Le trypanosome sud américain, Trypanosoma cruzi, transmis par un insecte hématophage de type triatome est le protozoaire connus pour causer la maladie de Chagas chez l’Homme. Le cycle de vie de ce parasite alterne à la fois sur le type d’hôte, insecte ou hôte vertébré, et sur la forme :trypomastigote pour la forme quiescente et amastigote et épimastigote pour les formes prolifératives. Concernant la forme, seuls les parasites épimastigotes évoluent et prolifèrent dans le tube digestif des triatomes et possèdent un système endocytaire actif nécessaire à leur besoin énergétique. Toutefois, cette endocytose est restreinte à deux sites membranaires, la poche flagellaire et le cytostome, à partir desquels se créent des cargos endocytaires. Ces cargos endocytaires fusionnent ensuite avec un réseau vésiculaire endosomal qui délivre son contenu dans des réservosomes, compartiments similaires aux lysosomes.Chez le trypanosome africain (Trypanosoma brucei brucei), l’endocytose ne se réalise qu’au niveau de la poche flagellaire. Certaines protéines appartenant à cette voie endocytaire sont modifiées par de longues chaines de résidus poly-N-acétyllactosamine (pNAL) de manière post-traductionnelle. Initialement, il a été proposé que ces résidus puissent agir en tant que signal de tri dans le processus d’endocytose chez ces parasites.En nous basant sur les travaux qui ont été réalisés chez le trypanosome africain, nous nous sommes proposés d’approfondir les connaissances sur la voie endocytaire du trypanosome sud américain (Trypanosoma cruzi) qui est beaucoup moins étudié. Pour ce faire, à l’aide de deux lectines, la tomatolectine et la lectine de Griffonia simplicifolia qui présentent respectivement une affinité pour les résidus pNAL et les résidus N-acétylglucosamine (GlcNAc) en fin de chaine, nous avons pu enrichir et caractériser par LC-MS², 173 glycoprotéines putatives dont plus de 13% sont localisées dans la voie endocytaire. Parmi les protéines identifiées, en plus des nombreuses hydrolases lysosomiales, nous avons pu identifier une lectine de type C localisée dans la partie antérieure des parasites, au niveau des principaux sites endocytaires. Cette dernière possédant de nombreux résidus en commun avec les récepteurs de type scavengers, elle pourrait donc jouer un rôle important dans la fixation et l’endocytose de certains nutriments.Nos travaux ont ainsi permis d’établir que similairement aux trypanosomes africains, Trypanosoma cruzi possèdent des glycoprotéines modifiées par des N-glycanes contenant des pNAL. Nos travaux ont également permis d’établir que ces résidus s’associent préférentiellement aux glycoprotéines de la voie endocytaire (au niveau du cytostome et du réservosome) de la forme épimastigote. L’ensemble des résultats obtenus durant cette thèse tendent à montrer que les résidus pNAL des glycoprotéines présentes dans la voie endocytaire ont été conservées entre les deux parasites étudiés (Trypanosoma cruzi et Trypanosoma brucei brucei). / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les glucides, les biomolécules les plus abondants dans la nature, sont essentiels pour le maintien et la survie des organismes. Certaines molécules à base de glucides à la fois d'origine synthétique et naturelle sont connues pour leurs activités pharmacologiques et utilisés pour traiter différentes maladies. Deux projets ont été réalisés sur la base d'oligosaccharides biologiquement actifs dans cette thèse. Sialyl Lewis X (sLex), un tétrasaccharide exprimé sur la surface cellulaire d'un tissu embryonnaire et un tissu tumoral, a été découvert pour jouer un rôle important dans l'adhésion entre les cellules tumorales et les cellules endothéliales dans les métastases tumorales à travers la liaison de E-sélectine avec une forte affinité et sélectivité. E-sélectine est un membre de famille des sélectines des molécules d'adhésion cellulaire, exprimée sur l'endothélium tumoral qui est activé par la cytokine. En conséquence, un type de sonde moléculaire basé sur sLex a été conçu et synthétisé dans le but de diagnostiquer le cancer. Aminoéthyle glycoside de sLex 36 a été efficacement préparé à partir de quatre synthons monosaccharidiques par sept étapes d’une manière hautement stéréosélective et régiosélective avec un rendement total de 18%. La sonde moléculaire conjuguant 36 avec une protéine marqué a été montré capable de visualiser une tumeur implantée chez la souris par imagerie in vivo, alors que le contrôle négatif a donné seulement un profil de circulation vasculaire. Ce travail est actuellement dans le processus d'un dépot de brevet. D'autre part, le mécanisme de traitement des N-glycanes par l'endo-α-mannosidase et les inhibiteurs de cette enzyme ont été étudiés. Décorations de N-glycane existent sur la plupart des protéines eucaryotes et leurs compositions aberrantes sont associés à diverses maladies, y compris les infections virales et le cancer. Endo-α-mannosidase hydrolyse la liaison α-1,2-mannosidique entre le mannose en glucose-substitué et le reste de la N-glycane dans l'appareil de Golgi, en fournissant une voie indépendante de glucosidase I et II pour la maturation de N-glycane. Le but de présente étude est de confirmer le mécanisme catalytique nouvellement proposé pour endo-α-mannosidase: la participation du groupe voisin via un 1,2-anhydro intermédiaire de sucre. Etant donné que cet intermédiaire est instable, le glycosyl-1,2-β-anhydro carbasucre 53 a été conçu et synthétisé pour soutenir l'existence de cet intermédiaire pour l'hydrolyse catalysée par glycosidase. De plus, le glycosyl-1,2-β-aziridine carbasucre 64 et le glycosyl-1,2-oléfinique carbasucre 63 ont été conçus et synthétisés comme nouveaux inhibiteurs d’endo-α-mannosidase pour le développement d'agents thérapeutiques. En outre, le glycosyl-1,2-α-anhydro carbasucre 52 et le glycosyl-1,2-α-aziridine carbasucre 65 ont également été synthétisés comme références. Tous les composés cibles ont été préparés de manière efficace à partir du même glycosyl-carbasucre qui a été obtenu de D-(+)-maltose par plusieurs étapes de protections, de modifications, du réarrangement de Claisen. L'évaluation biologique est en cours. / As the most abundant biomolecules found in nature, carbohydrates are essential for sustaining and survival of organisms. Some carbohydrate-based molecules both of synthetic and natural origin are known for their pharmacological activities and used to treat different diseases. Two projects were carried out based on biologically active oligosaccharides in this thesis. Sialyl Lewis X (SLex), which is a tetrasaccharide expressed on cell surface of embryonic tissue and tumor tissue, was discovered to play an important role in the adhesion between tumor cells and blood endothelial cells in tumor metastases through binding E-selectin with high affinity and selectivity. E-selectin is a member of the selectin family of cell adhesion molecules, expressed on cytokine activated tumoral endothelium. Accordingly, a kind of molecular probe based on sLex was designed and synthesized for the purpose of diagnosing cancer. Aminoethyl glycoside of sLex 36 was efficiently prepared from four monosaccharide building blocks through seven steps with high stereoselectivity and regioselectivity in 18% total yield. The molecular probe conjugating 36 with a labelled scaffold protein was shown to visualize an implanted tumor in the mouse by in vivo imaging, whereas the negative control gave only a vascular circulation profile. This work is currently in the process of a patent application. On the other hand, N-linked glycans processing mechanism by endo-α-mannosidase and this enzyme’s inhibitors were studied. N-glycan decorations exist on the majority of eukaryotic proteins and their aberrant compositions are associated with various diseases, including viral infection and cancer. Endo-α-mannosidase hydrolyzes the α-1,2-mannosidic bond between the glucose-substituted mannose and the rest of the N-glycan in Golgi apparatus, providing a glucosidase I and II independent pathway for the maturation of N-glycan. The aim of the present study is to confim the newly proposed catalytic mechanism for endo-α-mannosidase: neighboring group participation via a 1,2-anhydro sugar intermediate. Since this intermediate is unstable, glycosyl-1,2-β-anhydro carbasugar 53 was designed and synthesized to support the existence of this intermediate for glycosidase-catalyzed hydrolysis. Moreover, glycosyl-1,2-β-aziridine carbasugar 64 and glycosyl-1,2-olefinic carbasugar 63 were designed and synthesized as novel endo-α-mannosidase inhibitors for the development of therapeutic agents. In addition, glycosyl-1,2-α-anhydro carbasugar 52 and glycosyl-1,2-α-aziridine carbasugar 65 were also synthesized as compare. All the target compounds were prepared efficiently from the same glycosyl-carbasugar that was obtained from D-(+)-maltose through several steps of protections, modifications, and Claisen rearrangement. The biological evaluation is underway.

La barrière épidermique du stratum corneum (SC) est doublée par une barrière secondaire des jonctions serrées (JS) qui influent sur la formation de barrière principale. Dans mes travaux, je me suis concentré sur l'étude de la présence et l'évolution des éléments jonctionnelles composants ces deux barrières ; les cornéodesmosomes au niveau du SC et les JSs au niveau de la granuleuse. En plus, je me suis intéressé à l'implication des glycanes dans la fonction barrière épidermique. Ces travaux ont été réalisés soit dans un contexte physiologique soit par la modulation de la barrière épidermique par des facteurs intrinsèques et extrinsèques. Nos résultats confirment que les JSs jouent un rôle subalterne par rapport à la barrière du SC et montrent que les glycanes persistent à la surface des cornéocytes humains. La composition et la répartition utlrastructurale des glycanes évoluent à travers les assises du SC jusqu'à la desquamation d'une manière concordante avec la répartition des cornéodesmosomes. Certaines modifications intrinsèques naturelles lors du vieillissement ou pathologiques notamment l'état pelliculaire et la dermatite atopique, ont permis d'appréhender le rôle de ces composants dans la cohésion du SC et la prestance d'une barrière fonctionnelle. Les modifications extrinsèques de la barrière par l'application de solvants, d'excipients ou de formulations perméabilisantes montrent l'importance de l'organisation utlrastructurale des composants jonctionnelles et non jonctionnelles du SC dans le maintien d'une barrière efficace / The stratum corneum (SC) barrier is doubled by the secondary barrier of tight junctions which influences the formation of the main barrier. In my work, I focused on the study of the junctional elements composing those two barriers; corneodesmosomes in the SC and the tight junction at the granular layer level. In addition, I got interested in the involvement of glycans in the epidermal barrier function. This work was carried out either in skin physiological conditions or by the modulation of the epidermal barrier by intrinsic or extrinsic factors. Our results confirm that tight junctions play a subordinate role compared to the SC barrier and that glycans remain present at the surface of human corneocytes. The composition and the ultrastructure distribution of glycans evolve from the SC compactum to the SC disjunctum, towards desquamation in a comparable manner to the repartition of corneodesmosomes. Natural intrinsic changes during aging and pathological changes, including dandruff and atopic dermatitis, helped us to understand the role of those components in the cohesion of the SC and the conservation of functional barrier. Extrinsic modulation of the barrier by the application of solvents, excipients or topical formulations shows the importance of the ultrastructural organization of junctional and non-junctional SC components in maintaining an effective barrier

Structure and functional study of glycans are highly challenging due to the difficulties in analyzing glycans and limited availability of samples for study. These limitations could be resolved by attaching DNA barcode to the glycan, which virtually represent glycan in further application, by increasing the sensitivity of detection by polymerase chain reaction (PCR), requiring minimal samples for analysis. Assuming bigger arena of DNA Encoded Glycan Libraries (DEGL) in future, we propose here a method for uniquely coding all glycans using computer program that can convert the structural information of glycans to DNA barcode. A unique and universal coding for glycans will benefit both synthesis and analysis of DEGLs. As a proof of principle study, a small DNA Encoded Glycan Library (DEGL) of blood and globo series glycan antigen and its application was demonstrated in detecting blood group and breast cancer from plasma.

For a long time, the scientific community underestimated the value of carbohydrates and the approach of most scientists to the complex world of glycans was apprehensive. The scenario, however, has changed today. With the development of new research tools and methodologies the study of carbohydrates and glycoconjugates has progressed rapidly, increasing our understanding of these molecules. Carbohydrates are most abundant amongst biological polymers in nature and vital for life processes. In their simplest form, they serve as a primary source of energy to most living organisms. In generalis, they exist as complex structures (glycans), and as conjugates of protein (glycoproteins, proteoglycans), lipids (glycolipids) and nucleosides (UDP-Glucose). Defined in the broadest sense, the study of glycans in all their forms and their interacting partners is termed “Glycobiology”. Glycans are ubiquitously found in nature decorating cells of almost all types with a “sugar coat”. They are also present within the cytoplasm, as well as in the extra-cellular matrix. They have key roles in a broad range of biological processes, including signal transduction, cell development and immune responses. All living organisms have evolved to express proteins that recognize discrete glycans and mediate specific physiological or pathological processes. One major class of such proteins is “Lectins”. Found in all forms of life, they are characterized by their ability to recognize carbohydrates. They are proteins of non-immune origin that bind glycans reversibly with a high degree of stereo-specificity in a non-catalytic manner. It must be emphasized that they are a different class from glycan-specific antibodies. Lectins were first discovered in plants and a large amount of work has been carried out on plant lectins to decipher their structural organization, mode of interaction with substrate and as models to study protein stability and folding. Study on animal and microbial lectins, on the other hand, gathered momentum only recently. In spite of this, more is known about their function in animals and micro-organisms rather than in plants. Lectin-glycan binding is implicated in several important biological processes such as protein folding, trafficking, host-pathogen interactions, immune cell responses and in malignancy and metastasis. Most lectins have one or more carbohydrate recognition domains (CRDs) which often share either 3-D structural features or amino acid sequence. New members of a family can be identified using either sequence or structural homology. Interestingly, it turns out that several plant and microbial lectins have structural or sequential similarity with animal lectins , revealing that these CRDs are evolutionarily related. This thesis, entitled “Structural, Biophysical and Biochemical Studies on Mannose-specific Lectins”, focuses on three lectins, Banana lectin (Banlec), Calreticulin (CRT) and Peptide-N-Glycanase (PNGase). Although all three lectins have distinct biological functions, they share a common ligand specificity at the monosaccharide level i.e. mannose. This thesis, besides characterizing these lectins, studies in detail, the difference in the mode of interaction with their ligands. Chapter 1 is a general introduction on lectins, glycan-lectin interactions and the various techniques that are employed to characterize these interactions. Several principles have emerged about the nature of glycan–lectin interactions. It has been observed that the binding sites for low molecular weight glycans are of relatively low affinity (Kd values in the high micromolar to low millimolar range). Selectivity is mostly achieved via a combination of hydrogen bonds and by van der Waals packing of the hydrophobic faces of monosaccharide rings against aromatic amino acid side chains. Further selectivity and enhanced affinity can be achieved by additional contacts between the glycan and the protein. It is notable that the actual region of contact between the saccharide and the polypeptide typically involves only one to three monosaccharide residues. As a consequence of all of the above, these lectin-binding sites tend to be of relatively low affinity, although they can exhibit high specificity. It is intriguing to observe that such low-affinity sites have the ability to mediate biologically relevant interactions. There are many different ways to study binding of glycans to proteins, and each approach has its advantages and disadvantages in terms of thermodynamic rigor, amounts of protein and glycan needed, and the speed of analysis. In examining these interactions, two broad categories of techniques are applied: (1) kinetic and near-equilibrium methods, such as titration calorimetry; and (2) non-equilibrium methods such as glycan microarray screening and ELISA-based approaches. Two of the most widely used biophysical approaches for examining glycan-lectin interactions at the molecular level are X-ray crystallography and nuclear magnetic resonance (NMR). However, as small molecules often co-crystallize with a lectin better than large molecules, a lot of our knowledge about glycan–lectin interactions at the atomic level is based on co-crystals of lectins with unnatural ligands. Thus, a great challenge exists in attempting to understand glycan–lectin interactions in the context of natural glycans present as glycoproteins, glycolipids, or proteoglycans. Chapter 2 introduces Banana lectin and describes the stability studies carried out. The unfolding pathway of Banlec was determined using GdnCl induced denaturation. Analysis of isothermal denaturation provided information on its conformational stability and the high values of ΔG of unfolding at various temperatures indicated the strength of inter-subunit interactions. It was found that Banlec is a very stable protein and denatures only at high chaotrope concentrations. The basis of the stability may be attributed to strong hydrogen bonds at the dimeric interface along with the presence of water bridges. This is a very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions. High temperature molecular dynamics simulations have been utilized to monitor and understand early stages of thermally induced unfolding of Banlec. The present study investigates the behavior of the dimeric protein at four different temperatures. The process of unfolding was monitored by monitoring the radius of gyration, the rms deviation of each residue, change in relative solvent accessibility and the pattern of inter- and intra-subunit interactions. The overall study demonstrates that the Banlec dimer is a highly stable structure, the stability in most part contributed by interfacial interactions. The pattern of hydrogen bonding within the subunits and at the interface across different stages has been analyzed and has provided the rationale for its intrinsic high stability. In Chapter 3 the conformational and dynamic behaviour of three mannose containing oligosaccharides, a tetrasaccharide with α1→2, and α1→3, and a penta- and a heptasaccharide with α1→2, α1→3, and α1→6 linkages has been evaluated. Molecular mechanics, molecular dynamics simulations and NMR spectroscopy methods were used for evaluation. It is found that they display a fair amount of conformational freedom, with one major and one minor conformation per glycosidic linkage. The evaluation of their recognition by Banlec has been performed by STD NMR methods and a preliminary view of their putative interaction mode has been carried out by means of docking procedures. In Chapter 4 the conformational behaviour of three mannose containing oligosaccharides, namely, the α1→3[α1→6] trisaccharide, the heptasaccharide with α1→2, α1→3, and α1→6 linkages and the tetrasaccharide consisting of α1→3 and α1→2 linkages, when bound to Banlec has been evaluated by trNOE NMR methods and docking calculations. It is found that the molecular recognition event involves a conformational selection process, with only one of the conformations, among those available to the sugar in free state, being recognised at the lectin binding site. It is known that many proteins, including members of the Jacalin-related lectin family (of which Banlec is a member), bind the high-mannose saccharides found on the surface of the HIV-associated envelope glycoprotein, gp120, thus interfering with the viral life cycle, potentially providing a manner of controlling a variety of infections, including HIV. These proteins are thought to recognize the high-mannose type glycans with subtly different structures, although the precise specificities are yet to be clarified. This study was carried out to gain a better understanding of these protein-carbohydrate recognition events. Chapter 5 reports interactions of Calreticulin (CRT) with the trisaccharide Glcα1-3Manα1-2Man. Previously in our laboratory it was established using modeling studies the residues in CRT important for sugar binding. Here, the relative roles of Trp-319, Asp-317 and Asp-160 for sugar binding have been explored by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in calnexin (CNX) are known to play important roles in sugar binding. The present study demonstrates that Asp-160 is not involved in sugar binding, while Asp-317 plays a crucial role. Further, it is also validated that hydroxyl-pi interactions of the sugar with Trp-319 dictate sugar binding in CRT. This study defines further the binding site of CRT and also highlights its subtle differences with that of CNX. Additionally, mono-deoxy analogues of the trisaccharide unit Glcα1-3Manα1-2Man have been used to determine the role of various hydroxyl groups of the sugar substrate in sugar-CRT interactions. Using the thermodynamic data obtained by carrying out ITC of CRT with these analogues, it is demonstrated that the 3-OH group of Glc1 plays an important role in sugar-CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. Therefore, the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT are further delineated. Chapter 6 introduces Peptide-N-Glycanase (PNGase) and delineates the various interactions involved in the binding of oligomannose structures of glycoproteins to the C-terminal domain (the carbohydrate recognition module) of PNGase. ITC is used to characterize the interaction to oligosaccharides in terms of affinity, stoichiometry, enthalpy, entropy and heat capacity changes with the mouse PNGase C-terminal domain. Using the thermodynamic data obtained, it was determined that PNGase requires the tri-mannoside moiety of the native glycan on glycoproteins as the basic minimum entity for recognition and binding. Additional mannose moieties on the glycan do not significantly interact with PNGase and therefore no enhancement in binding affinity is observed (unlike CRT) which is in concordance with its role of stripping glycans from misfolded glycoproteins targeted for degradation via the ERAD (Endoplasmic reticulum assisted degradation) pathway. Chapter 7 briefly summarizes all the findings of the research carried out and presents a comparative analysis of the three lectins studied. Appendix A: Protein folding in the ER is assisted by molecular chaperones. Lectin chaperones such as CRT and CNX assist the folding of glycoproteins by their N-linked oligosaccharide chains. Dynamic processing of the original glycan chain of (GlcNAc)2(Man)9(Glc)3 to remove the terminal glucose moieties is essential for accurate folding. Proteins that attain their native conformation are then transported to the Golgi complex for further glycan modifications. In case of aberrant folding the proteins are retrotranslocated into the cytosol, ubiquitinated, deglycosylated and degraded by the proteasome. Peptide-N-glycanase is a cytosolic enzyme that releases N-glycans from glycoproteins and glycopeptides. PNGase is now widely recognized as a major participant in protein quality control machinery for ERAD or the proteasomal degradation of retrotranslocated glycoproteins. It is therefore desirable to synthesize fluorescently labeled glycoprotein substrates which will provide direct understanding of how, when and where, the interaction between the substrate and the enzyme occurs. Towards this goal, cloning of GFP and RFP tagged full length mouse and human PNGase and CRT was carried out which is described in this section.

In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures. A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides. Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated. Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.

Longtemps considérée comme un simple support mécanique, la matrice extracellulaire (MEC) est un élément majeur dans le maintien de l’homéostasie. Ainsi l’élastine, principal constituant de la MEC des gros vaisseaux élastiques, est dégradée au cours du vieillissement, produisant ainsi des peptides d’élastine bioactifs (PE). Plusieurs études ont démontré l'implication des PE en physiopathologies tels que l’invasion tumorale, l’athérosclérose ou l’insulino-résistance (IRes). Ces effets s’expliquent par l’activation du complexe récepteur de l’élastine (CRE), composé par : une sous-unité extracellulaire liant les PE (EBP, elastin binding protein), la cathepsine A (dont le rôle reste inconnu), et la neuraminidase 1 (induisant la signalisation intracellulaire). L'IRes décrite, pourrait être associée à l’activité de désialylation de la neuraminidase-1 sur les chaines de N-glycosylation (Ng-c) du récepteur de l’insuline (RI). Sur la base de cette hypothèse, notre objectif a donc été de confirmer ce mécanisme et ses conséquences in silico (sur le RI), in vitro (pré-adipocytes 3T3-L1) et in vivo (aorte de souris). Nous montrons ainsi in vitro que les PE provoquent un dysfonctionnement de l’autophosphorylation du RI se répercutant sur plusieurs processus cellulaires comme l’entrée du glucose ou encore la différenciation adipocytaire. In silico, nous montrons pour la première fois le rôle des acides sialiques sur le comportement des Ng-c d'une part et sur le RI d'autre part. Enfin, in vivo, cette interaction CRE / IR engendre une hypertension artérielle par une diminution de la vasorelaxation des cellules endothéliales. / Often considered as a simple mechanical support, the extracellular matrix (ECM) is a major element of homeostasis regulation. Thus, elastin, the main constituent of large elastic vessels, is degraded during aging, producing bioactive elastin-derived-peptides (EDP). Several studies have demonstrated the EDP effects in physiopathologies such as tumor invasion, atherosclerosis, or insulin resistance (IRes) development. Those effects are explained by the activation of the elastin receptor complex (CRE), composed of: an extracellular subunit binding EDP (EBP, elastin binding protein), cathepsin A (its role is still unknown) and the sialidase neuraminidase-1 (Neu-1, involved in signaling pathway induction). Interestingly, the lab suggested that IRes may be induced by the desialylation of the N-glycan chains (Ng-c) on the insulin receptor (IR). The aim of this study was to confirm this hypothesis by demonstrating the consequence of desialylation on the IR in silico, on a 3T3-L1 pre-adipocyte cell in vitro, and on vascular complications in vivo. We show that EDP induce in vitro an impairment of IR autophosphorylation, affecting glucose uptake and adipocyte differentiation. In silico approach demonstrates the role of sialic acids on the behavior of Ng-c in the one hand and in other hand of IR. Finally, the IRes induced by ERC-IR interaction increase the vascular complication such as arterial hypertension by endothelial cell impairment. To conclude, Ng-c alteration would likely be responsible for structural changes in the IR at the origin of insulin resistance.

Speichelglykane können einerseits spezifisch an bakterielle Lektine binden und damit deren Adhäsion an orale Oberflächen vermitteln, andererseits eine Antiadhäsion bedingen. Sie stellen ein Schutzsystem für orale Oberflächen dar. Bei vorhandener Karies bzw. Parodontitis ist die Konzentration bestimmter Glykokonjugate verändert. Ziel dieser Studie war es, die Reaktivität der Glandulae majores bzgl. ihrer Sekretion von Glykanen in Abhängigkeit einer experimentellen Gingivitis zu ermitteln. 14 gesunde Probanden enthielten sich 9 Tage der Mundhygiene. Neben der Erhebung des PBI und QH wurde drüsenspezifisch Speichel gewonnen. Die Konzentrationen an die Lektine PNA, GS1, VVA, SNA und AAA bindender Komponenten und deren drüsenspezifische Sekretionsraten wurden bestimmt. Bei allen Probanden stiegen PBI und QH im Versuchsverlauf signifikant an. Gleiches galt für die Speichelmenge nach Stimulation sowie zum Ende der Kontrollreihe. Die Konzentrationen der verschiedenen Glykane verhielten sich unabhängig von der Speichelmenge und unabhängig voneinander. Meist ergab sich eine erhöhte Glykansekretion spezifisch für das untersuchte Lektin. Neben dem Konzentrationsgefälle der einzelnen Drüsen war auch eine Verschiebung nach erfolgter Stimulation zu beobachten. Da genetische und externe Einflüsse für diese Studie weitgehend ausgeschlossen werden konnten bzw. als konstant einzuordnen waren, darf die Veränderung als Reaktion auf die orale Bakterienbelastung angesehen werden. Der Rückgang bestimmter terminaler Strukturen könnte als Folge der vermehrten Synthese anderer, in Bezug auf die veränderte Bakterienflora effektiverer Speichelbestandteile eingeordnet werden. Basierend auf dem Modell, dass freie Glykane die Adhäsion von Mikroorganismen inhibieren können, ließe sich die gemessene Reaktion der Speicheldrüsensekretion als ein gesteigerter Schutzmechanismus im Sinne einer ´first line of defence´ interpretieren. Dieser könnte z.B. in Bezug auf Prophylaxe und Therapie genutzt werden. / Salivary glycans can bind specificly to bacterial lectins. Consequently, bacterial adhesion to oral surfaces is mediated or inhibited by glycans. It is known that the concentration of certain glycans changes in the presence of caries or periodontitis. Therefore this study examines the reactivity of the major salivary glands with respect to the secretion of glycans as conditioned by an experimentally induced gingivitis. 14 healthy subjects refrained from all oral hygiene measures for 9 days. On 5 days a plaque and bleeding index as well as pure glandula saliva with and without stimulation were obtained. The collected salivary samples were examined for their concentration of certain structures that bind to the lectins ´PNA´, ´GS1´, ´VVA´, ´SNA´ and ´AAA´. All subjects developed a gingivitis as measured by the plaque and bleeding index. Salivary flow increased after stimulation and compared to baseline at the end of the trial. The concentration of glycans was neither related to one of the glands nor to the salivary flow. Besides to the differentials of concentration after stimulation there was no symmetrical development between the concentrations of salivary lectin-specific components compared one lectin to another. Genetic and external influences could be largely excluded or considered to be stable during the trial. Therefore the observed results can be regarded as a reaction to the increased bacterial load. The decrease of certain terminal structures in saliva might be explained by a raised synthesis of other components, which are more effective in defending the body against bacterial adhesion. The observed changes in salivary secretion might be interpreted as a mechanism in order to protect the human organism within the meaning of a ´first line of defence´. This mechanism would be able to respond more quickly than the immune system and might be used in future, for example, for preventive and therapeutical strategies.

The sujet de cette these portait sur la caractérisation de la machinerie originale utilisée par les Mimiviridae pour glycosyler les fibrilles entourant leurs capsides en travaillant sur les prototypes des 3 lignées connues, Mimivirus (A), Megavirus chilensis (B) et Moumouvirus australensis (C). Les fibrilles de Mimivirus sont décorées par 2 polysaccharides différents :l’un est caractérisé par la répétition d’un disaccaride linéaire fait de 3)-α-L-Rha-(1→3)-β-D-GlcNAc-(1→, avec un pyruvate branché en position 4,6 du GlcNAc ; l’autre présente une unité répétée branchée de séquence 2)-α-L-Rha-(1→3)-β-D-GlcNAc-(1→ pour le squelette linéaire et du rhamnose branché en position 3 par de 2OMeVioNAc. Nous avons suggéré que les fibrilles de Megavirus sont décorées par plus d'une espèce de polysaccharides/oligosaccharides, donc l’un ayant présentant un trisaccharide de RhaNAc:α-L-4OMe-RhaNAc-(1→3)-α-L-RhaNAc-(1→3)-α-L-RhaNAc-(1→. Les fibrilles de Moumouvirus sont décorées de glucosamine, quinovosamine et bacillosamine. A partir de ces données expérimentales il devenait possible de rechercher de nouveaux gènes responsables de ces glycosylations spécifiques. Le cluster de 9 gènes déjà publié de Mimivirus a pu être étendu à 13 gènes. Un cluster de 14 gènes a été d’autre part identifié dans le génome de Moumouvirus, le premier cluster de gènes de la glycosylation identifié dans la lignée B. Parmi les gènes de glycosylation, l’analyse fonctionnelle in vitro de la protéine L142 a permis de démontrer qu’il s’agit d’une N-acétyltransferase. En conclusion, les fibrilles des Mimiviridae sont lourdement glycosylées and le type de sucres et leur organisation dépend de la lignée considérée. / The aim of this thesis is the study of the innovative glycosylation machinery used by the Mimiviridae family for the glycosylation of the fibrils sourrounding their capsid, using Mimivirus, Moumouvirus australensis and Megavirus chilensis as prototypes of lineages A, B and C, respectively. Mimivirus fibrils are decorated with two distinct polysaccharide: one is characterized by a linear disaccharide repeating unit made of 3)-α-L-Rha-(1→3)-α-D-GlcNAc-(1→, with a pyruvic acid branched at position 4,6 of GlcNAc.; the other has a branched repeating unit with the sequence 2)-α-L-Rha-(1→3)-β-D-GlcNAc-(1→ in the linear backbone and rhamnose further branched at position 3 by viosamine methylated at position 2 and acetylated at position 4. We suggested that Megavirus chiliensis fibrils are decorated by more than one polysaccharides/oligosaccharide species, one having this trisaccharide: α-L-4OMe-RhaNAc-(1→3)-α-L-RhaNAc-(1→3)-α-L-RhaNAc-(1→. Moumouvirus australensis fibrils are decorated with glucosamine and quinovosamine in addition to the rare sugar, bacillosamine. Starting from this experimental data, it was possible to identify new genes involved in glycosylation. As a result, the published nine-gene cluster of Mimivirus was extended to thirteen genes. A different cluster of fourteen genes was identified in Moumouvirus australensis, representing the first glycosylation gene cluster identified for the B lineage.Among the glycosylation genes, the function of L142 was investigated in vitro, demonstrating that it is an N-acetyltransferase. To conclude, the fibrils of Mimiviridae are heavily glycosylated and the type of sugars and their organization depends on their lineage.

Mass spectrometry (MS) is a power analytical tool which is capable of analyzing biomolecules in great detail, both structurally and quantitatively. With regards to glycans, special considerations regarding sample preparation are necessary in order to achieve reproducible identification and relative quantification of these analytes. A workflow for isolation at the glycopeptide level and subsequent detection at the glycan level with phenylhydrazine, demonstrated that monoclonal antibodies (mAbs) containing a specific amino acid mutation were able to express approximately an additional 50% of the α2,6 disialylated glycan compared to their non-mutant analogues. In a second experiment using mAbs, an azide modified glycan (Ac4ManAz) was introduced both metabolically and enzymatically during mAb production. This glycan is a precursor in the sialic acid pathway and the azide moiety allows for specific chemistry post-production including the potential for highly specific enrichment. The results of this workflow demonstrated that [100 μM] of Ac4ManAz precursor added to the cell media was necessary for metabolic expression. More complex samples however, may contain multiple sites of glycosylation. To conserve the site of attachment, these molecules are often studied at the glycopeptide level, and require enrichment of glycopeptides to improve the lower signal intensity observed in the presence of co-eluting peptides. Carboxymethyl chitosan (CMCH) as well as amine-functionalized magnetic-nanoparticles (MNP) were developed as novel materials for this purpose. CMCH is naturally occurring, and therefore is cost-effective and readily available. In a 12 protein mixture CMCH demonstrated the bulk enrichment of glycopeptides yielding an approximately 20% higher enrichment of sialylated species as compared to a commercially available glycopeptide kit through the use of tandem mass tags for relative quantification. In the same approach, amine functionalized MNP were produced and used to enrich glycopeptides from tryptic digests. This approach was fast (about 10 mins) and quantitatively demonstrated improved retention for sialylated species. Examples of these techniques and their applications are reported in this work. / October 2015