Alice Tamburrini, Silvia Achilli, Francesca Vasile, Sara Sattin, Corinne Vivès, Cinzia Colombo,
Franck Fieschi, Anna Bernardi
The synthesis and conformational analysis of pseudo-thio-1,2-dimannoside are described. This molecule
mimics mannobioside (Mana(1,2)Man) and is an analog of pseudo-1,2-dimannoside, with expected
increased stability to enzymatic hydrolysis. A short and efficient synthesis was developed based on an
epoxide ring-opening reaction by a mannosyl thiolate, generated in situ from the corresponding thioacetate.
NMR-NOESY studies supported by MM3⁄ calculations showed that the pseudo-thio-1,2-dimannoside
shares the conformational behavior of the pseudo-1,2-dimannoside and is a structural mimic of
the natural disaccharide. Its affinity for DC-SIGN was measured by SPR and found to be comparable to
the corresponding O-linked analog, offering good opportunities for further developments.
Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH–p Stacking Interactions by Different Fluorination Patterns
Luca Unione, María Alcalá, Begoña Echeverria, Sonia Serna, Ana Ardá, Antonio Franconetti, F. Javier Cañada, Tammo Diercks, Niels-Christian Reichardt, Jesús Jiménez-Barbero
We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar–protein interactions by simple 1H and/or 19F NMR spectroscopy methods. In this process, we have chosen the binding of N,N’-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2FCONHgroup and the natural molecule (with the CH3CONH- fragment). In contrast, the molecule with the CF3CONH- isoster displayed the weakest intermolecular interaction (one order
of magnitude weaker). Because sugar–aromatic CH–p interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.
João T. Monteiro, Bernd Lepenies
Recognition of viral glycans by pattern recognition receptors (PRRs) in innate immunity contributes to antiviral immune responses. C-type lectin receptors (CLRs) are PRRs capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, antigen processing and presentation, and subsequent T cell activation. The ability of CLRs to elicit and shape adaptive immunity plays a critical role in the inhibition of viral spread within the host. However, certain viruses exploit CLRs for viral entry into hosT cells to avoid immune recognition. To block CLR interactions with viral glycoproteins, antiviral strategies may involve the use of multivalent glycan carrier systems. In this review, we describe the role of CLRs in antiviral immunity and we highlight their dual function in viral clearance and exploitation by viral pathogens.
NMR and Molecular Recognition of N-Glycans: Remote Modifications of the Saccharide Chain Modulate Binding Features
Ana Gimeno, Niels-Christian Reichardt, F. Javier Cañada, Lukas Perkams, Carlo Unverzagt, Jesús Jiménez-Barbero, Ana Ardá
Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate–protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore their importance for the development of biomedical applications.
Specific anti-glycan antibodies are sustained during and after parasite clearance in Schistosoma japonicum-infected rhesus macaques
Y. Y. Michelle Yang, Xiao Hong Li, Katarzyna Brzezicka, Niels-Christian Reichardt, R. Alan Wilson, Angela van Diepen, Cornelis H. Hokke
Schistosomes express many glycan antigens to which antibodies are raised by the infected host. These glycans may therefore form potential vaccine targets. Unlike humans where the disease persists chronically if not treated, schistosome-infected rhesus macaques are able to elicit a self-cure process naturally. To find out if anti-glycan responses could contribute to the natural clearance process, we followed the dynamics of anti-glycan serum antibodies in Schistosoma-infected macaques in a longitudinal study starting from the onset of infection until 22 weeks post-infection, when the macaques had eliminated most of the parasites. We found that sera of macaques taken after 22 weeks of infection contained high IgG titres towards specific schistosome glycan epitopes highly abundant on schistosome larvae. Moreover, infected macaque serum at week 22 was able to kill schistosomula in vitro. Our results suggest that anti-glycan antibodies play an important role in the self-cure process and the acquired resistance to re-infection in Schistosoma infected macaques.
A. Berzi, S. Ordanini, B. Joosten, D. Trabattoni, A. Cambi, A. Bernardi and M. Clerici
DC-SIGN, a C-type lectin mainly expressed by DCs, mediates antigen uptake and can induce specific immune responses, depending on the ligand involved. Owing to these properties, DC-SIGN is an attracting target for approaches aimed at tailoring the immune response towards specific immunologic outcomes. A multivalent DC-SIGN ligand (Polyman26), containing at its core a fluorescent “rod-like” spacer and able to inhibit DC-SIGN mediated HIV infection in nanomolar concentration, has been recently developed by our group. We investigated the internalization pattern and the ability of Polyman26 to elicit innate immune responses. Results obtained by confocal microscopy indicate that Polyman26 is internalized by DCs via receptor- mediated endocytosis and is then routed to endolysosomal compartments, thus being presented together with MHC class II molecules, with important implications for the development of vaccines. Moreover, Polyman26 up-regulated the production of β-chemokines and pro-inflammatory cytokines (including IL-1β, IL-6, IL-12, and TNFα) as well as the expression of TLR9 and CD40L. These results indicate that glycomimetic DC-SIGN ligands should be further investigated and suggest that these compounds could be used to differentially stimulate immune responses.
Influence of Core β-1,2-Xylosylation on Glycoprotein Recognition by Murine C-type Lectin Receptors and Its Impact on Dendritic Cell Targeting
Brzezicka K, Vogel U, Serna S, Johannssen T, Lepenies B, Reichardt NC
Targeting antigens to dendritic cell subsets is a promising strategy to enhance the efficacy of vaccines. C-type lectin receptors (CLRs) expressed by dendritic cells are particularly attractive candidates since CLR engagement may promote cell uptake and may further stimulate antigen presentation and subsequent T cell activation. While most previous approaches have involved antibody-mediated CLR-targeting, glycan-based CLR targeting has become more and more attractive in recent years. In the present study, we show that small structural glycan modifications may markedly influence CLR recognition, dendritic cell targeting, and subsequent T cell activation. A biantennary N-glycan (G0) and its analogous O-2 core xylosylated N-glycan (XG0) were synthesized, covalently conjugated to the model antigen ovalbumin, and analyzed for binding to a set of murine CLR-Fc fusion proteins using lectin microarray. To evaluate whether the differential binding of G0 and XG0 to CLRs impacted dendritic cell targeting, uptake studies using murine dendritic cells were performed. Finally, effects of the ovalbumin glycoconjugates on T cell activation were measured in a dendritic cell/T cell cocultivation assay. Our results highlight the utility of glycan-based dendritic cell targeting and demonstrate that small structural differences may have a major impact on dendritic cell targeting efficacy.
Niels-Christian Reichardt, Manuel Martín-Lomas and Soledad Penadés
In this feature article we discuss the particular relevance of glycans as components or targets of functionalized nanoparticles (NPs) for potential applications in personalized medicine but we will not enter into descriptions for their preparation. For a more general view covering the preparation and applications of glyconanomaterials the reader is referred to a number of recent reviews. The combination of glyco- and nanotechnology is already providing promising new tools for more personalized solutions to diagnostics and therapy. Current applications relevant to personalized medicine include drug targeting, localized radiation therapy, imaging of glycan expression of cancer cells, point of care diagnostics, cancer vaccines, photodynamic therapy, biosensors, and glycoproteomics.
José J. Reina, Antonio Di Maio, Javier Ramos-Soriano, Rute C. Figueiredo and Javier Rojo
α(1,2)mannobiosides with different substituents at the reducing end have been synthesized by a common strategy using benzoyls as the permanent protecting groups and an acetyl as the orthogonal protecting group at position C2 of the glycosyl acceptor. The new synthetic strategy has been performed remarkably reducing the number of purification steps, the time of synthesis (less than 72 hours) and improving the overall yield at least three times with respect to the best procedure described in the literature at the moment. Additionally, this protecting group strategy is compatible with the presence of azido groups and the use of Cu catalyzed azide alkyne cycloaddition (CuAAC) also called “click chemistry” for conjugating the α(1–2)mannobiosides to different scaffolds for the preparation of mannosyl multivalent systems.
Monitoring Glycan-Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH- Interactions
Luis P. Calle, Begoña Echeverria, Antonio Franconetti, Sonia Serna, M. Carmen Fernández-Alonso, Tammo Diercks, F. Javier Cañada, Ana Ardá, Niels-Christian Reichardt, Jesús Jiménez-Barbero
Detection of molecular recognition processes requires robust, specific, and easily implementable sensing
methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan–protein interactions that involve N-acetylated sugars. Although difluoroacetamide has been used previously as a substituent
in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc-containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal 1H and 19F atoms that allow both 1H and 19F NMR methods for easy and robust detection of molecular recognition processes involving
GlcNAc- (or GalNAc-) moieties over a range of binding affinities. The CHF2CONH- moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic-sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF3CONH- analogue differs more significantly.
Designing nanomolar antagonists of DC-SIGN-mediated HIV infection: ligand presentation using molecular rods
Authors: Stefania Ordanini, Norbert Varga, Vanessa Porkolab, Michel Thépaut, Laura Belvisi, Andrea Bertaglia, Alessandro Palmioli, Angela Berzi, Daria Trabattoni, Mario Clerici, Franck Fieschi and Anna Bernardi
Journal: Chem. Commun., 2015, 51, 3816
DC-SIGN antagonists were designed combining one selective monovalent glycomimetic ligand with trivalent dendrons separated by a rigid core of controlled length. The design combines multiple multivalency effects to achieve inhibitors of HIV infection, which are active in nanomolar concentration.
Authors: Katarzyna Brzezicka, Begoña Echeverria, Sonia Serna, Angela van Diepen, Cornelis H. Hokke, and Niels-Christian Reichardt
Journal: ACS Chem. Biol. 2015 DOI: 10.1021/cb501023u
The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosomeinfected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannosebinding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans. DC-SIGN antagonists were designed combining one selective monovalent glycomimetic ligand with trivalent dendrons separated by a rigid core of controlled length. The design combines multiple multivalency effects to achieve inhibitors of HIV infection, which are active in nanomolar concentration.
Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing
Authors: P. Both, A. P. Green, C. J. Gray, R. Šardzík, J. Voglmeir, C. Fontana, M. Austeri, M. Rejzek, D. Richardson, R. A. Field, G. Widmalm, S. L. Flitsch, & C. E. Eyers
Journal reference: Nature Chemistry, 6,65–74, (2014) doi:10.1038/nchem.1817
Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.