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The attachment of proteins to the cell membrane using a glycosylphosphatidylinositol (GPI) anchor is a ubiquitous process in eukaryotic cells. Deficiencies in the biosynthesis of GPIs and the concomitant production of GPI-anchored proteins lead to a series of rare and complicated disorders associated with inherited GPI deficiencies (IGDs) in humans. Currently, there is no treatment for patients suffering from IGDs. Here, we report the design, synthesis, and use of GPI fragments to rescue the biosynthesis of GPI-anchored proteins (GPI-APs) caused by mutation in genes involved in the assembly of GPI-glycolipids in cells. We demonstrated that the synthetic fragments GlcNAc-PI (1), Man-GlcN-PI (5), and GlcN-PI with two (3) and three lipid chains (4) rescue the deletion of the GPI biosynthesis in cells devoid of the PIGA, PIGL, and PIGW genes in vitro. The compounds allowed for concentration-dependent recovery of GPI biosynthesis and were highly active on the cytoplasmic face of the endoplasmic reticulum membrane. These synthetic molecules are leads for the development of treatments for IGDs and tools to study GPI-AP biosynthesis.

The correct distribution and trafficking of proteins are essential for all organisms. Eukaryotes evolved a sophisticated trafficking system which allows proteins to reach their destination within highly compartmentalized cells. One eukaryotic hallmark is the attachment of a glycosylphosphatidylinositol (GPI) anchor to C-terminal ω-peptides, which are used as a zip code to guide a subset of membrane-anchored proteins through the secretory pathway to the plasma membrane. In fungi, the final destination of many GPI-anchored proteins is their outermost compartment, the cell wall. Enzymes of the Dfg5 subfamily catalyze the essential transfer of GPI-anchored substrates from the plasma membrane to the cell wall and discriminate between plasma membrane-resident GPI-anchored proteins and those transferred to the cell wall (GPI-CWP). We solved the structure of Dfg5 from a filamentous fungus and used in crystallo glycan fragment screening to reassemble the GPI-core glycan in a U-shaped conformation within its binding pocket. The resulting model of the membrane-bound Dfg5•GPI-CWP complex is validated by molecular dynamics (MD) simulations and in vivo mutants in yeast. The latter show that impaired transfer of GPI-CWPs causes distorted cell-wall integrity as indicated by increased chitin levels. The structure of a Dfg5•β1,3-glycoside complex predicts transfer of GPI-CWP toward the nonreducing ends of acceptor glycans in the cell wall. In addition to our molecular model for Dfg5-mediated transglycosylation, we provide a rationale for how GPI-CWPs are specifically sorted toward the cell wall by using GPI-core glycan modifications.

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array‐based assay for the high‐throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl‐, fucosyl‐, and xylosyltransferases can transfer azido‐functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3‐dipolar cycloaddition reaction with an alkynyl‐modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research., Express and screen: A high‐throughput assay based on the application of azido‐functionalized sugar nucleotide donors on a synthetic glycan array for screening heterologously expressed plant glycosyltransferases was developed. The opportunity to express and screen large numbers of glycosyltransferases instead of rationally selected candidates will markedly accelerate the elucidation of plant cell wall biosynthetic pathways.

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell‐ and tissue‐specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one‐pot intein‐mediated ligation (OPL) to obtain GPI‐anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP119 were prepared. Glypiation did not alter the structure of eGFP and MSP119 proteins in solution, but it induced a strong pro‐inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections., All together now: An intein‐mediated one‐pot ligation enables the semisynthesis of glycosylphosphatidylinositol (GPI)‐anchored proteins. The process works efficiently with denaturated and folded proteins under diverse conditions. This method was applied to the synthesis of a natural GPI‐anchored protein from Plasmodium berghei that exhibits proinflammatory activity in vitro.

Glycosylphosphatidylinositols (GPIs) are complex glycolipids present on the surfaces of Plasmodium parasites that may act as toxins during the progression of malaria. GPIs can activate the immune system during infection and induce the formation of anti-GPI antibodies that neutralize their activity. Therefore, an antitoxic vaccine based on GPI glycoconjugates may prevent malaria pathogenesis. To evaluate the role of three key modifications on Plasmodium GPI glycan in the activity of these glycolipids, we synthesized and investigated six structurally distinct GPI fragments from Plasmodium falciparum. The synthetic glycans were conjugated to the CRM197 carrier protein and were tested for immunogenicity and efficacy as antimalarial vaccine candidates in an experimental cerebral malaria model using C57BL/6JRj mice. Protection may be dependent on both the antibody and the cellular immune response to GPIs, and the elicited immune response depends on the orientation of the glycan, the number of mannoses in the structure, and the presence of the phosphoethanolamine and inositol units. This study provides insights into the epitopes in GPIs and contributes to the development of GPI-based antitoxin vaccine candidates against cerebral malaria.

Glycosylphosphatidylinositols (GPIs) are membrane anchors of many eukaryotic cell surface proteins. Biosynthesis of GPIs is initiated at the cytosolic face of the endoplasmic reticulum (ER) and the second intermediate, glucosaminyl-phosphatidylinositol (GlcN-PI), is translocated across the membrane to the lumenal face for later biosynthetic steps and attachment to proteins. The mechanism of the lumenal translocation of GlcN-PI is unclear. We report that Cleft lip and palate transmembrane protein 1-like protein (CLPTM1L), an ER membrane protein of unknown function, is a lipid scramblase involved in GPI biosynthesis. Purified CLPTM1L scrambles GlcN-PI, PI, and several other phospholipids in vitro. Knockout of CLPTM1L gene in mammalian cultured cells partially decreased GPI-anchored proteins due to impaired usage of GlcN-PI, suggesting a major role of CLPTM1L in lumenal translocation of GlcN-PI.One-Sentence SummaryCLPTM1L translocates glucosaminyl-phosphatidylinositol across the membrane during glycosylphosphatidylinositol biosynthesis.

ChemPhysChem 22(8), 757 - 763 (2021). doi:10.1002/cphc.202100002, Glycosylphosphatidylinositols (GPIs) are complex glycolipids found in free form or anchoring proteins to the outer leaflet of the cell membrane in eukaryotes. GPIs have been associated with the formation of lipid rafts and protein sorting on membranes. The presence of a conserved glycan core with cell-specific modifications together with lipid remodelling during biosynthesis suggest that the properties of the glycolipids are being fine-tuned. We synthesized a series of GPI fragments and evaluated the interactions and arrangement of these glycolipids in monolayers as a 2-D membrane model. GIXD and IRRAS analyses showed the need of N-acetylglucosamine deacetylation for the formation of hydrogen bonds to obtain highly structured domains in the monolayers and an effect of the unsaturated lipids in formation and localization of the glycolipids within or between membrane microdomains. These results contribute to understand the role of these glycolipids and their modifications in the organization of membranes., Published by Wiley-VCH Verl., Weinheim

Carbohydrates are functional and structural biomolecules with structures ranging from monosaccharides to polysaccharides. They are naturally found as pure glycans or attached to lipids and proteins forming glycoconjugates. The biosynthesis of carbohydrates is not genetically controlled. The regulation takes place by the expression of enzymes that transfer and hydrolyze the glycan units, leading to glycocojugates having complex mixtures of glycan structures. Chemical synthesis emerged as the best strategy to obtain defined glycan and glycoconjugates and overcome the challenging purification processes. Here, we review the recent advances in the synthesis of oligosaccharides using manual and automated methods. The chapter covers the methods for the preparation of building blocks and control of stereoselectivity and regioselectivity during glycosylations. Finally, it also presents the strategies to obtain natural and non-natural glycoconjugates with lipids and proteins.

The natural abundance of coiled coil (CC) motifs in the cytoskeleton and the extracellular matrix suggests that CCs play a crucial role in the bidirectional mechanobiochemical signaling between cells and the matrix. Their functional importance and structural simplicity has allowed the development of numerous applications, such as protein-origami structures, drug delivery systems and biomaterials. With the goal of establishing CCs as nanomechanical building blocks, we investigated the importance of helix propensity and hydrophobic core packing on the mechanical stability of 4-heptad CC heterodimers. Using single-molecule force spectroscopy, we show that both parameters determine the force-induced dissociation in shear loading geometry; however, with different effects on the energy landscape. Decreasing the helix propensity lowers the transition barrier height, leading to a concomitant decrease in the distance to the transition state. In contrast, a less tightly packed hydrophobic core increases the distance to the transition state. We propose that this originates from a larger side chain dynamics, possible water intrusion at the interface as well as differences in solvation of the hydrophobic amino acids at the transition state. In conclusion, the different contributions of helix propensity and hydrophobic core packing need to be considered when tuning the mechanical properties of CCs for applications.

Trypanosoma brucei uses variant surface glycoproteins (VSGs) to evade the host immune system and ensure parasitic longevity in animals and humans. VSGs are attached to the cell membrane by complex glycosylphosphatidylinositol anchors (GPI). Distinguishing structural feature of VSG GPIs are multiple α- and β-galactosides attached to the conserved GPI core structure. T. brucei GPIs have been associated with macrophage activation and alleviation of parasitemia during infection, acting as disease onset delaying antigens. Literature reports that link structural modifications in the GPIs to changes in biological activity are contradictory. We have established a synthetic route to prepare structurally overlapping GPI derivatives bearing different T. brucei characteristic structural modifications. The GPI collection will be used to assess the effect of galactosylation and phosphorylation on T. brucei GPI immunomodulatory activity, and to perform an epitope mapping of this complex glycolipid as potential diagnostic marker for Trypanosomiasis. A strategy for the synthesis of a complete α-tetragalactoside using the 2-naphthylmethyl protecting group and for subsequent attachment of GPI fragments to peptides is presented.

Glycosylphosphatidylinositol anchored proteins (GPI-APs) are natural conjugates in the plasma membrane of eukaryotic cells that result from the attachment of a glycolipid to the C-terminus of many proteins. GPI-APs play a crucial role in cell signaling and adhesion and have implications in health and diseases. GPI-APs and GPIs without protein (free GPIs) are found in abundance on the surface of the protozoan parasite Toxoplasma gondii. The detection of anti-GPI IgG and IgM antibodies allows differentiation between toxoplasmosis patients and healthy individuals using serological assays. However, these methods are limited by their poor efficiency, cross-reactivity and need for sophisticated laboratory equipment and qualified personnel. Here, we established a label-free electrochemical glycobiosensor for the detection of anti-GPI IgG and IgM antibodies in serum from toxoplasmosis seropositive patients. This biosensor uses a synthetic GPI phosphoglycan bioreceptor immobilized on screen-printed gold electrodes through a linear alkane thiol phosphodiester. The antigen-antibody interaction was detected and quantified by electrochemical impedance spectroscopy (EIS). The resultant device showed a linear dynamic range of anti-GPI antibodies in serum ranging from 1.0 to 10.0 IU mL-1, with a limit of detection of 0.31 IU mL-1. This method also holds great potential for the detection of IgG antibodies related to other multiple medical conditions characterized by overexpression of antibodies.

Carbohydrates are functional and structural biomolecules with structures ranging from monosaccharides to polysaccharides. They are naturally found as pure glycans or attached to lipids and proteins forming glycoconjugates. The biosynthesis of carbohydrates is not genetically controlled. The regulation takes place by the expression of enzymes that transfer and hydrolyze the glycan units, leading to glycocojugates having complex mixtures of glycan structures. Chemical synthesis emerged as the best strategy to obtain defined glycan and glycoconjugates and overcome the challenging purification processes. Here, we review the recent advances in the synthesis of oligosaccharides using manual and automated methods. The chapter covers the methods for the preparation of building blocks and control of stereoselectivity and regioselectivity during glycosylations. Finally, it also presents the strategies to obtain natural and non-natural glycoconjugates with lipids and proteins.

Cerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αβ-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αβ-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection.

C-type lectin receptors (CLRs) play a pivotal role in pathogen defense and immune homeostasis. Langerin, a CLR predominantly expressed on Langerhans cells, represents a potential target receptor for the development of anti-infectives or immunomodulatory therapies. As mammalian carbohydrate binding sites typically display high solvent exposure and hydrophilicity, the recognition of natural monosaccharide ligands is characterized by low affinities. Consequently, glycomimetic ligand design poses challenges that extend to the development of suitable assays. Here, we report the first application of (19)F R2-filtered NMR to address these challenges for a CLR, i.e., Langerin. The homogeneous, monovalent assay was essential to evaluating the in silico design of 2-deoxy-2-carboxamido-α-mannoside analogs and enabled the implementation of a fragment screening against the carbohydrate binding site. With the identification of both potent monosaccharide analogs and fragment hits, this study represents an important advancement toward the design of glycomimetic Langerin ligands and highlights the importance of assay development for other CLRs.

Toxoplasmosis, while often an asymptomatic parasitic disease in healthy individuals, can cause severe complications in immunocompromised persons and during pregnancy. The most common method to diagnose Toxoplasma gondii infections is the serological determination of antibodies directed against parasite protein antigens. Here we report the use of a bead-based multiplex assay containing a synthetic phosphoglycan portion of the Toxoplasma gondii glycosylphosphatidylinositol (GPI1) for the detection of GPI1-specific antibodies in human sera. The glycan was conjugated to beads at the lipid site to retain its natural orientation and its immunogenic groups. We compared the response against GPI1 with that against the protein antigen SAG1, a common component of commercial serological assays, via the detection of parasite-specific human IgG and IgM antibodies, respectively. The GPI1-based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI1 test shows 97% specificity and 98% sensitivity for the assay. GPI1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.

© 2019. Published under the PNAS license., Cerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αβ-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αβ-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection., This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/SAU-OSM/099ALTF 357-2009 and BPD-81953-2011, ALTF 960-2009, and PD/BD/114099/2015724/2008 to A.P.) and European Research Council (CoG_646701 to B.S.-S.). We also acknowledge UID/ BIM/50005/2019, a project funded by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado. A.P. was supported by a Ciência 2008 position of the Portuguese Ministry of Science and Technology, supported by a FCT fellowship (SFRH/BPD/110380/2015), and currently holds a research position supported by FCT (under decree-law no. 57/2016 of July 19th, as amended by law no. 57/2017). J.C.R., R.N., V.Z.-L., L.M.-S., and S.M. were supported by individual fellowships from FCT and European Molecular Biology Organization Long-Term Fellowships (IF/00013/2014, SFRH/BI/51054/2010, SFRH/BPD/81953/2011, , respectively). L.M.-S. was also supported by the European Community’s Sev- enth Framework Programme (FP7/2007-2013) under Grant Agreement 242095 (EVIMalaR). Currently V.Z.-L. holds a research position supported by FCT (under decree-law no. 57/2016 of July 19th, as amended by law no. 57/2017), and J.C.R. holds a FCT investigator position (IF/00013/2014).

The European Chemical Biology Society (ECBS) and the International Chemical Biology Society (ICBS) recently organized a joint meeting in Berlin. This meeting had more than 250 participants. Four keynote lectures were given by Timothy Mitchison, David Tirrell, Carolyn Bertozzi and Jason Chin; in addition there were 13 invited speakers, 20 selected oral talks and 30 talks selected from 90 posters. The meeting was divided into six topics: chemoproteomics, epigenetics, conjugates for target delivering, anti-infectives, molecular imaging and probing the structure, and function of post-translational modifications. The highlights of the meeting are presented in this report.

Glycan identification and characterisation is essential to correlate glycoconjugate structure to biological function. The structural assignment of carbohydrates is often based on MS composition analyses and knowledge on well-studied glycosylation pathways. Nevertheless, many monosaccharide building blocks are indistinguishable by mass alone and detailed linkage information is also not easily obtained by MS/MS analyses, in particular when organisms are studied where the glycosylation pathways are less well defined. Here, we present a novel, simple and sensitive method using Reversed Phase (RP) Liquid Chromatography Electrospray ionisation tandem mass spectrometry (LC ESI MS/MS) for unambiguous identification and linkage determination of monosaccharides including N-acetylneuraminic acids. Sequential permethylation and reductive amination steps are employed prior and after acid hydrolysis to enable separation and differentiation of the various monosaccharides and their respective linkage positions. The well-established, monosaccharide specific methylation patterns allowed for the identification of the various derivatised monosaccharide alditols based on their retention time and tandem mass spectrometry fingerprint. Absolute quantitation can also be accomplished by including a set of internal standards, thus simultaneously providing qualitative and quantitative information on the monosaccharide residues present.

Geschatzte zwei Milliarden Menschen sind weltweit mit dem apikomplexen Parasiten Toxoplasma gondii infiziert, der die Ursache fur eine Reihe gesundheitlicher Probleme ist. So kann eine Primarinfektion wahrend der Schwangerschaft zum Tod des Fotus oder zu einer geistigen Behinderung des Kindes fuhren. Da die Medikamente gegen eine Infektion mit T. gondii potenziell schadlich fur das ungeborene Kind sind, ist eine verlassliche Diagnose akuter Infektionen von schwangeren Frauen unentbehrlich. Bessere, schnellere, zuverlassigere und gunstigere Diagnostika, die auf akkuraten serologischen Tests basieren und definierte Antigene verwenden, sind deshalb von grosem Interesse. Synthetische pathogenspezifische Glycosylphosphatidylinositol(GPI)-Glycan-Antigene sind solche diagnostischen Biomarker, die eine akkurate Unterscheidung verschiedener Toxoplasmose-Krankheitsstadien in menschlichen Seren ermoglichen.

Glycosylphosphatidylinositols (GPIs) are often attributed with the ability to associate with the organized membrane microdomains. GPI fragment 1 forms a highly ordered subgel-phase structure characterized by ordering of both headgroups and alkyl chains in thin layers. While investigating the driving forces behind the formation of these ordered monolayers, we have studied polymorphism of 1 under different conditions employing surface-sensitive X-ray diffraction methods. Three distinct polymorphs of 1 (I, II, and III) were identified and characterized by grazing incidence X-ray diffraction. Polymorphs II (a condensed monolayer structure) and III (highly ordered subgel phase) coexist on an 8 M urea solution subphase allowing for a detailed thermodynamic and kinetic analysis of the processes leading to the formation of these polymorphs. They are enantiotropic and can be directly interconverted by changes in temperature or lateral surface pressure. As a consequence, polymorph III nuclei of critical size (or larger) could be formed by density fluctuations in a multicomponent system, and they could continue to exist for a period of time even under conditions that would normally not allow for the nucleation of polymorph III. The processes described here could also lead to the formation of patches of highly ordered structures in a disordered environment of a cell membrane suggesting that GPIs may play a role in the formation of such domains.

Spezielle Glykolipide verankern eukaryotische Proteine an der auseren Zellmembran. Bislang ist jedoch wenig uber die Funktionen dieser Glykosylphosphatidylinositole bekannt. Erkenntnisse uber ihre biologischen Eigenschaften lassen sich mit synthetischen Molekulen gewinnen.

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics.

The current surge in defining glycobiomarkers by applying lectins rekindles interest in definition of the sugar-binding sites of lectins at high resolution. Natural complex-type N-glycans can present more than one potential binding motif, posing the question of the actual mode of interaction when interpreting, for example, lectin array data. By strategically combining N-glycan preparation with saturation-transfer difference NMR and modeling, we illustrate that epitope recognition depends on the structural context of both the sugar and the lectin (here, wheat germ agglutinin and a single hevein domain) and cannot always be predicted from simplified model systems studied in the solid state. We also monitor branch-end substitutions by this strategy and describe a three-dimensional structure that accounts for the accommodation of the α2,6-sialylated terminus of a biantennary N-glycan by viscumin. In addition, we provide a structural explanation for the role of terminal α2,6-sialylation in precluding the interaction of natural N-glycans with lectin from Maackia amurensis . The approach described is thus capable of pinpointing lectin-binding motifs in natural N-glycans and providing detailed structural explanations for lectin selectivity.

Glycosylphosphatidylinositol (GPI) glycolipids anchor a large number of proteins in the cell membrane of eukaryotic cells. Their conserved pseudopentasaccharide core carries additional phosphoethanolamine, saccharide and lipid substituents. These structural variations are characteristic for a species or a tissue but their functional significance remains largely unknown. Studies that would link a specific function to a structurally unique GPI rely on availability of homogeneous samples of these glycolipids. To address this need we have developed a general synthetic route to GPI glycolipids. Our convergent synthesis starts from common building blocks and relies on a fully orthogonal set of protecting groups that enables the regioselective introduction of phosphodiesters and efficient assembly of the GPI glycans. Here, we report on the development of this synthetic strategy, evaluation of the set of protecting groups with respect to phosphorylation methods, evaluation of the assembly plan for the GPI glycan, optimization of the glycosylation reactions, and the application of this strategy to the total syntheses of four structurally diverse branched GPI glycolipids.

In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1308-6) contains supplementary material, which is available to authorized users.

Seven in one blow: The efficient formation of mixed disulfides on the thiol-rich fusion protein A followed by subsequent intein cleavage gave the fragment B with all seven cysteines protected against oxidation. The native chemical ligation of B with synthetic glycopeptide thioesters provides glycoproteins.

Aktives RNase-Glycoprotein aus drei Segmenten: Das Glycoprotein-Enzym Ribonuclease C mit einem komplexen nonasaccharidischen N-Glycan wurde durch sequenzielle native chemische Ligation synthetisiert. Durch optimierte Ligations- und Isolierungsbedingungen konnte das aus 124 Aminosauren bestehende Enzym effizient aufgebaut und zuruckgefaltet werden.

Sieben auf einen Streich: Durch die effiziente Bildung von gemischten Disulfiden an dem thiolreichen Fusionsprotein A konnte nach Inteinabspaltung das Cys-Fragment B mit sieben oxidationsgeschutzten Cysteinen erhalten werden. Die native chemische Ligation von B mit synthetischen Glycopeptidthioestern ergibt Glycoproteine.

Vaccination against the ubiquitous parasite Toxoplasma gondii would provide the most efficient prevention against toxoplasmosis-related congenital, brain, and eye diseases in humans. We investigated the immune response elicited by pathogen-specific glycosylphosphatidylinositol (GPI) glycoconjugates using carbohydrate microarrays in a BALB/c mouse model. We further examined the protective properties of the glycoconjugates in a lethal challenge model using the virulent T. gondii RH strain. Upon immunization, mice raised antibodies that bind to the respective GPIs on carbohydrate microarrays, but were mainly directed against an unspecific GPI epitope including the linker. The observed immune response, though robust, was unable to provide protection in mice when challenged with a lethal dose of viable tachyzoites. We demonstrate that anti-GPI antibodies raised against the here described semi-synthetic glycoconjugates do not confer protective immunity against T. gondii in BALB/c mice.

Background Export from the ER is an essential process driven by the COPII coat, which forms vesicles at ER exit sites (ERESs) to transport mature secretory proteins to the Golgi. Although the basic mechanism of COPII assembly is known, how COPII machinery is regulated to meet varying cellular secretory demands is unclear. Results Here, we report a specialized COPII system that is actively recruited by luminal cargo maturation. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are luminal secretory proteins anchored to the membrane by the glycolipid GPI. After protein attachment in the ER lumen, lipid and glycan parts of the GPI anchor are remodeled. In yeast, GPI-lipid remodeling concentrates GPI-APs into specific ERESs. We found that GPI-glycan remodeling induces subsequent recruitment of the specialized ER export machinery that enables vesicle formation from these specific ERESs. First, the transmembrane cargo receptor p24 complex binds GPI-APs as a lectin by recognizing the remodeled GPI-glycan. Binding of remodeled cargo induces the p24 complex to recruit the COPII subtype Lst1p, specifically required for GPI-AP ER export. Conclusions Our results show that COPII coat recruitment by cargo receptors is not constitutive but instead is actively regulated by binding of mature ligands. Therefore, we reveal a novel functional link between luminal cargo maturation and COPII vesicle budding, providing a mechanism to adjust specialized COPII vesicle production to the amount and quality of their luminal cargos that are ready for ER exit. This helps to understand how the ER export machinery adapts to different needs for luminal cargo secretion., This work was supported by grants from the Spanish Ministry of Science and Innovation BFU2008-04119 (to M.M.), BFU2011-24513 (to M.M.), BFU2009-07290 (to V.G.), and BFU2010-21339 (to R.E.W.); Junta de Andalucia P09-CVI-4503 (to M.M.) and P11-CTS-7962 (to R.E.W.); Swiss National Science Foundation and the NCCR Chemical Biology (to H.R.); the Max-Planck Society (to P.H.S.); the RIKEN-Max-Planck Joint Center for Chemical Biology (to D.V.S.); by JSPS KAKENHI grant number 21580094 (to K.F.); University of Seville fellowships (to A.A.-R., J.M.-L., and A.M.P.-L.); and Ramon y Cajal program (to V.G.). We are grateful to Servicios de Biología y Microscopía (CITIUS, Universidad de Sevilla).

Around 2 billion people worldwide are infected with the apicomplexan parasite Toxoplasma gondii which induces a variety of medical conditions. For example, primary infection during pregnancy can result in fetal death or mental retardation of the child. Diagnosis of acute infections in pregnant women is challenging but crucially important as the drugs used to treat T. gondii infections are potentially harmful to the unborn child. Better, faster, more reliable, and cheaper means of diagnosis by using defined antigens for accurate serological tests are highly desirable. Synthetic pathogen-specific glycosylphosphatidylinositol (GPI) glycan antigens are diagnostic markers and have been used to distinguish between toxoplasmosis disease states using human sera.

Building blocks: a new, general synthetic strategy, which allows the construction of branched glycosylphosphatidylinositols (GPIs), enables the synthesis of parasitic glycolipid 1 from Toxoplasma gondii. In addition, the structure is further confirmed by recognition of monoclonal antibodies.

Vaccinations provide an efficient and cost-effective way to combat devastating human diseases. Besides pathogenic protein markers, cell surface carbohydrates from biological sources are widely used as vaccines. Recently, synthetic immunogenic carbohydrate-protein conjugates have been advanced to vaccine candidates. Progress in the chemical synthesis of oligosaccharides and conjugation methods stimulated the development of novel carbohydrate-based vaccine candidates.

Active RNase glycoprotein from three pieces: The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N-glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.