Search

Assays which enable the detection of schistosome gut-associated circulating anodic (CAA) and cathodic (CCA) antigen in serum or urine are increasingly used as a diagnostic tool for schistosome infection. However, little is known about the production and clearance of these circulating antigens in relation to the sex and reproductive maturity of the parasite. Here we describe CAA and CCA excretion patterns by exploring a mouse model after exposure to 36 male-only, female-only and mixed (male/female) Schistosoma mansoni cercariae. We found that serum and urine CAA levels, analysed at 3 weeks intervals, peaked at 6 weeks post-infection. Worms recovered after perfusion at 14 weeks were cultured ex vivo. Male parasites excreted more circulating antigens than females, in the mouse model as well as ex vivo. In mixed infections (supporting egg production), serum CAA levels correlated to the number of recovered worms, whereas faecal egg counts or Schistosoma DNA in stool did not. No viable eggs and no inflammation were seen in the livers from mice infected with female worms only. Ex vivo, CAA levels were higher than CCA levels. Our study confirms that CAA levels reflect worm burden and allows detection of low-level single-sex infections. [ABSTRACT FROM AUTHOR]

Vaccination represents the most effective way to prevent invasive pneumococcal diseases. The glycoconjugate vaccines licensed so far are obtained from capsular polysaccharides (CPSs) of the most virulent serotypes. Protection is largely limited to the specific vaccine serotypes, and the continuous need for broader coverage to control the outbreak of emerging serotypes is pushing the development of new vaccine candidates. Indeed, the development of efficacious vaccine formulation is complicated by the high number of bacterial serotypes with different CPSs. In this context, to simplify vaccine composition, we propose the design of new saccharide fragments containing chemical structures shared by different serotypes as cross-reactive and potentially cross-protective common antigens. In particular, we focused on Streptococcus pneumoniae(Sp) 19A and 19F. The CPS repeating units of Sp 19F and 19A are very similar and share a common structure, the disaccharide ManNAc-β-(1→4)-Glc (A-B). Herein, we describe the synthesis of a small library of compounds containing different combinations of the common 19F/19A disaccharide. The six new compounds were tested with a glycan array to evaluate their recognition by antibodies in reference group 19 antisera and factor reference antisera (reacting against 19F or 19A). The disaccharide A-B, phosphorylated at the upstream end, emerged as a hit from the glycan array screening because it is strongly recognized by the group 19 antisera and by the 19F and 19A factor antisera, with similar intensity compared with the CPSs used as controls. Our data give a strong indication that the phosphorylated disaccharide A-B can be considered a common epitope among different Sp 19 serotypes.

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody.

Schistosomiasis treatment relies on the use of a single drug, praziquantel, which is insufficient to control transmission in highly endemic areas1. Novel medicines and vaccines are urgently needed2,3. An experimental human model for schistosomiasis could accelerate the development of these products. We performed a dose-escalating clinical safety trial in 17 volunteers with male Schistosoma mansonicercariae, which do not produce eggs (clinicaltrials.gov NCT02755324), at the Leiden University Medical Center, the Netherlands. The primary endpoints were adverse events and infectivity. We found a dose-related increase in adverse events related to acute schistosomiasis syndrome, which occurred in 9 of 17 volunteers. Overall, 5 volunteers (all 3 of the high dose group and 2 of 11 of the medium dose group) reported severe adverse events. Worm-derived circulating anodic antigen, the biomarker of the primary infection endpoint, peaked in 82% of volunteers at 3–10 weeks following exposure. All volunteers showed IgM and IgG1 seroconversion and worm-specific cytokine production by CD4+T cells. All volunteers were cured with praziquantel provided at 12 weeks after exposure. Infection with 20 Schistosoma mansonicercariae led to severe adverse events in 18% of volunteers and high infection rates. This infection model paves the way for fast-track product development for treatment and prevention of schistosomiasis.

Here, we describe a strategy for the rapid preparation of pure positional isomers of complex N-glycans to complement an existing array comprising a larger number of N-glycans and smaller glycan structures. The expanded array was then employed to study context-dependent binding of structural glycan fragments by monoclonal antibodies and C-type lectins. A partial enzymatic elongation of semiprotected core structures was combined with the protecting-group-aided separation of positional isomers by preparative HPLC. This methodology, which avoids the laborious chemical differentiation of antennae, was employed for the preparation of eight biantennary N-glycans with Galβ1,4GlcNAc (LN), GalNAcβ1,4GlcNAc (LDN), and GalNAcβ1,4[Fucα1,3]GlcNAc (LDNF) motifs presented on either one or both antennae. Screening of the binding specificities of three anti-LeXmonoclonal IgM antibodies raised against S. mansoniglycans and three C-type lectin receptors of the innate immune system, namely DC-SIGN, DC-SIGNR, and LSECtin, revealed a surprising context-dependent fine specificity for the recognition of the glycan motifs. Moreover, we observed a striking selection of one individual positional isomer over the other by the C-type lectins tested, underscoring the biological relevance of the structural context of glycan elements in molecular recognition.

Biofunctionalized gold nanoparticles (AuNPs) enable innovative translational research and development in biomedicine. Biomolecules such as peptides, proteins, lipids, and carbohydrates can be assembled onto AuNPs to yield nanomaterials with unique properties for applications in imaging, photothermal therapy, vaccination strategies, and drug delivery. The characterization of functionalized AuNPs still remains an analytical challenge that normally requires the combination of multiple techniques. Laser desorption/ionization (LDI) and matrix-assisted LDI (MALDI) have been applied successfully in combination with time-of-flight (TOF) mass spectrometry (MS) for the analysis of the surface chemistry of AuNPs functionalized with synthetic ligands, however only for ligands with a molecular mass limited to 1000 Da. TOF-MS-based approaches in addition exhibit limited performance in terms of mass resolution and MS/MS possibilities. To overcome these limitations, we designed an approach for the analysis of AuNPs based on ultrahigh resolution Fourier transform ion cyclotron resonance (FTICR) MS and a combination of LDI and MALDI. To illustrate the performance of the method, we present a comprehensive characterization of the surface chemistry of AuNPs conjugated viaa thiol-ending linker to either the ovalbumin peptide (OVA 323-339), the Lewis X antigen (Galβ1-4[Fucα1-3]GlcNAcβ1) trisaccharide, the tetramannoside Manα1-2Manα1-2Manα1-3Manα1, or a mixture of both carbohydrates. Collision-induced dissociation (CID) was used to characterize the structure of pseudomolecular ions generated by LDI/MALDI in-depth. These included [M + H]+and [M + Na]+, and importantly also [M + Au]+and [M + 2Au–H]+ions. This first observation of gold-containing pseudomolecular ions provides direct evidence for the Au-conjugation of ligands. In addition, we show the applicability of the method to monitor proteolytic cleavage of peptides that are conjugated to the AuNP surface. The presented LDI/MALDI–FTICR–MS and MS/MS approach will be applicable to the characterization of a wide range of functionalized AuNPs.

Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica(FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepaticategumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepaticategument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepaticaand lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepaticainfection and the development of an effective vaccine.

ABSTRACTSchistosomiasis is a tropical disease affecting over 230 million people worldwide. Although effective drug treatment is available, reinfections are common, and development of immunity is slow. Most antibodies raised during schistosome infection are directed against glycans, some of which are thought to be protective. Developing schistosomula are considered most vulnerable to immune attack, and better understanding of local antibody responses raised against glycans expressed by this life stage might reveal possible glycan vaccine candidates for future vaccine research. We used antibody-secreting cell (ASC) probes to characterize local antiglycan antibody responses against migrating Schistosoma japonicumschistosomula in different tissues of rats. Analysis by shotgun Schistosomaglycan microarray resulted in the identification of antiglycan antibody response patterns that reflected the migratory pathway of schistosomula. Antibodies raised by skin lymph node (LN) ASC probes mainly targeted N-glycans with terminal mannose residues, Galβ1-4GlcNAc (LacNAc) and Galβ1-4(Fucα1-3)GlcNAc (LeX). Also, responses to antigenic and schistosome-specific glycosphingolipid (GSL) glycans containing highly fucosylated GalNAcβ1-4(GlcNAcβ1)nstretches that are believed to be present at the parasite's surface constitutively upon transformation were found. Antibody targets recognized by lung LN ASC probes were mainly N-glycans presenting GalNAcβ1-4GlcNAc (LDN) and GlcNAc motifs. Surprisingly, antibodies against highly antigenic multifucosylated motifs of GSL glycans were not observed in lung LN ASC probes, indicating that these antigens are not expressed in lung stage schistosomula or are not appropriately exposed to induce immune responses locally. The local antiglycan responses observed in this study highlight the stage- and tissue-specific expression of antigenic parasite glycans and provide insights into glycan targets possibly involved in resistance to S. japonicuminfection.

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoniinduce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1–4(Fucα1–3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1–4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1–3(Galβ1–6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.

The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoniinfection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world's three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayirevealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayiglycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayiglycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoniseropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoniseropositivity, binds to S. mansonicercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development.

Cooperia oncophorais one of the most common intestinal parasitic nematodes in cattle worldwide. To date, C. oncophorainfections are treated using broad-spectrum anthelmintics. However, during the past decade, reports of anthelmintic resistance in this parasite species have emerged worldwide, necessitating new avenues for its control, possibly through vaccination. In this frame, we analyzed the adult-stage C. oncophoraexcretome/secretome (ES), covering both the protein and glycan components, since this fraction constitutes the primary interface between parasite and host and may hold potential vaccine candidates. Two-dimensional gel electrophoretic separation of the ES material enabled the MALDI-TOF mass spectrometry (MS)-directed identification of 12 distinct proteins, grouped in three separate molecular weight fractions: (i) a high molecular weight fraction consisting of a double-domain activation-associated secreted protein (ASP), (ii) a midmolecular weight fraction predominantly containing a single-domain ASP, a thioredoxin peroxidase and innexin, and (iii) a low molecular weight protein pool essentially holding two distinct low molecular weight antigens. Further MS-driven glycan analysis mapped a variety of N-glycans to the midmolecular weight single-domain ASP, with Man6GlcNAc2oligomannosyl glycans as the major species. The predominance of the nonglycosylated double-domain ASP in the high-molecular weight fraction renders it ideal for advancement toward vaccine trials and development.

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

Schistosome infections in humans are characterized by the development of chronic disease and high re-infection rates after treatment due to the slow development of immunity. It appears that anti-schistosome antibodies are at least partially mediating protective mechanisms. Efforts to develop a vaccine based on immunization with surface-exposed or secreted larval or worm proteins are ongoing. Schistosomes also express a large number of glycans as part of their glycoprotein and glycolipid repertoire, and antibody responses to those glycans are mounted by the infected host. This observation raises the question if glycans might also form novel vaccine targets for immune intervention in schistosomiasis. This review summarizes current knowledge of antibody responses and immunity in experimental and natural infections with Schistosoma, the expression profiles of schistosome glycans (the glycome), and antibody responses to individual antigenic glycan motifs. Future directions to study anti-glycan responses in schistosomiasis in more detail in order to address more precisely the possible role of glycans in antibody-mediated immunity are discussed. [ABSTRACT FROM AUTHOR]

Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered.

Earlier studies have suggested that complexes of the human IgA receptor FcalphaRI/CD89 with mouse IgA are pathogenic upon deposition in the renal mesangium. Transgenic mice expressing FcalphaRI/CD89 on macrophages/monocytes developed massive mesangial IgA deposition and a clinical picture of IgA nephropathy (IgAN). Based on these findings, the purpose of this study was to design an experimental model of IgAN by injection of human CD89 in mice. The interaction of mouse IgA with CD89 was investigated further.

KLH (keyhole limpet haemocyanin), the oxygen-carrying molecule of the marine snail Megathura crenulata, is often used as an adjuvant or as a hapten carrier for immunizations with peptides, oligosaccharides or other low-molecular-mass organic compounds. KLH exhibits several carbohydrate determinants, at least some of which are immunogenic: it shares an antigenic Fuc(α1-3)GalNAc-determinant with schistosomes and contains unique Gal-(β1-6)Man-structural motifs on its N-glycans. This study reveals the presence of N-glycans with unusual ±Gal(β1-4)Gal(β1-4)Fuc- units (α1-6)-linked to the reducing end N-acetylglucosamine residue. The following novel structures of KLH N-glycans were deduced by linkage analysis, exoglycosidase digestion, matrix-assisted laser-desorption ionization-tandem MS and nano-LC-ESI-IT-MS (where LC stands for liquid chromatography, ESI for electrospray ionization and IT for ion trap): Man(α1-6)[±Man(α1-3)]Man(β1-4)GlcNAc(β1-4)[Gal(β1-4)Fuc(α1-6)]GlcNAc and Man(α1-6)Man(β1-4)GlcNAc(β1-4)[Gal(β1-4)Gal(β1-4)Fuc(α1-6)]GlcNAc. The Gal(β1-4)Fuc- and Gal(β1-4)Gal(β1-4)Fuc- core modifications are expected to be immunogenic, similar to other non-mammalian-type core modifications, and to contribute to the immunostimulatory properties of KLH.

By the use of surface plasmon resonance spectroscopy, immunoglobulin G (IgG) subclass and IgM antibodies against three schistosome-derived carbohydrate structures, FLDN (Fucalpha1-3GalNAcbeta1-4GlcNAcbeta1-3Galalpha1), LDN-DF [GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1], and LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galalpha1], were measured in 184 previously unexposed Kenyan immigrants who moved into the Masongaleni area, where Schistosoma mansoni is endemic. They were sampled within their first year of exposure and again 2 years later. A cohort selected out of the original residents of the area, who had been exposed for many years, served as controls. Associations with responses to S. mansoni worm, egg (SEA), and cercarial (CERC) antigens were examined. In addition, we measured responses to keyhole limpet hemocyanin, a glycoprotein which carries glycan epitopes that are also expressed by schistosomes. Specific IgG1 responses were most pronounced against FLDN and LDN-DF and strongly associated with those previously measured to SEA and CERC. Similarly to previously published age profiles of IgG1 and IgG2 responses to SEA, levels of IgG1 against LDN-DF decreased with age. In contrast, specific IgM responses against the three schistosome-derived carbohydrate structures were most marked against LDNF. Our results indicate that, of the three glycan structures tested, the acute response against schistosome glycoconjugate antigens in young children is mainly directed against the LDN-DF epitope. The response to LDN-DF in older individuals and the responses to the two other epitopes were similar in the two cohorts, suggesting that these antigens are recognized in the early stages of infection and that the immune response persists. The biological significance of these observations needs further elucidation.

ABSTRACTWe report here that sera of children and adults infected withSchistosoma mansoni, S. haematobium, or S. japonicumcontain antibodies against GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF) and to a lesser extent to Galβ1-4(Fucα1-3)GlcNAc (Lewisx) and GalNAcβ1-4GlcNAc (LDN). Surface plasmon resonance (SPR) spectroscopy was used to monitor the presence of serum antibodies to neoglycoconjugates containing these carbohydrate epitopes and to define the immunoglobulin M (IgM) and IgG subclass distribution of the antibodies. The serum levels of antibodies to LDN-DF are high related to LDN and Lewisxfor all examined groups ofSchistosoma-infected individuals. A higher antibody response to the LDN-DF epitope was found in sera of infected children than in sera of infected adults regardless of the schistosome species. With respect to the subclasses, we found surprisingly that individuals infected with S. japonicumhave predominantly IgG antibodies, while individuals infected with S. mansonimainly show an IgM response; high levels of both isotypes were measured in sera of individuals infected with S. haematobium. These data provide new insights in the human humoral immune response to schistosome-derived glycans.

ABSTRACTThe lipopolysaccharide (LPS) of Helicobacter pyloriexpresses the Lewis x (Lex) and/or Leyantigen. We have shown previously that H. pyloriLPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lexplus Leyor nonfucosylated polylactosamine. H. pylorihas two α3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pyloriLPS phase variation and demonstrate that the on or off status of α3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the α3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of α3-fucosyltransferase knockout mutants. The data also show that the two α3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futAand futBto designate the orthologs of the H. pylori26695 α3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the α3-fucosylation in H. pyloriprecedes α3-fucosyltransferase, an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host.

HPLC analysis of sialic acid released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of N-acetylneuraminic and N-glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz 1H-NMR spectroscopy, of the enzymatically released N-linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that α2-3 linked N-glycolylneuraminic acid can occur in different N-acetyllactosamine type antennary structures.

The disaccharide Manβ1-4GlcNAc and the trisaccharide Manβ1-4GlcNAcβ1-4GlcNAc were each incubated with CMPNeuAc and rat liver α2-6-sialyltransferase (CMP-NeuAc: Galβ1-4GlcNAc α2-6- N-acetylneuraminyl transferase). The resulting mixtures were fractionated by HPLC on Partisil 10 SAX, and the fractions obtained were investigated by TLC, GLC (monosaccharide analysis) and 500-MHz 1H-NMR spectroscopy. The followingproducts were identified: NeuAcα2-6Manβ1-4GlcNAc and NeuAcα2-6Manβ1-4GlcNAcβ1-4GlcNAc in yields of 4% and 27%, respectively. The sialylation of Manβ-4GlcNAc-R by Galβ-4GlcNAc α2-6-sialyltransferase contrasts the reported high specificity of this enzyme for the structural element Galβ1-4GlcNAc of N-linked carbohydrate chains of glycoproteins and related oligosaccharides.

An approach is presented for the determination of the branch location of 1 or 2 extra N-acetyllactosamine units in sialo N-linked carbohydrate chains from glycoproteins. Tetraantennary oligosaccharides containing extra N-acetyllactosamine units were digested with endo-β-galactosidase, followed by treatment with N-acetyl-β-glucosaminidase, yielding products which could be analysed by 1H-NMR spectroscopy, thereby giving conclusive data about the location of the extra units in the intact structures.

Three glycoprotein N-glycans, namely, a disialylated diantennary carbohydrate chain linked to Asn. a monosialylated, fucosylated diantennary glycopeptide with bisecting N-acetylglucosamine, and a tetrasialylated, fucosylated tetra-antennary oligosaccharide. have been investigated by two-dimensional NOE and HOHAHA spectroscopy in 1H 2O as solvent. The amide protons of all N-acetylglucosamine and sialic acid residues could readily be assigned. The large chemical-shift dispersion of the amide resonances of the N-acetylglucosamine residues, allowed the unambiguous assignment of every N-acetyl methyl signal, via strong NOEs. Subspectra could be obtained of all N-acetylglucosamine residues in HOHAHA spectra. These results have as main implication that several biologically important large glycans, will not become amenable for conformational studies by multidimensional NMR in 1H 2O solution.

Three glycoprotein N‐glycans, namely, a disialylated diantennary carbohydrate chain linked to Asn. a monosialylated, fucosylated diantennary glycopeptide with bisecting N‐acetylglucosamine, and a tetrasialylated, fucosylated tetra‐antennary oligosaccharide. have been investigated by two‐dimensional NOE and HOHAHA spectroscopy in 1H2O as solvent. The amide protons of all N‐acetylglucosamine and sialic acid residues could readily be assigned. The large chemical‐shift dispersion of the amide resonances of the N‐acetylglucosamine residues, allowed the unambiguous assignment of every N‐acetyl methyl signal, via strong NOEs. Subspectra could be obtained of all N‐acetylglucosamine residues in HOHAHA spectra. These results have as main implication that several biologically important large glycans, will not become amenable for conformational studies by multidimensional NMR in 1H2O solution.

An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specificO-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role ofO-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminalN-acetylglucosaminyl or, alternatively, a terminal α-galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit thein vitrobinding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAcβ1,4GlcNAcβ1,4GlcNAc,was inactive (1–72 μmrange). In contrast, a β4-galactosyl-capped trisaccharide (Galβ1,4GlcNAcβ1, 4GlcNAc) and an α3-galactosyl-capped trisaccharide (Galα1,3Galβ1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50= 42 μmand 5.3 μm, respectively). The addition of an α3-fucosyl residue to each of these two competitive inhibitors, forming Galβ1,4[Fucα1,3] GlcNAcβ1,4GlcNAc or Galα1,3Galβ1, 4[Fucα1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50= 500 and 430 nm). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the α3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated β-galactosyl-capped trisaccharide.

Antibody effector functions have been shown to be influenced by the structure of the Fc N-glycans. Here we studied the changes in plasma or serum IgG Fc N-glycosylation upon vaccination of 10 Caucasian adults and 10 African children. Serum/plasma IgG was purified by affinity chromatography prior to and at two time points after vaccination. Fc N-glycosylation profiles of individual IgG subclasses were determined for both total IgG and affinity-purified anti-vaccine IgG using a recently developed fast nanoliquid chromatography-electrospray ionization MS (LC-ESI-MS) method. While vaccination had no effect on the glycosylation of total IgG, anti-vaccine IgG showed increased levels of galactosylation and sialylation upon active immunization. Interestingly, the number of sialic acids per galactose increased during the vaccination time course, suggesting a distinct regulation of galactosylation and sialylation. In addition we observed a decrease in the level of IgG1 bisecting N-acetylglucosamine whereas no significant changes were observed for the level of fucosylation. Our data indicate that dependent on the vaccination time point the infectious agent will encounter IgGs with different glycosylation profiles, which are expected to influence the antibody effector functions relevant in immunity.

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcβ1–4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galβ1–4(Fucα1–3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoniare mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansonieggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcβ1–4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galβ1–4(Fucα1–3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.

To study the biology of ALL, primary leukemic cells were cultured in a longterm serum-free culture system. In 10 out of 25 primary precursor B-ALL cells we succeeded to establish ALL cell lines, phenotypically identical to their original clone. The cultured leukemic cells proliferated for more than two years with a constant doubling time of 2.0 ± 0.3 days, but only in cultures of moderate to high densities, suggesting the production of an autocrine proliferation inducing factor. To study the possible autocrine production of a proliferation inducing factor low cell concentrations (1,000 cells/ml/well) were cultured in the absence or presence of autologous conditioned medium(CM, 20%v/v). In the absence of CM, no proliferation was observed as determined by 3H-thymidine uptake as well as in a limiting dilution assay in 9 out of 10 ALL samples. In the presence of CM, proliferation was induced in all ALL cell lines. CM generated from a variety of other unrelated cell sources, as well as CM derived from lysed cells also induced identical proliferation in all ALL samples demonstrating that the samples proliferated and responded to a factor present in all cell populations.To characterize this autocrine factor CM was prepared from ALL cell lysates. Chemical analysis of this CM revealed that the factor was a heat stable molecule with a molecular size less than 500 Dalton. The factor was further purified on a carbograph column and finally on a Superdex Peptide size-exclusion column. The active fractions were analysed by NMR spectroscopy and the pattern of the spectrum strongly suggested the presence of a heterocycle purine. CE-MS analysis confirmed this observation by detecting the presence of a molecule with a mass of 136.05, identical to the theoretical mass of Hypoxanthine. By MS/MS fragmentation analysis of both the isolated compound and synthetic Hypoxanthine the compounds were found to be identical. Addition of the chemically synthetised Hypoxanthine (0.45 μM) to low cell concentrations of ALL cell cultures as well as to cultures of primary ALL cells induced proliferation comparable to cell cultures in the presence of CM. Proliferation of ALL cells could also be induced by Deoxyinosine or Inosine comparable to Hypoxanthine but only moderate to low proliferation was observed after addition of Adenine or Adenosine and no proliferation in the presence of Guanine, Guanosine or Inosinemonophosphate. In conclusion, we developed a culture system allowing identification of the requirements of ALL for proliferation in vitro to study the biology of these leukemic cells. Using this culture system, we illustrated that ALL cells appeared to be highly dependent on an exogenous source of Hypoxanthine or its metabolic component. This dependency may open new perspectives in the development of treatment for ALL, and may result in a more specific control of the different metabolic pathways of purines in malignant cells.

Sex-specific gene expression by Schistosoma mansoni worms has been demonstrated at the transcriptome as well as the proteome levels. In view of the important role of glycans in the biology of schistosomes and the interaction with their human host, we have investigated the sex-specific protein glycosylation. Mass spectrometric profiling and structural characterization of PNGase F-released N-glycans revealed the following gender-specific glycosylation patterns: Complex-type N-glycans of females mainly carried Gal(β1-4)GlcNAc (LacNAc) and Gal(β1-4)[Fuc(α1-3)]GlcNAc (Lewis x) antennae structures, whereas GalNAc(β1-4)GlcNAc- (LacdiNAc; LDN) and GalNAc(β1-4)[Fuc(α1-3)]GlcNAc (LDN-F) were prevalent in N-glycans from males. LDN(-F) motifs were found to occur as repeats on the antennae of large N-glycans that contained up to seven LDN(-F) units. The female complex-type glycans were mostly di-antennary and tri-antennary, whereas male structures were predominantly of the mono-antennary and di-antennary type. Oligomannosidic N-glycans were expressed at similar levels in females and males. The localization of the sex-biased glycan motifs was studied by immunofluorescence microscopy using defined anti-glycan monoclonal antibodies (mAbs). The Lewis x element was strongly expressed in the gut of both males and females, but with respect to tegument localization, the females expressed this structure, while Lewis x seemed to be almost completely absent from the male tegument. The expression of LDN-F was predominantly detected in the parenchyma of both male and female worms as well as in the tegument of the male ventral cavity facing the female. LDN was detected in the tegument of male and female worms at similar levels. The sex-specific expression and differential localization of these antigenic glycan motifs in schistosomes may play a role in male–female interactions during conjugal biology and may lead to a differential immune reaction of the host to the two sexes.

Schistosomes, major parasitic helminths, express numerous glycoconjugates that provoke humoral and cellular immune responses in the infected human host. The main pathology in schistosomiasis is due to the formation of granulomas around tissue-trapped eggs and the resulting organ damage. By using a mouse model of induction of granulomas by hepatic implantation of antigen-coated beads, it has been determined that the glycan part of schistosomal soluble egg antigens (SEA) initiates granulomogenesis. To identify which individual glycan elements in this complex SEA mixture are granulomogenic, we have tested in the same mouse model conjugates of various synthetic oligosaccharides characteristic for schistosome eggs, including GalNAcβ1-4GlcNAc (LacdiNAc, LDN), Galβ1-4(Fucα1-3)GlcNAc (Lewisx), Fucα1-2Fucα1-3GlcNAc (DF-Gn), and Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F). Ribonuclease (RNase) A and B, and different fetuin glycoforms were included as controls. Only beads that carry glycoconjugates with terminal LacdiNAc or Galβ1-4GlcNAc (LacNAc, LN) elements gave rise to granulomas, with macrophage, lymphocyte, and eosinophil levels similar to the granulomatous lesions caused by schistosome eggs in a natural infection. Uncoated beads, and beads coated with fucosylated glycoconjugates or glycoconjugates lacking terminally exposed Gal or GalNAc, only attracted a monolayer of macrophages. These results indicate that the formation of hepatic granulomas is triggered specifically by glycoconjugates which carry terminal LacNAc or LacdiNAc, both constituents of the schistosome egg.