Ultra-high sensitivity mass spectrometry analysis of glycans released from such probes along with glycoproteomic analysis demonstrate the presence of both the sialyl Lewis-X (sLe(X)) and the di-sialylated T-antigen (NeuAcα2,3Galß1,3(NeuAcα2,6)GalNAc) at the PSGL-1 N-terminus. Overexpression of glycoprotein-specific ST6GalNAc-transferases (ST6GalNAc1, -2, or -4) in human promyelocytic HL-60 cells altered glycan structures and cell adhesion properties. In particular, ST6GalNAc2 overexpression abrogated cell surface HECA-452/CLA expression, reduced the number of rolling leukocytes on P- and L-selectin-bearing substrates by ~85%, and increased median rolling velocity of remaining cells by 80-150%. Cell rolling on E-selectin was unaltered although the number of adherent cells was reduced by 60%. ST6GalNAc2 partially co-localizes in the Golgi with the core-2 ß(1,6)GlcNAc-transferase C2GnT-1. Overall, the data describe the glycan microheterogeneity at the PSGL-1 N-terminus. They suggest that a competition between ST6GalNAc2 and C2GnT-1 for the core-1/Galß1,3GalNAc glycan may regulate leukocyte adhesion under fluid shear.

Biological evaluation of multivalent lewis X-MGL-1 interactions.

Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are pattern-recognition receptors involved in the recognition of pathogens as well as of self-antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin-carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le(X) ) epitope on its interaction with the CLR macrophage galactose-type lectin-1 (MGL-1). Glycan arrays, including N-glycan structures with terminal Le(X) , were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL-1-hFc fusion protein showed up to tenfold increased binding to multiantennary N-glycans displaying Le(X) structures, compared to monovalent Le(X) trisaccharide. Multivalent presentation of Le(X) on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le(X) -OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate-lectin interactions can be exploited to modulate immune responses.

Paradigms for glycan-binding receptors in cell adhesion.

Diverse glycans found on the surfaces of mammalian cells provide a basis for selective adhesion between cells mediated by glycan-specific receptors. Well-understood examples of cell adhesion based on such interactions include selectin-mediated rolling of leukocytes on endothelia. Other receptors with similar selectivity for specific sugar epitopes on cell surfaces are being characterised. However, the simple paradigm of adhesion resulting from receptors on one cell binding to glycans on another cell applies in only a limited number of systems. Instead, glycans and receptor-glycan interactions often modulate adhesion in indirect ways, such as by changing the organisation of cell surface glycoproteins and by antagonising the effect of protein adhesion systems.

Synthesis and screening of a library of Lewisx deoxyfluoro-analogues reveals differential recognition by glycan-binding partners.

Glycan-mediated interactions play a crucial role in biology and medicine, influencing signalling, immune responses, and disease pathogenesis. However, the use of glycans in biosensing and diagnostics is limited by cross-reactivity, as certain glycan motifs can be recognised by multiple biologically distinct protein receptors. To address this specificity challenge, we report the enzymatic synthesis of a 150-member library of site-specifically fluorinated Lewisx analogues ('glycofluoroforms') using naturally occurring enzymes and fluorinated monosaccharides. Subsequent incorporation of a subset of these glycans into nanoparticles or a microarray revealed a striking spectrum of distinct binding intensities across different proteins that recognise Lewisx. Notably, we show that for two proteins with unique binding sites for Lewisx, glycofluoroforms exhibited enhanced binding to one protein, whilst reduced binding to the other, with selectivity governed by fluorination patterns. We finally showcase the potential diagnostic utility of this approach in glycofluoroform-mediated bacterial toxin detection by lateral flow.

Regulation of the metastatic cell phenotype by sialylated glycans.

Tumor cells exhibit striking changes in cell surface glycosylation as a consequence of dysregulated glycosyltransferases and glycosidases. In particular, an increase in the expression of certain sialylated glycans is a prominent feature of many transformed cells. Altered sialylation has long been associated with metastatic cell behaviors including invasion and enhanced cell survival; however, there is limited information regarding the molecular details of how distinct sialylated structures or sialylated carrier proteins regulate cell signaling to control responses such as adhesion/migration or resistance to specific apoptotic pathways. The goal of this review is to highlight selected examples of sialylated glycans for which there is some knowledge of molecular mechanisms linking aberrant sialylation to critical processes involved in metastasis.

Fluorescent conjugate of sLe(x)-selective bisboronic acid for imaging application.

Carbohydrate-based biomarkers such as sialyl Lewis X are known to correlate with cancer formation and progression. By targeting sialyl Lewis X, we have developed a boronolectin-fluorophore conjugate, which was able to selectively label and image xenograft (sc) tumor. This represents the very first example that a small molecule capable of recognizing a carbohydrate biomarker was used for optical imaging application.

Multiparameter flow cytometry for the characterization of human embryonic stem cells.

Using multiparameter staining methods and flow cytometry to investigate the pluripotency of HUES7 human embryonic stem cell cultures, it was found that the multidimensional approach of marker co-expression allowed the different cell populations to be easily identified and demonstrated cross reactivity between the SSEA 4 and SSEA 1 antibodies, resulting in a substantial false positive SSEA 1 population. It is the accepted norm to apply control gates at a 95 % confidence level of the isotype control; however, this study found that adjusting the control gate to a 99 % confidence level significantly reduced the effect of this cross reactivity. Though conversely, this gating shift also decreased the positive marker expression of SSEA 4 and Tra-1-60, indicating that there is a need for strongly expressing markers coupled with increased optimization of fluorophore/antibody combinations before a gating strategy of 99 % can be implemented on a more routine basis.

Regulation of catch bonds by rate of force application.

The current paradigm for receptor-ligand dissociation kinetics assumes off-rates as functions of instantaneous force without impact from its prior history. This a priori assumption is the foundation for predicting dissociation from a given initial state using kinetic equations. Here we have invalidated this assumption by demonstrating the impact of force history with single-bond kinetic experiments involving selectins and their ligands that mediate leukocyte tethering and rolling on vascular surfaces during inflammation. Dissociation of bonds between L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) loaded at a constant ramp rate to a constant hold force behaved as catch-slip bonds at low ramp rates that transformed to slip-only bonds at high ramp rates. Strikingly, bonds between L-selectin and 6-sulfo-sialyl Lewis X were impervious to ramp rate changes. This ligand-specific force history effect resembled the effect of a point mutation at the L-selectin surface (L-selectinA108H) predicted to contact the former but not the latter ligand, suggesting that the high ramp rate induced similar structural changes as the mutation. Although the A108H substitution in L-selectin eliminated the ramp rate responsiveness of its dissociation from PSGL-1, the inverse mutation H108A in P-selectin acquired the ramp rate responsiveness. Our data are well explained by the sliding-rebinding model for catch-slip bonds extended to incorporate the additional force history dependence, with Ala-108 playing a pivotal role in this structural mechanism. These results call for a paradigm shift in modeling the mechanical regulation of receptor-ligand bond dissociation, which includes conformational coupling between binding pocket and remote regions of the interacting molecules.

Preferences for uptake of carbohydrate-coated liposomes by C-type lectin receptors as antigen-uptake receptors.

We evaluated the carbohydrate preferences of the C-type lectin receptors (CLRs) SIGNR1, SIGNR3, and Langerin as pathogen-uptake receptors based on uptake of liposomes consisting of cholesterol, DPPC, and various neoglycolipids at molar ratios of 10:10:1 and 10:7:4, respectively, using non-phagocytic CHO cells that express these receptors transiently. SIGNR1-expressing cells ingested liposomes coated with neoglycolipids with terminal mannose residues, such as Man2-, Man3-, and Man5-DPPE, and with a terminal N-acetylglucosamine. SIGNR1 mediated uptake of Man3-DPPE-coated liposomes most efficiently. Uptake of liposomes with lower neoglycolipid content by SIGNR3- or Langerin-expressing cells was slight or negligible, but uptake into these cells was detected for liposomes with higher neoglycolipid content. SIGNR1-expressing cells clearly ingested liposomes coated with Lewis X antigen, whereas SIGNR3- or Langerin-expressing cells barely ingested these liposomes, even at the higher neoglycolipid content. In contrast, SIGNR3 or Langerin, but not SIGNR1, mediated uptake of liposomes coated with blood group H antigen. These results indicate that CLRs with similar carbohydrate-recognition characteristics have distinct properties as pathogen-uptake receptors for carbohydrate-decorated particles.

Interaction of Helicobacter pylori with C-type lectin dendritic cell-specific ICAM grabbing nonintegrin.

In this study we asked whether Helicobacter pylori whole cells and lipopolysaccharide (LPS) utilize sugar moieties of Lewis (Le) antigenic determinants to interact with DC-SIGN (dendritic cell specific ICAM grabbing nonintegrin) receptor on dendritic cells (DCs). For this purpose the soluble DC-SIGN/Fc adhesion assay and the THP-1 leukemia cells with induced expression of DC-SIGN were used. We showed that the binding specificity of DC-SIGN with H. pylori Le(X/Y) positive whole cells and H. pylori LPS of Le(X/Y) type was fucose dependent, whereas in Le(XY) negative H. pylori strains and LPS preparations without Lewis determinants, this binding was galactose dependent. The binding of soluble synthetic Le(X) and Le(Y) to the DC-SIGN-like receptor on THP-1 cells was also observed. In conclusion, the Le(XY) dependent as well as independent binding of H. pylori whole cells and H. pylori LPS to DC-SIGN was described. Moreover, we demonstrated that THP-1 cells may serve as an in vitro model for the assessment of H. pylori-DC-SIGN interactions mediated by Le(X) and Le(Y) determinants.

Detection and isolation of dendritic cells using Lewis X-functionalized magnetic nanoparticles.

dendritic cell (DC)-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) is a receptor found on DCs that recognizes antigens bearing mannose-rich or fucosylated glycans, including Lewis X (Le(X)). Here, we report the fabrication of magnetic nanoparticles coated with multivalent Le(X) glycans using Cu (I)-catalyzed azide-alkyne cycloaddition. The resulting nanoparticles are selective and biocompatible, serving as a highly efficient tool for DC detection and enrichment.