Structural base for the transfer of GPI-anchored glycoproteins into fungal cell walls.

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•ß1,3-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.

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

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 ß-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.

Zwitterionic Character and Lipid Composition Determine the Behaviour of Glycosylphosphatidylinositol Fragments in Monolayers.

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.

Semisynthesis of Functional Glycosylphosphatidylinositol-Anchored Proteins.

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.

A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases.

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.

Detection of Anti-Toxoplasma gondii Antibodies in Human Sera Using Synthetic Glycosylphosphatidylinositol Glycans on a Bead-Based Multiplex Assay.

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.

Structural determinants of coiled coil mechanics.

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.

Synthesis of Galactosylated Glycosylphosphatidylinositol Derivatives from Trypanosoma brucei.

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 ß-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.

ECBS & ICBS 2015 Joint Meeting: Bringing Chemistry to Life.

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.

Investigation of the protective properties of glycosylphosphatidylinositol-based vaccine candidates in a Toxoplasma gondii mouse challenge model.

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.

Defining the Interaction of Human Soluble Lectin ZG16p and Mycobacterial Phosphatidylinositol Mannosides.

ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analysis of the interaction of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs) by glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation transfer difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interact with ZG16p by using the mannose residues. The binding site of PIM was identified by chemical-shift perturbation experiments with uniformly (15)N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan; this will help to elucidate the physiological role of ZG16p.

Versatility of a glycosylphosphatidylinositol fragment in forming highly ordered polymorphs.

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.

Diagnosis of toxoplasmosis using a synthetic glycosylphosphatidylinositol glycan.

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.

Molecular recognition of complex-type biantennary N-glycans by protein receptors: a three-dimensional view on epitope selection by NMR.

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.

Synthetic phosphoethanolamine-modified oligosaccharides reveal the importance of glycan length and substitution in biofilm-inspired assemblies.

Bacterial biofilm matrices are nanocomposites of proteins and polysaccharides with remarkable mechanical properties. Efforts understanding and tuning the protein component have been extensive, whereas the polysaccharide part remained mostly overlooked. The discovery of phosphoethanolamine (pEtN) modified cellulose in E. coli biofilms revealed that polysaccharide functionalization alters the biofilm properties. To date, the pattern of pEtN cellulose and its mode of interactions with proteins remains elusive. Herein, we report a model system based on synthetic epitomes to explore the role of pEtN in biofilm-inspired assemblies. Nine pEtN-modified oligosaccharides were synthesized with full control over the length, degree and pattern of pEtN substitution. The oligomers were co-assembled with a representative peptide, triggering the formation of fibers in a length dependent manner. We discovered that the pEtN pattern modulates the adhesion of biofilm-inspired matrices, while the peptide component controls its stiffness. Unnatural oligosaccharides tune or disrupt the assembly morphology, revealing interesting targets for polysaccharide engineering to develop tunable bio-inspired materials.

A general method for synthesis of GPI anchors illustrated by the total synthesis of the low-molecular-weight antigen from Toxoplasma gondii.

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.

Advances in the Chemical Synthesis of Carbohydrates and Glycoconjugates.

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.

Rescue of Glycosylphosphatidylinositol-Anchored Protein Biosynthesis Using Synthetic Glycosylphosphatidylinositol Oligosaccharides.

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.

Phosphoglycan-sensitized platform for specific detection of anti-glycan IgG and IgM antibodies in serum.

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.

Immunological Evaluation of Synthetic Glycosylphosphatidylinositol Glycoconjugates as Vaccine Candidates against Malaria.

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.