Publisher Correction: Imaging single glycans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Imaging single glycans.

Imaging of biomolecules guides our understanding of their diverse structures and functions1,2. Real-space imaging at sub-nanometre resolution using cryo-electron microscopy has provided key insights into proteins and their assemblies3,4. Direct molecular imaging of glycans-the predominant biopolymers on Earth, with a plethora of structural and biological functions5-has not been possible so far6. The inherent glycan complexity and backbone flexibility require single-molecule approaches for real-space imaging. At present, glycan characterization often relies on a combination of mass spectrometry and nuclear magnetic resonance imaging to provide insights into size, sequence, branching and connectivity, and therefore requires structure reconstruction from indirect information7-9. Here we show direct imaging of single glycan molecules that are isolated by mass-selective, soft-landing electrospray ion beam deposition and imaged by low-temperature scanning tunnelling microscopy10. The sub-nanometre resolution of the technique enables the visualization of glycan connectivity and discrimination between regioisomers. Direct glycan imaging is an important step towards a better understanding of the structure of carbohydrates.

Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides.

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of ß-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

A modular synthetic route to size-defined immunogenic Haemophilus influenzae b antigens is key to the identification of an octasaccharide lead vaccine candidate.

The first glycoconjugate vaccine using isolated glycans was licensed to protect children from Haemophilus influenzae serotype b (Hib) infections. Subsequently, the first semisynthetic glycoconjugate vaccine using a mixture of antigens derived by polymerization targeted the same pathogen. Still, a detailed understanding concerning the correlation between oligosaccharide chain length and the immune response towards the polyribosyl-ribitol-phosphate (PRP) capsular polysaccharide that surrounds Hib remains elusive. The design of semisynthetic and synthetic Hib vaccines critically depends on the identification of the minimally protective epitope. Here, we demonstrate that an octasaccharide antigen containing four repeating disaccharide units resembles PRP polysaccharide in terms of immunogenicity and recognition by anti-Hib antibodies. Key to this discovery was the development of a modular synthesis that enabled access to oligosaccharides up to decamers. Glycan arrays containing the synthetic oligosaccharides were used to analyze anti-PRP sera for antibodies. Conjugates of the synthetic antigens and the carrier protein CRM197, which is used in licensed vaccines, were employed in immunization studies in rabbits.

Pushing the limits of automated glycan assembly: synthesis of a 50mer polymannoside.

Automated glycan assembly (AGA) enables rapid access to oligosaccharides. The overall length of polymers created via automated solid phase synthesis depends on very high yields at every step to obtain full length products. The synthesis of long polymers serves as the ultimate test of the efficiency and reliability of synthetic processes. A series of Man-(1 → 6)-α-Man linked oligosaccharides up to a 50mer, the longest synthetic sequence yet assembled from monosaccharides, has been realized via a 102 step synthesis. We identified a suitable mannose building block and applied a capping step in the final five AGA cycles to minimize (n - 1) deletion sequences that are otherwise difficult to remove by HPLC.

Automated glycan assembly of branched ß-(1,3)-glucans to identify antibody epitopes.

ß-(1,3)-Glucans exhibit immunomodulatory and anti-tumor effects. Since the isolation of pure ß-(1,3)-glucan oligosaccharides from natural sources is complicated, especially when certain branching patterns are desired, chemical synthesis is frequently the only means of accessing these molecules. We report the iterative automated glycan assembly (AGA) of conjugation-ready linear and branched ß-(1,3)-glucan oligosaccharides.

A traceless photocleavable linker for the automated glycan assembly of carbohydrates with free reducing ends.

We report a traceless photocleavable linker for the automated glycan assembly of carbohydrates with free reducing ends. The reductive-labile functionality in the linker tolerates all commonly used reagents and protocols for automated glycan assembly, as demonstrated with the successful preparation of nine plant cell wall-related oligosaccharides, and is cleaved by hydrogenolysis.

Integrated on-chip mass spectrometry reaction monitoring in microfluidic devices containing porous polymer monolithic columns.

Chip-based microfluidics enable the seamless integration of different functions into single devices. Here, we present microfluidic chips containing porous polymer monolithic columns as a means to facilitate chemical transformations as well as both downstream chromatographic separation and mass spectrometric analysis. Rapid liquid phase lithography prototyping creates the multifunctional device economically.

Distinguishing N-acetylneuraminic acid linkage isomers on glycopeptides by ion mobility-mass spectrometry.

Differentiating the structure of isobaric glycopeptides represents a major challenge for mass spectrometry-based characterisation techniques. Here we show that the regiochemistry of the most common N-acetylneuraminic acid linkages of N-glycans can be identified in a site-specific manner from individual glycopeptides using ion mobility-mass spectrometry analysis of diagnostic fragment ions.

High-Throughput Synthesis of Diverse Compound Collections for Lead Discovery and Optimization.

Small-molecule intervention of protein function is one central dogma of drug discovery. The generation of small-molecule libraries fuels the discovery pipeline at many stages and thereby resembles a key aspect of this endeavor. High-throughput synthesis is a major source for compound libraries utilized in academia and industry, seeking new chemical modulators of pharmacological targets. Here, we discuss the crucial factors of library design strategies from the perspective of synthetic chemistry, giving a brief historic background and a summary of current approaches. Simple measures of success of a high-throughput synthesis such as quantity or diversity have long been discarded and replaced by more integrated measures. Case studies are presented and put into context to highlight the cross-connectivity of the various stages of the drug discovery process.

Synthesis of glycosylphosphatidylinositol (GPI)-anchor glycolipids bearing unsaturated lipids.

2-Naphthyl-methyl ethers as permanent protecting groups are readily removed under acidic conditions and are key to the synthesis of complex glycosylphosphatidylinositol anchors containing unsaturated lipids. The total synthesis of the GPI pseudo-disaccharide core found on the surface of the Trypanosoma cruzi parasite serves to illustrate the power of the strategy.

Identification of carbohydrate anomers using ion mobility-mass spectrometry.

Carbohydrates are ubiquitous biological polymers that are important in a broad range of biological processes. However, owing to their branched structures and the presence of stereogenic centres at each glycosidic linkage between monomers, carbohydrates are harder to characterize than are peptides and oligonucleotides. Methods such as nuclear magnetic resonance spectroscopy can be used to characterize glycosidic linkages, but this technique requires milligram amounts of material and cannot detect small amounts of coexisting isomers. Mass spectrometry, on the other hand, can provide information on carbohydrate composition and connectivity for even small amounts of sample, but it cannot be used to distinguish between stereoisomers. Here, we demonstrate that ion mobility-mass spectrometry--a method that separates molecules according to their mass, charge, size, and shape--can unambiguously identify carbohydrate linkage-isomers and stereoisomers. We analysed six synthetic carbohydrate isomers that differ in composition, connectivity, or configuration. Our data show that coexisting carbohydrate isomers can be identified, and relative concentrations of the minor isomer as low as 0.1 per cent can be detected. In addition, the analysis is rapid, and requires no derivatization and only small amounts of sample. These results indicate that ion mobility-mass spectrometry is an effective tool for the analysis of complex carbohydrates. This method could have an impact on the field of carbohydrate synthesis similar to that of the advent of high-performance liquid chromatography on the field of peptide assembly in the late 1970s.

Carbohydrate coating reduces adhesion of biofilm-forming Bacillus subtilis to gold surfaces.

The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion--the first step in colonization and biofilm formation--is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future.

A comparative structural study in monolayers of GPI fragments and their binary mixtures.

Glycosylphosphatidylinositols (GPIs), natural complex glycolipids essential for a range of biological functions, are poorly understood with regard to their interactions and arrangements in cellular membranes. To evaluate the role of the head group in the structure formation in 2D model membranes (monolayers formed at the soft air/liquid interface), we employed the highly surface sensitive grazing incidence X-ray diffraction technique to investigate three GPI-fragments bearing the same hydrophobic part but different head groups. Condensed monolayers of simple GPI fragments are defined only by ordered alkyl chains. The monolayers of more complex fragments are additionally characterized by highly ordered head groups. Due to the strong H-bond network formed by the head groups, GPI-fragment both segregates and induces order into a model membrane phospholipid (POPC) that mimics the liquid-disordered phase of cell membranes. Here, we show that the strong van der Waals interactions between hydrophobic chains overcome the head group interactions and dominate the structure formation in mixtures of GPI-fragment with lipids that form liquid-condensed phases. This behaviour can be linked to the GPIs affinity for the lipid rafts.

The C-type lectin-like domain containing proteins Clec-39 and Clec-49 are crucial for Caenorhabditis elegans immunity against Serratia marcescens infection.

Caenorhabditis elegans exhibits protective immunity against a variety of fungal and bacterial pathogens. Since C. elegans lacks an adaptive immune system, pathogen recognition is mediated entirely by innate immunity. To date, little is known about the involvement of pattern recognition receptors (PRRs) in pathogen sensing as part of the C. elegans immunity. C-type lectin-like domain (CTLD) containing proteins represent a superfamily of PRRs. A large number of genes encoding for CTLD proteins are present in the C. elegans genome, however the role of CTLD proteins in bacterial recognition and antibacterial immunity has not yet been determined. In this study, we investigated the function of selected C. elegans CTLD proteins during infection with the Gram-negative bacterium Serratia marcescens. Wild-type and CTLD gene-deficient C. elegans strains were compared in their susceptibility to S. marcescens infection. Interestingly, survival and egg laying were significantly reduced in strains deficient for clec-39 and clec-49 indicating a role for both CTLD proteins in C. elegans immune defense against bacteria as evidenced by using S. marcescens infection. Binding studies with recombinantly expressed Clec-39-Fc and Clec-49-Fc fusion proteins revealed that both CTLD proteins recognized live bacteria in a Ca(2+)-independent manner. This study provides insight into the role of CTLD proteins in C. elegans immunity and demonstrates their function during bacterial infection.

Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides.

Mass spectrometry has made possible the field of proteomics and become an invaluable tool for identifying and quantifying post-translational modifications of proteins from complex mixtures. Because PTMs are recognized as key factors of biological activity, reliable PTM characterization is essential to understanding the relationship between protein isoform and activity. Protein glycosylations, in particular, can be especially difficult to characterize due to the range of different oligosaccharide entities that may be attached at any particular site. In this month' Special Feature Dr Daniel Kolarich of the Dept. of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces and his collaborators point out that quantitative label-free glycoproteomics can yield information about glycoprotein microand macroheterogeniety if the tools are sufficiently accurate. To understand and characterize the performance capability of MS tools they synthesized a panel of peptides and their glycopeptide derivatives as references and used these to investigate the qualitative and quantitative results from various ionization techniques and mass analyzers.

Characterization of annexin A1 glycan binding reveals binding to highly sulfated glycans with preference for highly sulfated heparan sulfate and heparin.

Annexin A1 is a multifunctional, calcium-dependent phospholipid binding protein involved in a host of processes including inflammation, regulation of neuroendocrine signaling, apoptosis, and membrane trafficking. Binding of annexin A1 to glycans has been implicated in cell attachment and modulation of annexin A1 function. A detailed characterization of the glycan binding preferences of annexin A1 using carbohydrate microarrays and surface plasmon resonance served as a starting point to understand the role of glycan binding in annexin A1 function. Glycan array analysis identified annexin A1 binding to a series of sulfated oligosaccharides and revealed for the first time that annexin A1 binds to sulfated non-glycosaminoglycan carbohydrates. Using heparin/heparan sulfate microarrays, highly sulfated heparan sulfate/heparin were identified as preferred ligands of annexin A1. Binding of annexin A1 to heparin/heparan sulfate is calcium- but not magnesium-dependent. An in-depth structure-activity relationship of annexin A1-heparan sulfate interactions was established using chemically defined sugars. For the first time, a calcium-dependent heparin binding protein was characterized with such an approach. N-Sulfation and 2-O-sulfation were identified as particularly important for binding.

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains.

AIMS: Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium. METHODS AND RESULTS: The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide--anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate-protein conjugates containing the synthetic tetrasaccharide, an anthrose-rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested. CONCLUSIONS: Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.

Microreactors as tools in the hands of synthetic chemists.

Recent developments in the construction of microstructured reaction devices and their wide-ranging applications in many different areas of chemistry suggest that microreactors may significantly impact the way chemists conduct experiments. Miniaturizing reactions offers many advantages for the synthetic organic chemist: high-throughput scanning of reaction conditions, precise control of reaction variables, the use of small quantities of reagents, increased safety parameters, and ready scale-up of synthetic procedures. A wide range of single and multiphase reactions has been performed in microfluidic-based devices. Certainly, microreactors cannot be applied to all chemistries yet and microfluidic systems also have disadvantages. Limited reaction time ranges, high sensitivity to precipitating products, and analytical challenges have to be overcome. An overview of microfluidic devices available for chemical synthesis is provided and some specific examples, mainly from our laboratory, are discussed to illustrate the potential of microreactors.

Automated solid-phase synthesis of a branched Leishmania cap tetrasaccharide.

[reaction--see text] Described is the first automated solid-phase synthesis of a branched oligosaccharide by stepwise assembly from monosaccharides. Cap tetrasaccharide 1, found as part of the cell surface lipophosphoglycan (LPG) of the protozoan parasite Leishmania, was readily prepared using glycosyl phosphate and glycosyl trichloroacetimidate building blocks.