Molecular basis of the differences in binding properties of the highly related C-type lectins DC-SIGN and L-SIGN to Lewis X trisaccharide and Schistosoma mansoni egg antigens

The dendritic cell-specific C-type lectin DC-SIGN functions as a pathogen receptor that recognizes Schistosoma mansoni egg antigens through its major glycan epitope Galbeta1,4(Fucalpha1,3)GlcNAc (Lex). Here we report that L-SIGN, a highly related homologue of DC-SIGN found on liver sinusoidal endothelial cells, binds to S. mansoni egg antigens but not to the Lex epitope. L-SIGN does bind the Lewis antigens Lea, Leb, and Ley, similar as DC-SIGN. A specific mutation in the carbohydrate recognition domain of DC-SIGN (V351G) abrogates binding to all Lewis antigens. In L-SIGN Ser363 is present at the corresponding position of Val351 in DC-SIGN. Replacement of this Ser into Val resulted in a "gain of function" L-SIGN mutant that binds to Lex, and shows increased binding to the other Lewis antigens. These data indicate that Val351 is important for the fucose specificity of DC-SIGN. Molecular modeling and docking of the different Lewis antigens in the carbohydrate recognition domains of L-SIGN, DC-SIGN, and their mutant forms, demonstrate that Val351 in DC-SIGN creates a hydrophobic pocket that strongly interacts with the Fucalpha1,3/4-GlcNAc moiety of the Lewis antigens. The equivalent amino acid residue Ser363 in L-SIGN creates a hydrophilic pocket that prevents interaction with Fucalpha1,3-GlcNAc in Lex but supports interactions with the Fucalpha1,4-GlcNAc moiety in Lea and Leb antigens. These data demonstrate for the first time that DC-SIGN and L-SIGN differ in their carbohydrate binding profiles and will contribute to our understanding of the functional roles of these C-type lectin receptors, both in recognition of pathogen and self-glycan antigens.     

The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding. In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galbeta1-4GlcNAc, and binding to neoglycoconjugates containing only alpha-fucose or oligosaccharides with a terminal alpha1-2-linked fucose is low. These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity. Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs. By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120. These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD. Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.     

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)<2muM to a neoglycoconjugate in which Galbeta1-4(Fucalpha1-3)GlcNAc (Le(x)) trisaccharides are expressed multivalently. A lower selective binding was observed to oligomannose-type N-glycans, diantennary N-glycans expressing Le(x) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LacdiNAc-fucose), whereas no binding was observed to N-glycans expressing core-fucose linked either alpha1-6 or alpha1-3 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.     

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 Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(X)) and Fucalpha1-3Galbeta1-4(Fucalpha1-3)GlcNAc (pseudo-Lewis(Y)) 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 Lewis(X) 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-Lewis(Y) to DC-SIGN is not directly compatible with the model described. To fit pseudo-Lewis(Y) into the model, the orientation of the side chain of Phe(313) in the secondary binding site of DC-SIGN was slightly changed, which results in a perfect stacking of Phe(313) with the hydrophobic side of the galactose-linked fucose of pseudo-Lewis(Y). 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.     

Characteristic structural features of schistosome cercarial N-glycans: expression of Lewis X and core xylosylation

Schistosomal egg N-glycans are the only examples in nature that have been structurally shown to contain beta2-xylosylation, alpha6-fucosylation, and alpha3-fucosylation on the N,N'-diacetyl chitobiose core. We present evidence that core difucosylated and xylosylated N-glycans are characteristics of Schistosoma japonicum eggs but not of the cercariae and adults, for which neither core xylosylation nor alpha3-fucosylation could be readily detected. In contrast, a majority of the N-glycans from Schistosoma mansoni cercariae but not the adults are core xylosylated. Tandem mass spectrometry analysis coupled with chromatographic mapping, sequential exoglycosidase digestion, and methylation analysis were employed to unambiguously define the structures of core beta2-xylosylated, alpha6-fucosylated N-glycans from S. mansoni cercariae. Unexpectedly, a majority of these N-glycans were found to carry Lewis X determinant, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->, on the nonreducing termini of mono- and biantennary structures. The Lewis X-containing glycoproteins were found to be distinct from those carrying the complex, multifucosylated glycocalyx O-glycans reported previously. The corresponding N-glycans from S. japonicum cercariae are likewise dominated by Lewis X termini but without the core xylosylation. We concluded that the invading cercariae present an important and abundant source of Lewis X antigens, which may contribute to the induced humoral response upon infection. Following transformation and development into the adults, the N-glycans synthesized comprise a significantly larger amount of high mannose and fucosylated pauci-mannose structures in comparison with the cercarial N-glycans. A portion of the mono- and biantennary complex types were identified to carry Lewis X and fucosylated LacdiNAc termini, which could also be detected by mass spectrometry analysis on larger, complex-type structures.     

Glycans of Schistosoma mansoni and keyhole limpet haemocyanin induce hepatic granulomas in vivo

Schistosoma mansoni eggs trapped in the liver of an infected host cause the major pathological manifestations of schistosomiasis. Miracidia within the deposited eggs secrete soluble egg antigens (SEA) that induce periovular granuloma formation, which may lead to severe hepatic fibrosis. Several reports have highlighted the immunomodulatory capacities of carbohydrate determinants present in the glycoproteins of SEA. These glycans contain among others the immunogenic Galbeta1-4(Fucalpha1-3)GlcNAc (LewisX) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) elements. Due to cross-reactivity with schistosomal glycan antigens, keyhole limpet haemocyanin (KLH) has been used extensively for diagnostic and therapeutic studies on schistosomiasis. In the present study, a granulomatous response with numerous eosinophils towards SEA- and KLH-coated beads implanted in the liver by mesenteric injection was observed. Immunophenotyping of these experimentally induced granulomas for cellular recruitment, chemokines, adhesion and extracellular matrix proteins revealed very close resemblance with hepatic lesions evoked by native schistosome eggs, hence demonstrating the usefulness of the bead model, in general, as well as of KLH as a model antigen to study the immunopathological mechanisms of schistosome infections. While trypsin digestion of KLH did not alter its antigenic characteristics, beads coated with SEA or KLH treated with sodium periodate to destroy the immunological properties of their carbohydrate chains, yielded only a monolayer of macrophages similar to negative control beads. Up-regulation of ICAM-1, LFA-1 and fibronectin in SEA-induced granulomas and in native and trypsinised KLH-induced granulomas indicates a major role of the carbohydrate elements of SEA and KLH in the initiation and homeostasis of the inflammatory response. These data provide new insights in the complex and multifactorial carbohydrate-dependent host-parasite immunological interactions.     

Specific glycan elements determine differential binding of individual egg glycoproteins of the human parasite Schistosoma mansoni by host C-type lectin receptors

During infection with the blood fluke Schistosoma mansoni, glycan motifs present on glycoproteins of the parasite’s eggs mediate immunomodulatory effects on the host. The recognition of these glycan motifs is primarily mediated by C-type lectin receptors on dendritic cells and other cells of the immune system. However, it is not yet known which individual glycoproteins interact with the different C-type lectin receptors, and which structural components are involved. Here we investigated the structural basis of the binding of two abundant egg antigens, kappa-5 and IPSE/α1, by the C-type lectin receptor dendritic cell-specific ICAM3-grabbing non-integrin, macrophage galactose-type lectin and mannose receptor. In the natural soluble form, the secretory egg glycoprotein IPSE/α1 interacts with dendritic cells mainly via mannose receptors. Surprisingly, in plate-based assays mannose receptors preferentially bound to mannose conjugates, while in cell-based assays, IPSE/α1 is bound via the fucosylated Galβ1-4(Fucα1-3)GlcNAc (LeX) motif on diantennary N-glycans. Kappa-5, in contrast, is bound by dendritic cells via all three C-type lectin receptors studied and for a minor part also via other, non-C-type lectin receptors. Kappa-5 interacts with macrophage galactose-type lectins via the GalNAcβ1-4GlcNAc antenna present on its triantennary N-glycans, as well as the GalNAcβ1-4(Fucα1-3)GlcNAc antennae present on a minor N-glycan subset. Dendritic cell-specific ICAM3-grabbing non-integrin binding of kappa-5 was mediated via the GalNAcβ1-4(Fucα1-3)GlcNAc antennae, whereas binding of mannose receptors may involve either GalNAcβ1-4(Fucα1-3)GlcNAc antennae or the fucosylated and xylosylated chitobiose core. This study provides a molecular and structural basis for future studies of the interaction between C-type lectin receptors and other soluble egg antigen glycoproteins and their effects on the host immune response.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

Novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits

The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid B-subunit, but not CTB or LTB, to slowly migrating non-acid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glc NAcbeta3Galbeta4Glc NAcbeta3Galbeta4Glcbeta1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Galalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAc beta. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.     

Immunity to schistosomiasis: glycans are potential antigenic targets for immune intervention

The major humoral immune responses in animals infected with Schistosoma mansoni are directed toward carbohydrate antigens. Among these antigens are complex-type N-glycans expressing LDN [GalNAcbeta1-4GlcNAc-R], LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R], and polymeric Lewis x (Lex) [Galbeta1-4(Fucalpha1-3)GlcNAc]n-R epitopes. We have now evaluated the potential of the three glycan antigens as targets for immune-mediated intervention of infections and serodiagnosis. A variety of approaches were employed, including ELISA, Western blot, immunohistology, and in vitro complement lysis assays, to determine the immunogenicity of the glycans in infected humans, their localization on the parasites and their efficacy as targets for parasite lysis. Our results show that S. mansoni-infected patients, with either intestinal or hepatosplenic disease, generate predominantly IgM, but also IgG and IgA, antibodies to LDN, LDNF, and Lex. However, immune responses to Lex are generally lower than responses to LDN and LDNF and less specific to schistosome infections. Western blot analysis with monoclonal antibodies (mAb) to LDN, LDNF, and Lex determinants show that the glycan antigens occur on multiple glycoproteins from cercariae, 3-h, 48-h, and lung stage schistosomula, as well as adults and eggs. Immunohistological studies demonstrate that LDN, LDNF, and Lex are expressed on the parasite surface at all stages of development in the vertebrate host. Importantly, a mAb to LDN in the presence of complement efficiently kills schistosomula in vitro, as demonstrated by flow-cytometric assays that quantify cytolysis by propidium iodide uptake into damaged parasites. These findings raise the possibility that LDN and LDNF may be targets for vaccination and/or serodiagnosis of chronic schistosomiasis in humans.     

Various stages of schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1-4 (Fucalpha1-3)GlcNAc and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

Exquisite specificity of mitogenic lectin from <Emphasis Type="Italic">Cephalosporium curvulum</Emphasis> to core fucosylated N-glycans

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.     

Human gastric glycosphingolipids recognized by Helicobacter pylori vacuolating cytotoxin VacA

Many bacterial toxins utilize cell surface glycoconjugate receptors for attachment to target cells. In the present study the potential carbohydrate binding of Helicobacter pylori vacuolating cytotoxin VacA was investigated by binding to human gastric glycosphingolipids on thin-layer chromatograms. Thereby a distinct binding of the toxin to two compounds in the non-acid glycosphingolipid fraction was detected. The VacA-binding glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as galactosylceramide (Galbeta1Cer) and galabiosylceramide (Galalpha4Galbeta1Cer). Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources showed an additional recognition of glucosylceramide (Glcbeta1Cer), lactosylceramide (Galbeta4Glcbeta1Cer) and globotriaosylceramide (Galalpha4Galbeta4Glcbeta1Cer). No binding to the glycosphingolipids recognized by the VacA holotoxin was obtained with a mutant toxin with deletion of the 37 kDa fragment of VacA (P58 molecule). Collectively our data show that the VacA cytotoxin is a glycosphingolipid binding protein, where the 37 kDa moiety is required for carbohydrate recognition. The ability to bind to short chain glycosphingolipids will position the toxin close to the cell membrane, which may facilitate toxin internalization.     

Structural characterization of Schistosoma mansoni adult worm glycosphingolipids reveals pronounced differences with those of cercariae

Immune responses induced by glycans upon infection with Schistosoma mansoni may be mediated by either schistosomal glycoproteins or glycosphingolipids. In this study, we have elucidated the structural features of both carbohydrate moieties and respective ceramide units of complex glycosphingolipids from adult S. mansoni. Obtained data revealed a vast structural heterogeneity due to manifold combinations of different oligosaccharides and ceramide entities. Observed carbohydrate moieties included Lewis(X) (Le(X); Gal(β1-4)[Fuc(α1-3)]GlcNAc) as well as, in part, multiply fucosylated LacdiNAc (LDN; GalNAc(β1-4)GlcNAc) carbohydrate epitopes. Corresponding lipid portions comprised predominantly C18-sphingosine as well as C18- and C20-phytosphingosine derivatives. Intriguingly, glycosphingolipids carrying an Le(X) epitope contained predominantly C18-sphingosine, whereas LDN-based species exhibited mostly phytosphingosine derivatives, in addition to C18-sphingosine, indicating that the two classes of glycosphingolipids might be synthesized via different biosynthetic routes. Compared with literature data, adult worm glycosphingolipids with Le(X) epitopes revealed clear structural differences in comparison to corresponding cercarial species which have been shown to exhibit mainly sphinganine bases with 18-21 carbon atoms. Therefore, it may be hypothesized that the divergent structural features of the respective ceramide moieties are responsible for the published observation that only adult worm, but not cercarial glycosphingolipids are able to induce dendritic cell activation skewing the T-cell response toward a Th1 profile.     

Synthesis and antibody-binding studies of a series of parasite fuco-oligosaccharides

Complex multifucosylated oligosaccharides are structural elements of glycoprotein and glycolipid subsets of larval, egg, and adult stages of Schistosoma, the parasitic worms that cause schistosomiasis, a serious disease affecting more than 200 million people in the tropics. The fucosylated structures are thought to play an important role in the immunology of schistosomiasis. Defined schistosomal oligosaccharides that enable immunological studies are difficult to obtain from natural sources. Therefore, we have chemically synthesized spacer-linked GlcNAc, Fucalpha1-3GlcNAc, Fucalpha1-2Fucalpha1-3GlcNAc, and Fucalpha1-2Fucalpha1-2Fucalpha1-3GlcNAc. This series of linear oligosaccharides was used to screen a library of anti-schistosome monoclonal antibodies by surface plasmon resonance spectroscopy. Interestingly, the reactive antibodies could be grouped according to their specificity for the different oligosaccharides tested, showing that these oligosaccharides form different immunological entities based on the number and linkage of the fucose residues. Subsequently, the thus defined monoclonal antibodies were used to visualize the expression of the corresponding oligosaccharide epitopes by adult Schistosoma mansoni worms.     

Characteristic of carbohydrate components of chickens and human's fibronectins

The paper presents new information about the carbohydrate structures of 39-days chicken's fibronectin. It is found out that chicken fibronectin contains mainly biantennary N-glycans with a core fucose and fucosylated O-glycans. It is shown that N-glycans of chicken fibronectin are poorly sialated, since this protein exhibits affinity for the PNA and weak binding to sialospecific SNA. A comparative analysis of lectin-binding activity of chicken and human fibronectins has shown that both glycoproteins differ in glycan composition.     

Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen

Lambs respond to vaccination against bacteria and viruses but have a poor immunological response to nematodes. Here we report that they are protected against the parasitic nematode Haemonchus contortus after vaccination with excretory/secretory (ES) glycoproteins using Alhydrogel as an adjuvant. Lambs immunized with ES in Alhydrogel and challenged with 300 L3 larvae/kg body weight had a reduction in cumulative egg output of 89% and an increased percentage protection of 54% compared with the adjuvant control group. Compared to the adjuvant dimethyl dioctadecyl ammonium bromide, Alhydrogel induced earlier onset and significantly higher ES- specific IgG, IgA, and IgE antibody responses. In all vaccinated groups a substantial proportion of the antibody response was directed against glycan epitopes, irrespective of the adjuvant used. In lambs vaccinated with ES in Alhydrogel but not in any other group a significant increase was found in antibody levels against the GalNAcbeta1,4 (Fucalpha1,3)GlcNAc (fucosylated LacdiNAc, LDNF) antigen, a carbohydrate antigen that is also involved in the host defense against the human parasite Schistosoma mansoni. In lambs the LDNF-specific response increased from the first immunization onward and was significantly higher in protected lambs. In addition, an isotype switch from LDNF-specific IgM to IgG was induced that correlated with protection. These data demonstrate that hyporesponsiveness of lambs to H. contortus can be overcome by vaccination with ES glycoproteins in a strong T-helper 2 type response-inducing aluminum adjuvant. This combination generated high and specific antiglycan antibody responses that may contribute to the vaccination-induced protection.     

The N-terminal carbohydrate recognition domain of galectin-8 recognizes specific glycosphingolipids with high affinity

Galectin-8 is a member of the galectin family and has two tandem repeated carbohydrate recognition domains (CRDs). We determined the binding specificities of galectin-8 and its two CRDs for oligosaccharides and glycosphingolipids using ELISA and surface plasmon resonance assays. Galectin-8 had much higher affinity for 3'-O-sulfated or 3'-O-sialylated lactose and a Lewis x-containing glycan than for oligosaccharides terminating in Galbeta1-->3/4GlcNAc. This specificity was mainly attributed to the N-terminal CRD (N-domain), whereas the C-terminal CRD (C-domain) had only weak affinity for a blood group A glycan. The N-domain bound not only to oligosaccharides but also to glycosphingolipids including sulfatide (SM4 s), SM3, sialyl Lc4Cer, SB1a, GD1a, GM3, and sialyl nLc4Cer, suggesting that the N-domain recognizes a 3-O-sulfated or 3-O-sialylated Gal residue. The substitution of the C-3 of the Gal residue in lactose or N-acetyllactosamine with sulfate increased the degree of recognition by galectin-8 more potently than substitution with sialic acid. This is the first demonstration that galectin-8 binds to specific sulfated or sialylated glycosphingolipids with high affinity (KD approximately 10-8-10-9 M). When the Gln47 residue of the N-domain was converted to Ala47, the specific affinity for sulfated or sialylated glycans was selectively lost, indicating that this Gln47 plays important roles for binding to Neu5Acalpha2-->3Gal or SO3--->3Gal residues. The binding ability of galectin-8 to membrane-associated GM3 was confirmed using CHO cells, which predominantly express GM3. Binding of CHO cells to the mutein was significantly lower than to the N-domain.     

Novel fucogangliosides found in human colon adenocarcinoma tissues by means of glycomic analysis

The structures of acidic glycosphingolipids in colon adenocarcinoma have been analyzed extensively using a number of conventional methods, such as thin-layer chromatography and methylation analysis, and a variety of acidic glycosphingolipids present in the tissues have been reported. However, because of a number of limitations in the techniques used in previous studies in terms of resolution, quantification, and sensitivity, we employed a different method that could be applied to small amounts of tissue. In this technique, the carbohydrate moieties of acidic glycosphingolipids from approximately 20mg of colon adenocarcinoma were released by endoglycoceramidase II and were labeled by pyridylamination. They were separated and structurally characterized by a two-dimensional HPLC mapping technique, electrospray ionization tandem mass spectrometry (ESI-MS/MS), and enzymatic cleavage. A total of 22 major acidic glycosphingolipid structures were identified, and their relative quantities were revealed in detail. They are composed of 1 sulfated (SM3), 1 lacto-series (SLe(a)), 6 kinds of ganglio-series, and 14 kinds of neolacto-series glycosphingolipids. They include most of the acidic glycosphingolipids previously reported to be present in the tissues and two previously unknown fucogangliosides sharing the same terminal structure: NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, and NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3-Galbeta1-4Glc. Thus, this highly sensitive, high-resolution analysis enabled the identification of novel structures of acidic glycosphingolipids from small amounts of already comprehensively studied cancerous tissues. This method is a powerful tool for microanalysis of glycosphingolipid structures from small quantities of cancerous tissues and should be applicable to different types of malignant tissues.