In previous work we showed that Ag5, a major diagnostic antigen from the metacestode of Echinococcus granulosus, possesses a dominant sugar epitope that upon removal results in abolition of most of the antigen immunoreactivity with patient sera. Analysis of this glycan modification has now been performed by western blotting and mass spectrometry. Reactivity to both a specific monoclonal antibody (TEPC15) and human C-reactive protein as well as the presence of a modification of 165 mass units, as detected by mass spectrometry of both glycopeptides and released N-glycans, indicated that the immunodominant sugar epitope of the Ag5 38kDa subunit is a biantennary structure modified by phosphorylcholine. We believe this is the first time that such a modification has been proven in cestodes and provides the structural basis for understanding the antigenicity of this major E. granulosus component.

The in situ preparation of ethylene dimethacrylate porous polymer monoliths within 20 μL polypropylene pipette tips, bound via surface grafted methacrylate anchor sites, is reported. Gold nano-particles (AuNPs) were immobilised onto the monolith pore surface utilising azlactone chemistry and coverage verified using field emission scanning electron microscopy. Erythrina cristagalli lectin (ECL) was immobilised upon the attached AuNPs via a bio-functional linker. The ECL-modified tip was successfully applied for the enrichment of galactosylated protein (desialylated transferrin) versus a non-galactosylated protein (ribonuclease B) due to the specificity of ECL. Reversed-phase capillary HPLC was used to validate the efficiency and selectivity of the developed micro-extraction phase which resulted in an increase in extraction recovery of ∼95% due to the AuNP enhanced surface area. Further specificity of the ECL-modified tip was demonstrated with a complex mixture of non-glycosylated and glycosylated proteins with differing terminal sugar structures. Finally, the lectin affinity phase was applied to a galactosylated glycoproteins spiked Escherichia coli cell lysate to successfully demonstrate matrix tolerance.

The isolation and analysis of glycoproteins by coupling lectin affinity chromatography with MS has emerged as a powerful strategy to study the glycoproteome of mammalian cells. However, this approach has not been used extensively for the analysis of plant glycoproteins. As with all eukaryotes, N-glycosylation is a common post-translational modification for plant proteins traveling through the secretory pathway. Many such proteins are destined for the cell wall, or apoplast, where they play important roles in processes such as modifying cell wall structure, sugar metabolism, signaling, and defense against pathogens. Here, we describe a strategy to enrich for and identify secreted plant proteins based on affinity chromatography using the lectin Concanavalin A and two-dimensional liquid chromatography, together with matrix-assisted laser desorption/ionization MS analysis. The value of this approach is illustrated through the characterization of glycoproteins that are expressed in ripe tomato (Solanum lycopersicum) fruit, a developmental stage that is fundamentally linked with significant changes in cell wall structure and composition. This glycoprotein trap strategy allowed the isolation of a sub-proteome with an extremely high proportion of proteins that are predicted to be resident in the cell wall or secretory pathway, and the identification of new putative cell wall proteins.

Solid-phase extraction microtips are important devices in modern bioanalytics, as they allow miniaturized sample preparation for mass spectrometric analysis. Here we introduce the use of cotton wool for the preparation of filter-free HILIC SPE microtips. To this end, pieces of cotton wool pads (approximately 500 μg) were packed into 10 μL pipet tips. The performance of the tips was evaluated for microscale purification of tryptic IgG Fc N-glycopeptides. Cotton wool HILIC SPE microtips allowed the removal of salts, most nonglycosylated peptides, and detergents such as SDS from glycoconjugate samples. MALDI-TOF-MS glycopeptide profiles were very repeatable with different tips as well as reused tips, and very similar profiles were obtained with different brands of cotton wool pads. In addition, we used cotton HILIC microtips to purify N-glycans after N-glycosidase F treatment of IgG and transferrin followed by MALDI-TOF-MS detection. In conclusion, we establish cotton wool microtips for glycan and glycopeptide purification with subsequent mass spectrometric detection.

Lectins are proteins capable of recognizing and binding to specific oligosaccharide structures found on glycoproteins and other biomolecules. As such, they have utility for glycoanalytical applications. One common difficulty encountered in the application of these proteins, particularly in multiwell plate assay formats known as enzyme-linked lectin assays (ELLAs), is finding appropriate blocking solutions to prevent nonspecific binding with plate surfaces. Many commonly used blocking agents contain carbohydrates and generate significant background signals in ELLAs, limiting the utility of the assays. In this study, we examined the suitability of a range of blocking reagents, including protein-based, synthetic, and commercially available carbohydrate-free blocking reagents, for ELLA applications. Each blocking reagent was assessed against a panel of 19 commercially available biotinylated lectins exhibiting diverse structures and carbohydrate specificities. We identified the synthetic polymer polyvinyl alcohol (PVA) as the best global blocking agent for performing ELLAs. We ultimately present an ELLA methodology facilitating broad spectrum lectin analysis of glycoconjugates and extending the utility of ELLAs.

One common method used for analyzing the glycoproteome is chromatography using multiple lectins that display different affinities toward oligosaccharide structures. Much has been done to determine lectin affinity using standard glycoproteins with known glycosylation; however, a knowledge of the selectivity and specificity of lectins exposed to complex mixtures of proteins is required if they are to be used as a means of studying the glycoproteome. In the present study, three lectins (Concanavalin A, Jacalin, and Wheat Germ Agglutinin) were used to fractionate glycoproteins from two different complex environments:(1) cell membranes and (2) plasma. Reproducible enrichment of glycoproteins from these samples has been shown to result from the combined use of these lectins. However, the global glycan profiles of the released N- and O-linked oligosaccharides from the glycoproteins retained by the lectins, and from those glycoproteins that did not bind, using both these complex samples, were found to be very similar. That is, although the lectins selectively and reproducibly retained some glycoproteins, other proteins with the same attached oligosaccharide structures did not bind. Some small N- and O-glycan differences were observed in the bound fractions but there was little absolute specificity toward individual oligosaccharide structures known to have high affinity to these lectins. These data indicate that lectins are useful for fractionating glycoproteins from complex mixtures, but that the overall glycoproteome is not isolated by this approach.

HASH(0x2b330e67dba0)

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the alpha1,4-N-acetylgalactosaminyltransferase-1 (alpha4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of alpha4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that alpha4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by alpha4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL-protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.

The binucleate trophoblast cells (BNCs) in the ruminant placenta are a unique feature of this taxon. These cells produce several secretory proteins and transfer these across the fetomaternal barrier into the dam. We used lectin histochemistry with a panel of 24 lectins to characterise the glycosylation pattern of BNC secretory granules in a variety of ruminants. Seven species out of three ruminant families were thus investigated: greater malayan chevrotain (Tragulidae); fallow deer, red deer, chinese water deer (Cervidae); and domestic goat, springbok, impala (Bovidae). BNC granules in all species studied strongly expressed tri-/tetraantennary complex N-glycans and bisecting N-acetylglucosamine [GlcNAc] as shown by binding of leuco- and erythroagglutins of Phaseolus vulgaris respectively. The presence of terminal N-acetylgalactosamine [GalNAc]) in BNC granules is shown by intense staining with lectins from Dolichos biflorus, Vicia villosa and Wisteria floribunda. Terminal galactose or GalNAc was also present, bound by Glycine max agglutinin. Treatment of slides with neuraminidase strongly intensified staining of Erythrina cristagalli lectin (ECA) to terminal lactosamine in all species studied; this was otherwise absent except in goat. Sambucus nigra-1 lectin bound to BNC granules in all species except in Impala, indicating the presence of abundant alpha2,6 linked sialic acid. These results indicate that these unusual highly branched glycans, with bisecting GlcNAc and terminal GalNAc are a general feature of BNC granules in Ruminants, including the most basal Tragulid branch. It therefore appears that the specific glycosylation pattern of BNC granules evolved early in ruminant phylogenesis, together with the appearance of BNC. The conserved glycan structure in BNC secretory granules indicates that this pattern of glycosylation is likely to be of considerable functional importance for the secretory glycoproteins of ruminant BNC.

Epithelial ovarian cancer is diagnosed less than 25% of the time when the cancer is confined to the ovary, leading to 5 year survival rates of less than 30 %. Therefore, there is an urgent need for early diagnostics for ovarian cancer. Our study using glycotranscriptome comparative analysis of endometrioid ovarian cancer tissue and normal ovarian tissue led to the identification of distinct differences in the transcripts of a restricted set of glycosyltransferases involved in N-linked glycosylation. Utilizing lectins that bind to glycan structures predicted to show changes, we observed differences in lectin-bound glycoproteins consistent with some of the transcript differences. In the present study, we have extended our observations by the use of selected lectins to perform a targeted glycoproteomic analysis of ovarian cancer and normal ovarian tissues. Our results have identified several glycoproteins that display tumor-specific glycosylation changes. We have verified these glycosylation changes on glycoproteins from tissue using immunoprecipitation followed by lectin blot detection. The glycoproteins that were verified were then analyzed further using existing microarray data obtained from benign ovarian adenomas, borderline ovarian adenocarcinomas, and malignant ovarian adenocarcinomas. Those verified glycoproteins found to be expressed above control levels in the microarray datasets were then screened for tumor-specific glycan modifications in serum from ovarian cancer patients. Results obtained from two of these glycoprotein markers, periostin and thrombospondin, have confirmed that tumor-specific glycan changes can be used to distinguish ovarian cancer patient serum from normal serum.

In previous work we showed that Ag5, a major diagnostic antigen from the metacestode of Echinococcus granulosus, possesses a dominant sugar epitope that upon removal results in abolition of most of the antigen immunoreactivity with patient sera. Analysis of this glycan modification has now been performed by western blotting and mass spectrometry. Reactivity to both a specific monoclonal antibody (TEPC15) and human C-reactive protein as well as the presence of a modification of 165 mass units, as detected by mass spectrometry of both glycopeptides and released N-glycans, indicated that the immunodominant sugar epitope of the Ag5 38kDa subunit is a biantennary structure modified by phosphorylcholine. We believe this is the first time that such a modification has been proven in cestodes and provides the structural basis for understanding the antigenicity of this major E. granulosus component.

Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data has been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and HPLC data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man(5)GlcNAc(2)) with α1,2- mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species.

Planarial species are of especial interest to biologists due to the phenomenon of pluripotency and, in comparison to other developmental processes, it can be hypothesised that glycan-lectin interactions may play a role. In order to examine the N-glycans of one of these organisms, Dugesia japonica, peptide:N-glycosidase A was employed and the released glycans were subject to pyridylamination, HPLC and mass spectrometric analysis. A range of oligomannosidic glycans was observed with a trimethylated Man(5) GlcNAc(2) structure being the dominant species. Three glycans were also observed to contain deoxyhexose; in particular, a glycan with the composition Hex(4) HexNAc(2) Fuc(1) Me(2) was revealed by exoglycosidase digestion, in combination with MS/MS, to contain a galactosylated core α1,6-fucose residue, whereas this core modification was found to be capped with a methylhexose residue in the case of a Hex(5) HexNAc(2) Fuc(1) Me(3) structure. This is the first report of these types of structures in a platyhelminth and indicates that the 'GalFuc' modification of N-glycans is not just restricted to molluscs and nematodes.

Here we present a comparative structure-function study of a nematode and a plant core α1,3-fucosyltransferase, based on deletion and point mutations of the coding regions of Caenorhabditis elegans FUT-1 and Arabidopsis thaliana FucTA (FUT11). In particular our results reveal a novel 'first cluster motif' shared by both core and Lewis-type α1,3-fucosyltransferases of the GT10 family. To evaluate the role of the conserved serine within this motif, this residue was replaced with alanine in FucTA (S218) and FUT-1 (S243). The S218A replacement completely abolished the enzyme activity of FucTA, while the S243A mutant of FUT-1 retained 20% of the 'wild-type' activity. Based on the results of homology modelling of FucTA, other residues potentially involved in the donor substrate binding were examined and mutations of N219 and R226 dramatically affected enzymatic activity. Finally, as both FucTA and FUT-1 were shown to be N-glycosylated, we examined the putative N-glycosylation sites. While alanine replacements at single potential N-glycosylation sites of FucTA resulted in loss of up to 80% of the activity, a triple glycosylation site mutant still retained 5%, as compared to the control. In summary, our data indicate similar trends in structure-function relationships of distantly-related enzymes which perform similar biochemical reactions and form the basis for future work aimed at understanding the structure of α1,3-fucosyltransferases in general.

Both helminth infections and contact with allergens result in development of a Th2 type of immune response in the affected individual. In this context, the hygiene hypothesis suggests that reduced prevalence of parasitic infections and successful vaccination strategies are causative for an increase of allergies in industrialized countries. It is therefore of interest to study glycans and their role as immunogenic structures in both parasitic infections and allergies. In the present paper we review information on the different types of glycan structure present in proteins from plant and animal food, insect venom and helminth parasites, and their role as diagnostic markers. In addition, the application of these glycan structures as immunomodulators in novel immunotherapeutic strategies is discussed.

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.

Determining the exact nature of N-glycosylation in Caenorhabditis elegans, a nematode worm and genetic model organism, has proved to have been an unexpected challenge in recent years; a wide range of modifications of its N-linked oligosaccharides have been proposed on the basis of structural and genomic analysis. Particularly mass spectrometric studies by a number of groups, as well as the characterisation of recombinant enzymes, have highlighted those aspects of N-glycosylation that are conserved in animals, those which are seemingly unique to this species and those which are shared with parasitic nematodes. These data, of importance for therapeutic developments, are reviewed.

In many invertebrates and plants, the N-glycosylation profile is dominated by truncated paucimannosidic N-glycans, i.e., glycans consisting of a simple trimannosylchitobiosyl core often modified by core fucose residues. Even though they lack antennal N-acetylglucosamine residues, the biosynthesis of these glycans requires the sequential action of GlcNAc transferase I, Golgi mannosidase II and, finally, beta-N-acetylglucosaminidases. In Drosophila, the recently characterised enzyme encoded by the fused lobes (fdl) gene specifically removes the non-reducing N-acetylglucosamine residue from the alpha1,3-antenna of N-glycans. In the present study, we examined the products of five beta-N-acetylhexosaminidase genes from Caenorhabditis elegans (hex-1 to hex-5, corresponding to reading frames T14F9.3, C14C11.3, Y39A1C.4, Y51F10.5 and Y70D2A.2) in addition to three from Arabidopsis thaliana (AtHEX1, AtHEX2 and AtHEX3, corresponding to reading frames At1g65590, At3g55260 and At1g05590). Based on homology, the Caenorhabditis HEX-1 and all three Arabidopsis enzymes are members of the same sub-family as the aforementioned Drosophila fused lobes enzyme, but either act as chitotriosidases or non-specifically remove N-acetylglucosamine from both N-glycan antennae. The other four Caenorhabditis enzymes a members of a distinct sub-family; nevertheless, two of these enzymes displayed the same alpha1,3-antennal specificity as the fused lobes enzyme. Furthermore, a deletion of part of the Caenorhabditis hex-2 gene drastically reduces the native N-glycan-specific hexosaminidase activity in mutant worm extracts and results in a shift in the N-glycan profile, which is a demonstration of its in vivo enzymatic relevance. Based on these data, it is hypothesised that the genetic origin of paucimannosidic glycans in nematodes, plants and insects involves highly-divergent members of the same hexosaminidase gene family.

In recent years, the glycoconjugates of many parasitic nematodes have attracted interest due to their immunogenic and immunomodulatory nature. Previous studies with the porcine roundworm parasite Ascaris suum have focused on its glycosphingolipids, which were found, in part, to be modified by phosphorylcholine. Using mass spectrometry and western blotting, we have now analyzed the peptide N-glycosidase A-released N-glycans of adults of this species. The presence of hybrid bi- and triantennary N-glycans, some modified by core alpha1,6-fucose and peripheral phosphorylcholine, was demonstrated by LC/electrospray ionization (ESI)-Q-TOF-MS/MS, as was the presence of paucimannosidic N-glycans, some of which carry core alpha1,3-fucose, and oligomannosidic oligosaccharides. Western blotting verified the presence of protein-bound phosphorylcholine and core alpha1,3-fucose, whereas glycosyltransferase assays showed the presence of core alpha1,6-fucosyltransferase and Lewis-type alpha1,3-fucosyltransferase activities. Although, the unusual tri- and tetrafucosylated glycans found in the model nematode Caenorhabditis elegans were not found, the vast majority of the N-glycans found in A. suum represent a subset of those found in C. elegans; thus, our data demonstrate that the latter is an interesting glycobiological model for parasitic nematodes.

The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) bind a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analysed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognise a range of glycoproteins and neoglycoconjugates containing beta1,2-xylose and core alpha1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients' sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients' IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1 using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein as compared to the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Due to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein, therefore, explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically-relevant allergen.