Rudolf Geyer

GlycoWorkbench: A Tool for the Computer-Assisted Annotation of Mass Spectra of Glycans†

Mass spectrometry is the main analytical technique currently used to address the challenges of glycomics as it offers unrivalled levels of sensitivity and the ability to handle complex mixtures of different glycan variations. Determination of glycan structures from analysis of MS data is a major bottleneck in high-throughput glycomics projects, and robust solutions to this problem are of critical importance. However, all the approaches currently available have inherent restrictions to the type of glycans they can identify, and none of them have proved to be a definitive tool for glycomics. GlycoWorkbench is a software tool developed by the EUROCarbDB initiative to assist the manual interpretation of MS data. The main task of GlycoWorkbench is to evaluate a set of structures proposed by the user by matching the corresponding theoretical list of fragment masses against the list of peaks derived from the spectrum. The tool provides an easy to use graphical interface, a comprehensive and increasing set of structural constituents, an exhaustive collection of fragmentation types, and a broad list of annotation options. The aim of GlycoWorkbench is to offer complete support for the routine interpretation of MS data. The software is available for download from: http://www.eurocarbdb.org/applications/ms-tools.

The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector

The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.

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

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

Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain

Among the large set of cell surface glycan structures, the carbohydrate polymer polysialic acid (polySia) plays an important role in vertebrate brain development and synaptic plasticity. The main carrier of polySia in the nervous system is the neural cell adhesion molecule NCAM. As polySia with chain lengths of more than 40 sialic acid residues was still observed in brain of newborn Ncam−/− mice, we performed a glycoproteomics approach to identify the underlying protein scaffolds. Affinity purification of polysialylated molecules from Ncam−/− brain followed by peptide mass fingerprinting led to the identification of the synaptic cell adhesion molecule SynCAM 1 as a so far unknown polySia carrier. SynCAM 1 belongs to the Ig superfamily and is a powerful inducer of synapse formation. Importantly, the appearance of polysialylated SynCAM 1 was not restricted to the Ncam−/− background but was found to the same extent in perinatal brain of WT mice. PolySia was located on N-glycans of the first Ig domain, which is known to be involved in homo- and heterophilic SynCAM 1 interactions. Both polysialyltransferases, ST8SiaII and ST8SiaIV, were able to polysialylate SynCAM 1 in vitro, and polysialylation of SynCAM 1 completely abolished homophilic binding. Analysis of serial sections of perinatal Ncam−/− brain revealed that polySia-SynCAM 1 is expressed exclusively by NG2 cells, a multifunctional glia population that can receive glutamatergic input via unique neuron-NG2 cell synapses. Our findings sug-gest that polySia may act as a dynamic modulator of SynCAM 1 functions during integration of NG2 cells into neural networks.

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

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

Saccharide linkage analysis using methylation and other techniques.

Publisher Summary This chapter focuses primarily on a microscale version of methylation analysis that can be applied to (glycoprotein) glycans available in picomolar amounts. Examples for the allocation of outer substituents by a combination of methylation analysis and preparative exoglycosidase digestion, as well as for the identification of high mannose-type oligosaccharide isomers by use of acetolysis, are presented. Methylation of carbohydrates by methyltrifluoromethane sulfonate in trimethyl phosphate is also described. Acetylation of partially methylated alditols is achieved by addition of acetic anhydride. In the case of submicrogram quantities, the presence of pyridine in the reaction mixture turns out to be crucial for quantitative yields. Separation of partially methylated alditol acetates (PMAAs) and partially methylated and acetylated methylglycosides by gas chromatography (GC) has been achieved by a variety of glass or fused silica capillary columns. In comparison to wall-coated glass columns, however, fused silica-bonded phase columns can be handled more easily; they give a better resolution, lead to more reproducible retention times, and have a longer life time.

Methylation analysis of complex carbohydrates in small amounts: capillary gas chromatography―mass fragmentography of methylalditol acetates obtained from N-glycosidically linked glycoprotein oligosaccharides

A version of the methylation analysis of complex carbohydrates by gas chromatography-mass spectrometry of the methylalditol acetates (H. Björndal, C. G. Hellerquist, B. Lindberg, and S. Svensson (1970) Angew. Chem. 82, 643-674) is described. With this version 100- to 500-pmol samples of N-glycosidically linked glycoprotein oligosaccharides may be analyzed. The method is based on the use of capillary columns which allow the separation of all partially methylated alditol acetates potentially obtained from this group of oligosaccharides and on their selective and sensitive detection by mass fragmentography after chemical ionization with ammonia.

Impact of the Polysialyltransferases ST8SiaII and ST8SiaIV on Polysialic Acid Synthesis during Postnatal Mouse Brain Development

Polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule (NCAM), is the key regulator of NCAM-mediated functions and crucial for normal brain development, postnatal growth, and survival. Two polysialyltransferases, ST8SiaII and ST8SiaIV, mediate polySia biosynthesis. To dissect the impact of each enzyme during postnatal brain development, we monitored the developmental changes in NCAM polysialylation in wild-type, ST8SiaII-, and ST8SiaIV-deficient mice using whole brain lysates obtained at 10 time points from postnatal days 1 to 21 and from adult mice. In wild-type and ST8SiaIV-null brain, polySia biosynthesis kept pace with the rapid increase in brain weight until day 9, and nearly all NCAM was polysialylated. Thereafter, polySia dropped by ∼70% within 1 week, accompanied by the first occurrence of polySia-free NCAM-140 and NCAM-180. In ST8SiaII-null brain, polySia declined immediately after birth, leading to 60% less polySia at day 9 combined with the untimely appearance of polySia-free NCAM. Polysialyltransferase deficiency did not alter NCAM expression level or isoform pattern. In all three genotypes, NCAM-140 and NCAM-180 were expressed at constant levels from days 1 to 21 and provided the major polySia acceptors. By contrast, NCAM-120 first appeared at day 5, followed by a strong up-regulation inverse to the decrease in polySia. Together, we provide a comprehensive quantitative analysis of the developmental changes in polySia level, NCAM polysialylation status, and polysialyltransferase transcript levels and show that the predominant role of ST8SiaII during postnatal brain development is restricted to the first 15 days.

Structural elucidation and monokine-inducing activity of two biologically active zwitterionic glycosphingolipids derived from the porcine parasitic nematode Ascaris suum.

 The isolated neutral glycosphingolipid fraction from the pig parasitic nematode,Ascaris suum, was fractionated by silica gel chromatography to yield a neutral and a zwitterionic glycosphingolipid fraction, the latter of which mainly contained two zwitterionic glycosphingolipids termed components A and C. Preliminary chemical characterization with hydrofluoric acid treatment and immunochemical characterization with a phosphocholine-specific monoclonal antibody indicated that both components contained phosphodiester substitutions: phosphocholine for component A, and phosphocholine and phosphoethanolamine for component C. Both components were biologically active in inducing human peripheral blood mononuclear cells to release the inflammatory monokines tumor necrosis factor α, interleukin 1, and interleukin 6. Component A was the more bioactive molecule, and its biological activity was abolished on removal of the phosphocholine substituent by hydrofluoric acid. The glycosphingolipid components were structurally analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary ion mass spectrometry, methylation analysis, 1H NMR spectroscopy, exoglycosidase cleavage, and ceramide analysis. Their chemical structures were elucidated to be (see Structure I below),           phosphocholine 6 − ‖ Component A                   Gal ( α 1 – 3 ) GalNAc ( β 1 – 4 ) GlcNAc ( β 1 – 3 ) Man ( β 1 – 4 ) Glc ( β 1 – 1 )   ceramide           phosphocholine 6   _ ‖                 ‖ _   6 phosphoethanolamine   Component C                   Gal ( α 1 – 3 ) GalNAc ( β 1 – 4 ) GlcNAc ( β 1 – 3 ) Man ( β 1 – 4 ) Glc ( β 1 – 1 )   ceramide Structure I  The carbohydrate moiety oligosaccharide core was characterized as belonging to the arthro series of protostomial glycosphingolipids. The ceramide moiety was distinguished by (R)-2-hydroxytetracosanoic acid as the dominant fatty acid species and by the C17 iso-branched sphingosine and sphinganine bases, 15-methylhexadecasphing-4-enine and 15-methylhexadecasphinganine, respectively.

EUROCarbDB: An open-access platform for glycoinformatics.

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.