A Sweet Galactose Transfer: Metabolic Oligosaccharide Engineering as a Tool To Study Glycans in Plasmodium Infection.

The introduction of chemical reporter groups into glycan structures through metabolic oligosaccharide engineering (MOE) followed by bio-orthogonal ligation is an important tool to study glycosylation. We show the incorporation of synthetic galactose derivatives that bear terminal alkene groups in hepatic cells, with and without infection by Plasmodium berghei parasites, the causative agent of malaria. Additionally, we demonstrated the contribution of GLUT1 to the transport of these galactose derivatives, and observed a consistent increase in the uptake of these compounds going from naïve to P. berghei-infected cells. Finally, we used MOE to study the interplay between Plasmodium parasites and their mosquito hosts, to reveal a possible transfer of galactose building blocks from the latter to the former. This strategy has the potential to provide new insights into Plasmodium glycobiology as well as for the identification and characterization of key glycan structures for further vaccine development

Bio-orthogonal site-selective labelling of carbohydrates and proteins

Carbohydrates and proteins represent two large groups of biomolecules which are tremendously important for biological processes in health and disease state. Although protein-structures are encoded in the genome, cellular glycan structures are template independent and can only be addressed in an indirect manner. The development of metabolic oligosaccharide engineering (MOE) gave rise to new methods to study carbohydrate structures in the context of different disease settings and in different organisms. While in many cases mannose derivatives are used to study the sialic acid structures in cancer cells, this work presents the results on the metabolic incorporation of galactose derivatives into cell membrane glycans of human hepatic cells. Three unnatural galactose derivatives containing terminal alkene groups in C2 or C6 position were synthesized and their reaction rates in inverse electron demand Diels Alder reactions (iEDDA) were evaluated, by using a high-throughput screening method in 96-well plates. It was shown that none of the developed galactose derivatives exhibit any cell toxic effect in HepG2 or Huh7 cell lines. Furthermore, all monosaccharides could be successfully incorporated in cell membrane glycan structures of both cell lines and the localization on the cell membrane was confirmed by co-localization with a plasma membrane dye. After developing this incorporation and labeling strategy of unnatural galactose derivatives in the cell membrane of human hepatic cells, the change in incorporation during an infection of these cells with Plasmodium berghei sporozoites was investigated. By using different techniques, such as confocal microscopy, flow cytometry and imaging flow cytometry, only a small trend for an increased uptake of the unnatural galactose derivative in P. berghei infected cells was observed. To explain this result, the pathway for the diffusion of the unnatural galactose derivative was determined. The application of specific and non-specific inhibitors for the glucose transporter GLUT1 revealed that this transporter is involved in the delivery of galactose derivatives into cultured cells. The enhanced translocation of this transporter to the surface of infected hepatic cells explains the observed tendency for an increased incorporation of the unnatural galactose derivative in these cells. Apart from cell studies, MOE was applied for the first time to study a possible transfer of galactose monosaccharides from the mosquito host to the parasite. Biosynthetic pathways for glycan assembly in the parasite are poorly understood. Suggestions on the participation of the mosquito host in some of these pathways, led to the idea to apply MOE in this situation. It was possible to show an uptake of the presented galactose derivatives by the mosquito but only reduced transfer to the parasite seems to occur. In addition to the development of monofunctional galactose derivatives, also a bifunctional derivative containing two orthogonal reporter groups was synthesized. However, so far it was not possible to achieve a metabolic incorporation or labeling of this derivative on cell membrane glycans. After developing cellular tools to study carbohydrate structures, a site-selective method for protein modification was generated, to be used for the development of new glycoconjugate vaccine candidates. By introducing selectively two dehydroalanine residues in place of the disulfide bond C186-C201 of the immunogenic protein CRM197, a new chemical moiety for the conjugation of carbohydrate antigens was obtained. It was shown that these moieties can be used for the selective introduction of polysaccharide antigens from group B Streptococcus (GBS) or Streptococcus pneumoniae. Both types of glycoconjugates could be synthesized and first trials on the purification methods were undertaken. This concept will be developed further for future vaccine candidates. Finally, a synthetic method was developed which could facilitate the synthesis of defined antigenic oligosaccharide structures. This method uses the thiophilic promoter O-mesitylenesulfonylhydroxylamine (MSH) for the activation of thioglycoside donors. It was demonstrated that different thioglycoside donors are activated with different kinetics, depending on the presented protecting groups or the anomeric leaving group. Apart from applying the developed activation method for the synthesis of several glycosylation products, the sequential activation of S-alkyl before S-phenyl anomeric groups was shown during the synthesis of a model trisaccharide. Overall, bio-orthogonal methods were developed and applied for the investigation of carbohydrate structures in the context of malaria disease, and for the site-selective modification of protein carriers during the development of glycoconjugate vaccine candidates

Chemical tools for the study of N-glycosylation in protozoan parasites

Glycosylation is the most abundant post translational modification in eukaryotic cells and can be accredited to an enormous variety of functions. Yet the tools used to study rare or unusual glycosylations are limited. N-Glycosylation, glycosylation of an asparagine residue, begins in the endoplasmic reticulum where a glycan precursor is synthesised by glycosyltransferases before it is transferred to a protein by an oligosaccharide transferase. The protein is then transferred to the Golgi where the glycan is further modified by glycosidases or glycosyltransferases. During this process, the array of glycosyltransferases present in each organism serves to produce a range of glycans, many of which are organism specific. Their formation and functions provide valuable information as to how certain parasites proliferate and cause disease. Therefore, new tools to study glycans and glycan processing enzymes could pave the way towards new therapeutics. Within this thesis, three chemical methods to explore glycosylation in Trypanosoma brucei and Plasmodium falciparum are described. Although UDP-agarose serves to enrich glycosyltransferases from complex mixtures, it is not specific towards UDP-galactosyltransferases. This particular family of enzymes forms linkages in many unique glycans of T. brucei and represent interesting drug targets due to their absence in humans. Due to their unusual nature, no homologues have been identified. The majority of this PhD project was aimed at synthesising an activity based probe to selectively enrich galactosyltransferases from T. brucei using analogues of the donor sugar nucleotide, UDP-galactose. In a newly developed synthetic route, UDP-galactose and UDP-(4F)-galactose were attached to tentagel resin. To our knowledge, these are the first resin bound sugar-nucleotides. After initial method development with a commercially available galactosyltranferase along with other proteins, the resins were proven to bind with the desired selectivity. They were then used in an assay to enrich galactosyltransferases from T. brucei lysates. UDP-Galactose was unable to enrich galactosyltransferases from this complicated mixture, most likely due to a low affinity and the complexity of the proteins it was submitted to. UDP-(4F)-Galactose showed a higher affinity but was only able to enrich one galactosyltransferase: TbGT3. The assay was only performed once, therefore with repeated experiments this result may improve. The second tool described in this thesis is the use of the small molecule inhibiter of N-glycosylation, NGI-1. From published data, this small molecule was predicted to only inhibit the transfer of high mannose glycans in T. brucei. In doing so, the effect of reduced glycosylation could be studied without the need for RNAi knock down of STT3B. NGI-1 was toxic to T. brucei but had a very high IC50 of 75.73 μM. Therefore, at lower concentrations the effect of the drug could be observed. By lectin blots, there appeared to be an effect of the drug on N-glycosylation with an increase in complex glycans and a decrease in high mannose. However, the results were not clear so mass spectrometry analysis of T. brucei’s variant surface glycoprotein were sought to determine the exact N-glycosylation profile. The third tool described is to enrich glycoproteins from Plasmodium falciparum lysates using various lectin based methods and mass spectrometry analysis. Initially, glycoproteins were enriched using the FASP FACE protocol and lectins GLS II and WGA. Although the mass spectrometry results indicated the presence of glycoproteins, the method used was not accurate enough to determine their nature. An Orbitrap mass analyser was then used, which improved the accuracy so that the presence of glycopeptides was confirmed. Unfortunately, these peptides were not of high enough resolution to be identified. Magnetic bead bound GSL II and WGA enrichment was tested, but there were difficulties in conjugating the lectin to the beads. Chemically modifying the glycoproteins with a galactoslytransferase so that they could be enriched with ricin (instead of GSL II and WGA) was also tested. Mass spectrometry showed that the enrichment was not successful and alternate methods must be investigated. These new methods to study N-glycosylation in T. brucei and P. falciparum require some optimisation. However, since both parasites synthesise unique (and in the case of P. falciparum, disputed) N-glycans, tools such as the ones described will be the most effective way to profile their N-glycosylation.Part of the Marie Curie ITN funded by the EU Commission (GA. 608295

Pathogen detection based on carbohydrate adhesion

The rapid detection of pathogenic organisms to ensure appropriate administration of treatment remains a global healthcare challenge. This is becoming increasingly difficult, as identification of the organism alone is no longer enough, with the rise of drug resistance amongst many pathogens it is becoming increasingly important that both the pathogen and drug resistance are identified. Currently, rapid identification can be achieved through a variety of techniques. However, many of these techniques are expensive, require extensive sample preparation, or highly trained personnel to run with results often not rapidly available. This leaves health care professionals to make point-of-care treatment decisions based on symptoms without any indication of drug resistance. The use of carbohydrate microarrays for pathogen detection has been identified as both a method for detection but also as a basis for identifying new drug targets. This exploits the initial protein-carbohydrate interaction that many pathogens utilise in the initial stages of infection. However, the use of microarrays is also challenging, as highly sensitive identification of pathogens often requires expensive or synthetically challenging oligosaccharides or coupling with a highly sensitive detection method thus limiting its point of care application. Herein we describe the coupling of a facile surface chemistry for glycan addition with a powerful statistical algorithm to improve the sensitivity of a cheap monosaccharide functionalised surface without using expensive detection methodologies. This technique was then applied to the detection and identification of toxic lectins, bacterial samples and finally the life-stage specific detection of Plasmodium falciparum (one of the parasites responsible for human malaria). In this last case, drug resistance related to carbohydrate binding profile was also observed

Facile Enzymatic Synthesis of Ketoses and Chemoenzymatic Reporter Strategy for Probing Complex Glycans

Of all possible ketoses, only D-fructose occur large scale in nature. Therefore, all the ketoses with the exception of D-fructose are defined as “rare ketose”. Despite their lower accessibility, rare ketoses offer an enormous potential for applications in pharmaceutical, medicine, functional food and synthetic chemistry. However, studies of rare ketoses have been hampered by the lack of efficient preparation methods. Here, a convenient and efficient platform for the facile synthesis of rare ketoses is described. The introduced two-step strategies are based on a “phosphorylation/de-phosphorylation” cascade reaction. Rare ketoses were prepared from readily available starting materials as their ketose-1-phosphate forms in step 1 by one-pot multienzyme reactions, followed by the hydrolysis of the phosphate groups in acidic conditions to produce desired ketoses in step 2. By this strategy, 14 rare ketoses were obtained from readily available starting materials with high yield, high purity, and without having to undergo tedious isomer separation step. Sialic acids are typically linked a2-3 or a2-6 to the galactose that located at the nonreducing terminal end of glycans, playing important but distinct roles in a variety of biological and pathological processes. However, details about their respective roles are still largely unknown due to the lack of an effective analytical technique. Lectin and antibody binding have been the primary method to analyze glycans, but lectins and antibodies often suffer from weak binding affinity, limited specificity, and cross-reactivity. To address this issue, we develop a chmoenzymatic reporter strategy for rapid and sensitive detection of N-acetylneuraminic acid-a(2-3)-Galactose (Neu5Aca(2-3)Gal) glycans on cell surface

The unique glycoproteins of Cryptosporidium parvum and Toxoplasma gondii

Cryptosporidium parvum and Toxoplasma gondii are obligate intracellular parasites transmitted by ingestion of resilient walled structures called oocysts. Infection is self-limiting in adults with normal immune systems. However, severe disease can occur in immunocompromised individuals, or those without cellular immunity. Cryptosporidium is a leading cause of infant mortality in developing countries, due to diarrhea. There are no human vaccines and no broad effective drug treatments. Several vaccine candidates have been described: the glycoproteins Gp900, Gp40, and Gp15 and the protein Cp23, the immuno-dominant-antigen. Details about modifications to these proteins have not previously been reported. Using mass spectrometry, we identified 16 Cryptosporidium N-glycosylated proteins, including Gp900 and a possible oocyst wall protein. The observed N-glycan structures exhibited only two compositions: HexNAc2Hex5 and HexNAc2Hex6; these glycoforms had a single extended arm. The simplicity of Cryptosporidium N-glycans contrasts with the complexity of host N-glycans. Four heavily O-glycosylated proteins included Gp900, Gp40, Gp15, and a novel mucin-like protein, Gp20. Single O-HexNAc residues modified Ser/Thr in low density regions of Gp15 and Gp900, while attachment of O-HexNAc residues on tandem Ser/Thr repeats of Gp20 and Gp40 approached saturation. Identification of N-acetylgalactosamine (GalNAc) as the HexNAc released from proteins suggests that most Cryptosporidium O-glycans resemble the immunogenic Tn antigen (O-GalNAc). The immunodominant antigen Cp23, while not glycosylated, was discovered to be N-myristoylated and S-palmitoylated on the first and second residues, respectively. This is the first identification in Cryptosporidium of these modifications. Information about the N-glycans, O-glycans, and lipid modifications may be useful for design of better serodiagnostic reagents and more effective vaccines. To date, there are no vaccines against Toxoplasma infection, and the only available pharmaceutical therapies are expensive. In the second study, a novel O-fucose modification was discovered on nuclear pore-associated proteins including nucleoporins. This observation has profound implications on how the organism may regulate trafficking in/out of the nucleus by employing a system parallel to that described for O- linked N-acetylglucosamine in other organisms. In summary, the new details regarding the vaccine candidates of Cryptosporidium and the discovery of the novel O-fucose modifications in T. gondii provide information that could prove useful for development of effective drugs and vaccines.2018-11-01T00:00:00

An exploration of phosphorylases for the synthesis of carbohydrate polymers

Phosphorylases are interesting enzymes with regard to both their role in metabolism and their use in the in vitro synthesis of carbohydrates. The disaccharide phosphorylases have attracted attention because of their strict stereo- and regiospecificity and their tractability. The polymerising phosphorylases have received less attention due to heterogeneous product formation, requiring more complex analyses. In this work three polymerising carbohydrate phosphorylases have been studied. The plant α-1,4-glucan phosphorylase PHS2 is closely related to the well characterised mammalian glycogen phosphorylase. We present the first crystal structures of the plant enzyme which reveals a unique surface binding site. PHS2 allowed the production of novel starch like surface, both in two and three dimensions, which show some of the same properties as a native starch granule. This can now be used to study starch-active enzymes on an insoluble glucan surface which is analogous to the native starch granule. The bacterial β-1,4-glucan phosphorylase CDP is involved in degradation of cellulose. In the reverse direction this enzyme allows the rapid synthesis of cellulose polymers in solution and also allows the synthesis of hemicellulose-like materials. The substrate specificity can in part be probed in the crystal structure presented here, which represents the first structure of a polymerising, inverting phosphorylase. Together these data provide the foundation for further work with this enzyme in the synthesis of plant cell wall related glycans. The third enzyme studied was the β-1,3-glucan from the unsequenced alga Euglena gracilis, which was used for the facile enzymatic synthesis of β-glucosyl glycerols. In order to identify the sequence of this enzyme we obtained de novo transcriptome sequencing data from this alga, which has revealed unexpected metabolic diversity. Aside from complex carbohydrate metabolism, there are also many surprising features, including novel enzyme architectures, antioxidants only previously noted in human parasites and complex natural product synthases

Marine Glycomics

Marine creatures are rich sources of glycoconjugate-containing glycans and have diversified structures. The advance of genomics has provided a valuable clue for their production and developments. This information will encourage breeding and engineering functional polysaccharides with slime ingredients in algae. These glycans will have the potential for applications to antioxidant, anticancer, and antimicrobial drugs in addition to health supplements and cosmetics. The combination of both biochemical and transcriptome approaches of marine creatures will lead to the opportunity to discover new activities of proteins such as glycan-relating enzymes and lectins. These proteins will also be used for experimental and medical purposes, such as diagnostics and trial studies. The topic of marine glycomics is also focusing on understanding the physiological properties of marine creatures, such as body defense against pathogens and cancers. In the competitions for natural selection, living creatures have evolved both their glycans and their recognition. They have primitive systems of immunity, and few of their mechanisms are closely related to glycans. If we are able to describe the accumulation of data of glycans of creatures living in the seashore and the oceans, we may be able to anticipate a time when we can talk about the ecosystem with glycans. That knowledge will be useful for the development of drugs that cure our diseases and for an understanding of living systems in addition to the preservation of living environments

Facettes de glycobioinformatique (applications à l'étude des interactions protéines-sucres)

Le travail décrit dans ce manuscrit rassemble les résultats obtenus au cours de ma thèse de doctorat. Ils s'inscrivent dans le domaine de la glycobioinformatique. Ils ont impliqué des développements de bases de données structurales et des applications en modélisation moléculaire des interactions protéines-sucres. Les méthodes de modélisation moléculaire ont été utilisées dans la reconstruction et dans la prédiction des structures tridimensionnelles de polysaccharides et d'oligosaccharides, ces dernières étant également établies par une approche de type haut-débit par application d'un algorithme génétique à des fins de minimisation énergétique. Les données ainsi générées ont été organisées sous la forme de bases de données relationnelles, proprement annotées (PolySca3DB et BiOligo) qui sont en libre accès pour consultation sur internet. Ces méthodes de modélisation moléculaire ont été appliquées à la caractérisation, par RMN en solution, des conformations de basse énergie d'une souche pathogène d'un polysaccharide de la bactérie E. coli. D'autres bactéries pathogènes de type gram négatif, interagissent avec des oligosaccharides par l'intermédiaire de protéines secrétées, telles que des lectines. Nous avons testé, au travers de l'utilisation de méthodes d'amarrage moléculaire, la possibilité d'identifier de manière automatique, la nature de ces interactions, en prenant comme cibles des épitopes oligosaccharidiques fucosylés. Les résultats de ces recherches ont été comparés, de manière critique, à ceux issus de l'application de bio-puces à sucres et de calorimétrie isotherme de titration. Les conclusions et perspectives de ces travaux sont présentées dans un article de revue consacré à l'application des méthodes de chimie computationnelle dans l'étude des interactions protéines-glucides qui viennent compléter l'arsenal des outils dédiés au champs de recherche couvert par la glycobiologie structurale et moléculaire.This thesis presents an account of two important facets of glycobioinformatics, comprising database development and molecular modeling of 3D structures of carbohydrates alongside the simulation of protein-carbohydrate interactions. Classical molecular modeling techniques were used to reconstruct 3D polysaccharide structures from experimentally determined atomic coordinates, or known starting points about their structures were used as guidelines to model them. A genetic algorithm search was employed as a high-throughput technique to characterize low energy conformers of bioactive oligosaccharides. The data generated were organized into two open-access relational databases, namely, PolySac3DB and BiOligo, for use by the scientific community. The validation of the molecular techniques used were performed using solution phase NMR experiments on four entero aggregative pathogenic E. coli strains, and were found to be robust and realistic. Further, the impact of the presentation of human fucosylated oligosaccharide epitopes to lectins from opportunistic gram negative bacteria, was investigated in a screening study using molecular docking studies, which could help in evaluating the feasibility of using automated docking procedures in such instances as well as deciphering binding data from glycan array experiments and also correlated to isothermal calorimetry data. On comparison with high-resolution experimental crystal complexes, automated docking was found to delineate the present level of applicability, while emphasizing the need of constant monitoring and possible filtering of the results obtained. Finally, a review of the present status of the computational aspects of protein-carbohydrate interaction studies is discussed in the perspectives of using molecular modeling and simulation studies to probe this aspect of molecular and structural glycobiology.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Nano-probes for point of care diagnostics

The COVID-19 pandemic has exposed deep health inequalities between more economically developed and less economically developed countries: both in terms of diagnostics and vaccinations. Robust and low-cost point of care devices are needed to ease these diagnostic inequalities. Current point of care lateral flow immunoassays, utilise proteins, such as antibodies, to sense for analytes. This is epitomised by the malaria rapid diagnostic test and archetypal home pregnancy test. Glycans are emerging as alternative detection units due to their fundamental role in biological signalling and recognition events. Furthermore, the increased robustness, low-cost and synthetic possibilities offered by glycan-based systems, especially glycosylated polymers, make them a promising alternative to antibody-based biosensing and diagnostic systems. Chapter 1 discusses the current use of protein-based lateral flow and flow-through devices; their advantages and disadvantages versus non-point of care techniques, and the potential of glycan-based lateral flow devices. The concepts introduced in Chapter 1 are then applied in Chapters 2 through 5. Chapter 2 demonstrates the use of glycosylated polymer-coated nanoparticles, produced by controlled radical polymerisation techniques for the sensitive, label-free detection of lectins in lateral flow and flow-through. The systems produced use only glycans, not antibodies, to provide recognition – a “lateral flow glyco-assay.” The lessons learned in Chapter 2 are applied in Chapter 3 to probe the glycan-binding of the SARS-COV-2 spike protein in a “flow-through glyco-assay” and target a pseudovirus mimic of the target coronavirus in a lateral flow glyco-assay. Chapter 4 builds on Chapters 2 and 3, applying the fledgling glyco-assay technology to the “real-world” by sensing for the SARS-COV-2 virus in patient samples, alongside exploring the robustness of the devices themselves. Having explored the concept of glycosylated polymer-coated nanoparticles in lateral flow and flow-through setups; Chapter 5 changes focus and explores the use of polymeric anchors for the design of all-polymer (“vegan”) lateral flow and flow-through devices. This work completely removes proteins as either detecting units or anchors from lateral flow for the first time. Chapters 6 and 7 explore more fundamental Chemistry than the previous chapters. Chapter 6 considers the use of the Mannich reaction to produce monosaccharides with amine functionality at C2, ideal for polymer conjugation, while maintaining hydroxyl functionality at C2. Although unsuccessful with the reagents used, the chapter highlights a potential avenue of future chemical exploration in novel glycan synthesis. Chapter 7 pulls together the x-ray photoelectron analysis data and spectra collected across a range of studies, including data collected in previous chapters, and considers if x-ray photoelectron spectroscopy can be used to determine relative grafting density in glycosylated polymer-coated nanoparticle systems. In summary, the key components of the emerging technology of lateral flow glycoassays are introduced, interrogated and investigated. The prototype devices tested against model proteins, viral proteins and patient samples, are found to show specificities and sensitivities that rival lateral flow immunoassay systems. The understanding developed in this thesis could pave the way to the first generation of lateral flow glyco-assays that are low-cost, stable in a wide range of conditions, and able to target a wide range of analytes and diseases