Dystroglycan is a heterodimeric transmembrane glycoprotein protein of α and β subunits that links the extracellular matrix to the cytoskeleton. Recent data tend to suggest that the role of dystroglycan in non-muscle cells is for cell adhesion, and cytoskeleton reorganisation signalling. To study the relationship between dystroglycan signalling and ERK signalling in focal adhesions, an YFP-ERK construct was expressed in REF52 cells. YFP-ERK was expressed with ERK activity and formed adhesion-like structures in the REF52 cells, however, no ERK activity was detected in the adhesion-like structures in the REF52 cells. To determine the function of dystroglycan for adhesion or cytoskeleton organisation in non-muscle cells, a GFP-tagged full-length dystroglycan (αβDG-GFP) or its deletion mutants was expressed in REF52 cells. Expression of αβDG-GFP markedly altered cell phenotype on a laminin or fibronectin substrate, resulting in the induction of actin-rich filopodia. The β-dystroglycan cytoplasmic domain is determined as the mediator for the dystroglycan-dependent filopodia formation mediated partly by integrin signalling. The dystroglycan deletion mutants lacking α-dystroglycan failed to target to the plasma membrane. Expression of an alkaline phosphate-tagged β-dystroglycan cytoplasmic domain construct (AP-cβ), which targeted the β-dystroglycan cytoplasmic domain to the membrane without the α-dystroglycan, was sufficient to induce the filopodia phenotype, indicating that with the proper membrane localisation, β-dystroglycan can regulate filopodia formation independently of α-dystroglycan. However, α-dystroglycan might be necessary for β-dystroglycan to target to the plasma membrane. These distinct morphologies strongly implied that β-dystroglycan mediates Cdc42 regulated cytoskeleton reorganisation. By cotransfecting dominant negative Cdc42 (Cdc42N17) or constitutively activated Cdc42 (V12Cdc42) constructs with αβDG-GFP or the mutants, Cdc42 was determined to be a mediator for dystroglycan-dependent filopodia formation. Therefore a signalling cycle of dystroglycan-Cdc42-PAK-ezrin-dystroglycan is identified. With this signalling cycle, dystroglycan plays a role in inducing actin filopodia formation and, at the same time, might also inhibit focal adhesion and stress fibre formation.

The antiphospholipid syndrome (APS), is an autoimmune condition characterised by the occurrence of thrombosis (both arterial and venous) and obstetric morbidity along with the persistent production of antiphospholipid antibodies (aPL). At present the pathophysiology of APS is unclear although several hypothesis are available in the literature. This thesis has aimed to examine some of the processes behind the APS focusing on the interaction of aPL with components of the fibrinolytic system and the interaction of Beta2 glycoprotein I (β2GPI) with fibrinolytic and other proteolytic enzymes. A detailed examination of our patient cohort was undertaken and compared to new serological criteria for APS. The interaction of aPL on plasmin mediated cleavage of β2GPI was examined and found to be reduced in the presence of some aPL. Plasma kallikrein and Xa were tested for proteolytic activity on β2GPI and found to posses this although to a lesser degree to that seen for plasmin. The effect of domain V genetic polymorphisms of βGPI on the action of plasmin on this part of the molecule was examined and found to be reduced in the presence of these polymorphisms. An investigation into the effect of one of these polymorphisms (Cys306Gly) on aPL production was undertaken with negative results in this respect. An examination of the effect of aPL on the binding of plasminogen to endothelial cell surfaces was undertaken and in some patient samples found to have an effect on this process. Moreover, some aPL were found to reduce fibrinolysis in a plasma clot lysis assay.

Von Willebrand Factor (VWF) is a large multi-domain plasma glycoprotein that is critical for normal platelet tethering during haemostasis. ADAMTS13 is a plasma metalloprotease that regulates VWF multimeric size/function by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. Deficiency of ADAMTS13 causes microvascular thrombosis. The only reported cleavage of VWF by ADAMTS13 is that of the Tyr1605-Met1606 bond. How this high substrate specificity is conferred remains unclear. To date, all the interactions between these molecules that have been described involve VWF residues C-terminal to the scissile bond and the non-catalytic domains of ADAMTS13. I hypothesised that VWF also contains an interaction site for ADAMTS13 N-terminal of the cleavage site to aid both in positioning of the scissile bond in the active site and substrate specificity. Previous studies have suggested that the VWF sequence between Asp1596 and Val1604, N-terminal to the cleavage site, is essential for cleavage by ADAMTS13. My aim was to identify the residues in this region that are important determinants for ADAMTS13 proteolysis. A panel of mutations were introduced into the substrate VWF 115 (VWF residues 1554- 1668). The mutants were expressed purified and their proteolysis by ADAMTS13 analysed. It was found that the proteolysis of VWF 115 variants (L1603A, L1603S, L1603N or L1603K) were all substantially impaired (up to >400 fold reduction). The importance of VWF Leu1603 was confirmed using a synthetic peptide 1596DREQAPNLVY1605, which competitively inhibited proteolysis of VWF 115 by ADAMTS13. A mutant peptide containing the L1603A mutation, 1596DREQAPNAVY1605, had minimal effect. When the VWF L1603A substitution was introduced into the full-length recombinant VWF, proteolysis by ADAMTS13 was again substantially reduced. These findings implied the presence of a subsite (S3) in the ADAMTS13 metalloprotease (MP) domain that interacts with VWF Leu1603. Using molecular modelling, the distance between VWF Leu1603 and the scissile bond was estimated as ~10Å. Structural homology modelling of the MP domain, mutagenesis of 11 candidate residues and functional characterisation of these variants identified two clusters, Leu198/Leu232/Leu274 and Val195/Leu151, as possible subsites interacting with VWF. It is suggested that VWF Leu1603 interacts with Leu198/Leu232/Leu274, while Val195/Leu151 may interact with VWF Tyr1605. I propose a mechanism for VWF cleavage involving remote C-terminal domain interactions that assist initial orientation of the VWF scissile bond within the active site of ADAMTS13, but in which N-terminal hydrophobic interactions between VWF Leu1603 and the S3 subsite in the MP domain of ADAMTS13 are critical for the positioning required for cleavage of the Tyr1605-Met1606 scissile bond.

No description available.

Two galactose-binding receptors, the hepatic asialoglycoprotein receptor (ASGPR) and the macrophage galactose lectin (MGL) have been investigated. The ASGPR is believed to function in glycoprotein clearance from serum while MGL is involved in recognition of pathogens and tumours and in signalling and immunomodulation. This work describes the analysis of the specificity, structure and organisation of both receptors in humans and the two MGLs in mice. The ligand-binding properties of the two subunits of the ASGPR as well as MGL have been separately tested in glycan array analysis. The results show that primary binding to ligands in the human ASGPR occurs via the ASGPR-1 subunit. MGLs have different specificities even though they are highly similar in sequence and the two mouse MGLs differ markedly from the single MGL in humans and in rats. One of the mouse MGLs has a similar specificity to ASGPR-1 that evolved independently. Hydrodynamic studies of ASGPR-1 revealed that it can form homo-oligomers and circular dichroism analysis of the neck fragment showed that it has a coiled-coil structure. Hetero-oligomer formation was monitored using a mutant version of ASGPR1 that allows purification of the complex using double-affinity chromatography on galactose and mannose. Hetero-oligomers containing both types of subunits are more stable than homo-oligomers. The results suggest a model that can account for the variable subunit stoichiometries observed by various investigators. Hydrodynamic studies and circular dichroism of MGL suggest that the extracellular domain of the human protein is an oligomer not as stable as previously thought, and that its neck is a coiled-coil structure. For both receptors, transmembrane and cytoplasmic domains as well as glycosylation may have a role in their stability. The ability of MGL to recognise pathogen glycans was demonstrated using Trichinella spiralis secretions. It was found that similar glycoproteins are bound by the human and mouse receptors.

No description available.

Les mucines sont impliquées dans de nombreux mécanismes biologiques comme la cancérogenèse et les interactions hôte/pathogènes, faisant de ces glycoprotéines de potentielles cibles thérapeutiques ou diagnostiques. Il a, par exemple, été démontré une néoexpression de MUC5AC dans les tumeurs coliques et dans les foyers de cryptes aberrantes (FCA) qui apparaissent dès les premières étapes de développement du cancer du côlon. MUC5AC constitue ainsi un biomarqueur de choix pour le diagnostic et le pronostic du cancer du côlon. La première partie de notre travail de thèse a consisté à développer un produit de contraste adapté à l’imagerie du cancer du côlon. Pour cela, nous avons tout d’abord sélectionné, par la technique du phage display, un peptide affin vis-à-vis de MUC5AC qui a été préalablement purifiée à partir d’adénomes coliques humains. Ce peptide a ensuite été conjugué à la biotine et greffé à un produit de contraste pour l’imagerie par résonance magnétique (IRM) (USPIO : Ultrasmall Particles of Iron Oxide). Cela nous a permis d’apporter la preuve de l’efficacité de détection par IRM des polypes pré-cancéreux dans des modèles animaux et cellulaires qui surexpriment MUC5AC et dans des tissus coliques humains. Dans l’estomac, MUC5AC est exprimée de façon physiologique et est impliquée dans l’adhésion d’Helicobacter pylori à la muqueuse gastrique. Cette adhésion est un prérequis aux pathologies induites par H. pylori. Dans la deuxième partie de notre travail de thèse, nous avons démontré que parmi les différents oligosaccharides portés par les mucines gastriques humaines, un nouveau type de O-glycane portant un motif LacdiNAc (LDN) était impliqué dans l’adhésion de la bactérie à la muqueuse gastrique. Le LDN est exclusivement porté par la mucine MUC5AC gastrique et son expression est corrélée à la localisation de H. pylori. Toutes les souches de H. pylori testées adhèrent fortement au LDN. L’analyse protéomique et la construction de mutants ont permis d’identifier une nouvelle adhésine bactérienne, LabA (LacdiNAc antigen binding adhesin) qui reconnait spécifiquement le LDN. Cette découverte permet de mieux comprendre les mécanismes d’adhésion d’H. pylori et son tropisme d’organe. Ces résultats sont le point de départ d’études visant à étudier l’implication du LDN dans la physiologie gastrique et l’homéostasie, mais aussi pour le développement de stratégies alternatives pour le traitement de l’infection à H. pylori. / Mucins are implicated in different biological phenomena such as cancer development and host/pathogen interactions. Mucins have thus been identified as promising therapeutic targets and have been proposed as potential prognostic or diagnostic markers.Improved detection sensitivity of early colorectal cancer would have important clinical applications. In this PhD thesis work, we report the development of new peptides against MUC5AC, a good marker of aberrant crypt foci (ACF), for the early diagnostic of colorectal cancer. Peptides have been identified by screening phage display peptide libraries against MUC5AC purified from fresh human colonic adenomas. One heptapeptide has been selected and further conjugated with biotin for immunohistochemistry studies and USPIO (ultrasmall superparamagnetic iron oxides) for nuclear magnetic resonance imagery (MRI). Its efficiency to detect MUC5AC has been evaluated on cellular and animal models which overexpressed this mucin, as well as on human colonic tissues. In stomach, MUC5AC is physiologically expressed and is implicated in adhesion of Helicobacter pylori to the gastric mucosa. This adhesion is a necessary prerequisite for the pathogenesis of H. pylori related diseases. Here we demonstrated that, among all the oligosaccharides expressed on human gastric mucins, a new type of O-glycan carrying a LacdiNAc motif is implicated in the binding of the bacteria to the gastric mucosa. LacdiNAc was exclusively carried by gastric MUC5AC mucin and its expression correlated with H. pylori localization. All strains tested adhere strongly to LacdiNAc. Proteomic analysis and construction of mutants allowed us to identify a novel bacterial adhesin, LabA (LacdiNAc antigen binding adhesin), which specifically recognizes LacdiNAc. These findings give new insights into the mechanisms of adhesion of H. pylori and its specific tissue tropism. We anticipate our results to be a starting point to study implication of LDN in gastric physiology and homeostasis and for development of alternative strategies for treatment of H. pylori infection.

La mucoviscidose est la maladie génétique la plus fréquente dans les populations caucasiennes. Elle est causée par des mutations du gène codant pour la protéine CFTR. Elle se caractérise par une inflammation et une infection pulmonaire chronique avec hypersécrétion de mucus. P. aeruginosa y trouve avantage pour coloniser les poumons. Les objectifs de cette thèse ont été de caractériser, dans un modèle porcin, les altérations de la composition biochimique des mucines dès la naissance; d’identifier les mécanismes moléculaires responsables de ces altérations; d’évaluer l’impact sur la colonisation par P. aeruginosa. Nous avons montré que les mucines respiratoires des porcelets CFTR-/- étaient plus sialylées que celles des porcelets CFTR+/+ dès la naissance, avant toute infection ou inflammation. Ces altérations n’étaient pas corrélées à des changements d’expression des mucines, ni à des changements d’expression génique ou protéique des siayltransférases. Nous avons démontré un effet direct de l’activité du canal CFTR sur la sialylation des mucines, pouvant être expliqué par un stress du réticulum endoplasmique. Nous avons également démontré que le transport muco-ciliaire est incapable d’éliminer les bactéries. Cette hypersialylation favorise l’adhésion de P. aeruginosa aux mucines et engendre un environnement favorable à sa croissance. L’infection des porcelets CFTR-/- et CFTR+/+ par P. aeruginosa entraine les mêmes modifications de sialylation. Ce travail de thèse a permis de comprendre comment P. aeruginosa est capable de coloniser les poumons CFTR-/-. Il constitue la base de travaux futurs visant à développer de nouveaux glycomimétiques pour lutter contre ces infections. / Cystic fibrosis is the most common lethal genetic disease in Caucasian populations caused by mutations in the gene coding for the CFTR protein. It is characterized by inflammation, hypersecretion of respiratory mucins and colonization of the lungs by a variety of bacteria such as P. aeruginosa. The objectives of this thesis were to characterize the biochemical alterations of mucins at birth; to identify the molecular mechanisms responsible for these alterations; to evaluate the impact of these alterations on P. aeruginosa lung colonization. We have shown that the respiratory mucins of CFTR-/- piglets were more sialylated before any previous infection or inflammation. These alterations in mucin glycosylation were not correlated with changes in the expression or localization of the secreted mucins, nor with changes in the gene and protein expression of sialyltransferases. We demonstrated a direct effect of the CFTR channel activity on mucin sialylation, which could be explained by endoplasmic reticulum stress. We have shown that mucociliary transport was unable to eliminate bacteria. The changes in mucin sialylation promoted adhesion of P. aeruginosa to the secreted mucins and created a favorable environment for its growth. The same profiles of hypersialylated glycans, as those observed constitutively in CFTR-/- neonates, were detected after infection by P. aeruginosa. Our results prove that sialylation provides a favorable ecological niche for P. aeruginosa. Data gleaned from these study provide new leads for the design of sialylated glycomimetics and the development of therapeutic strategies for treating infections.

High concentrations of large very low density lipoproteins (VLDL1) give rise to atherogenic dyslipidaemia, which is usually associated with insulin resistant conditions (e.g. obesity) and increases the risk for cardiovascular disease (CVD). Isotopic tracer methods for determining VLDL1 kinetics are costly, time-consuming, labour intensive and need experience and skill to calculate the kinetic parameters. The aim of this thesis was to develop a simpler and cost-effective method of obtaining triglyceride-rich lipoproteins (TRL) kinetic data, based on the fact that chylomicrons (CM) or CM-like particles (e.g. Intralipid) compete with large VLDL1 for the same lipoprotein lipase (LPL)-mediated catalytic pathway. From this method, it was possible to determine VLDL1-triglyceride (TG) and -apolipoprotein (apo) B production rates and the Intralipid-TG clearance rate (as a surrogate measure of CM clearance). Kinetic data obtained from this method agreed with values and relationships obtained from stable isotope methods. The protocol is relatively quick, inexpensive, and transferable to non-specialist laboratories. As a first application, the ‘Intralipid method’ was used to investigate the effects of hyperinsulinaemia and hyperglycaemia due to glucose ingestion on VLDL1-TG and -apoB production rates and Intralipid-TG clearance rate. This showed that hepatic VLDL1 production is suppressed in response to hyperinsulinaemia and that the change in Intralipid-TG clearance rate with hyperinsulinaemia correlated significantly with HOMA-estimated insulin resistance (HOMA-IR). In addition, alanine aminotranferase (ALT) concentrations (a marker for liver fat), within normal range, predicted the extent of hepatic VLDL1 suppression. Secondly, the Intralipid method was used to investigate the mechanisms responsible for the hypotriglyceridaemic effect of a moderate exercise session (120 min walking at 50% VO2max) in overweight/obese middle-aged men; the section of the population at high risk of CVD in whom exercise-for-health guidelines are targeted. This showed that the exercise-induced reduction in plasma TG was due to increased VLDL1-TG and -apoB clearance, rather than decreased hepatic production. Exercise also increased Intralipid-TG clearance rate, but to a lesser extent than VLDL1, suggesting an increased affinity of VLDL1 for LPL-mediated lipolysis post-exercise. Taken together, the Intralipid method is a relatively simple, safe and cost-effective method to determine in VLDL1-TG and -apoB production rates and Intralipid-TG clearance rates. It is also sensitive enough to detect physiological changes in TRL kinetics.

La transcription du gène lactoferrine conduit à deux isoformes : une sécrétée, la lactoferrine (Lf) et une intracellulaire, la delta lactoferrine (∆Lf). La Lf est dotée de nombreuses propriétés parmi lesquelles des activités immunomodulatrice et anticancéreuse. La ∆Lf possède une activité anticancéreuse via son activité transcriptionnelle. Ce sont des suppresseurs de tumeur potentiels dont l’expression est sous-régulée dans le cas de cancer. Elles sont considérées comme des marqueurs sains car leur expression est corrélée à un facteur de bon pronostic dans le cancer du sein. Au cours de la thèse, j’ai développé la PCR quantitative Taqman afin de distinguer les deux isoformes et étudié leur expression dans le cas de cancer et dans un contexte inflammatoire. Puis j’ai étudié le protéome de cellules HEK-293 exprimant la ∆Lf par électrophorèse 2-D et spectrométrie de masse. J’ai ainsi mis en évidence une nouvelle cible de l’activité transcriptionnelle de la ∆Lf, DcpS, enzyme clé de la maturation des ARNm. En parallèle, ces travaux ont montré une régulation de la stabilité et de l’activité transcriptionnelle de la ∆Lf par la O-GlcNAcylation. Enfin, j’ai étudié les effets différentiels des isoformes de la Lf dans les cellules de glande mammaire cancéreuse MDA-MB-231 via une approche protéomique basée sur le SILAC et la spectrométrie de masse, en collaboration avec l’équipe du Dr. Monsarrat (IPBS, Toulouse). 5030 protéines ont pu être identifiées et quantifiées. Ces travaux ont permis d’identifier le gène SelH en tant que cible de l’activité transcriptionnelle de la Lf et de la ∆Lf, suggérant que la Lf sécrétée possède également un rôle de facteur de transcription. / Transcription of the lactoferrin gene results in the production of two isoforms: a secreted lactoferrin (Lf) and intracellular delta-lactoferrin (ΔLf). Lf has numerous functions including immunomodulatory and antitumoral activities. We showed that ΔLf is a transcription factor, the only activity it shares with Lf being its anticancer activity. Lf and ΔLf are potential tumor suppressors whose expression is downregulated in the case of cancer. They may be considered as healthy markers, the expression of which is correlated with a good prognosis in breast cancer. During my PhD thesis, We developed quantitative Taqman PCR assay to evaluate the level of expression of the transcripts of the two Lf isoforms in case of cancer or in an inflammatory context. We then studied the proteome profile of ΔLf-expressing cells using 2-D electrophoresis and mass spectrometry analyses. Our data established that DcpS, a key enzyme in mRNA decay, is a new target of ΔLf transcriptional activity. In parallel, we demonstrated that Lf stability and transcriptional activity are regulated by O-GlcNAcylation. Finally, We compare the differential effects of the two antitumoral isoforms on the cancerous mammary gland MDA-MB-231 cell line. We used a high-throughput proteomic approach (Stable Isotope Labelling with Amino acids in Cell culture, SILAC) coupled to mass spectrometry to carry out highly accurate quantitative and systematic proteome profiling in collaboration with the team of Dr. Monsarrat (IPBS, Toulouse). Our study allowed the identification of SelH, a thioredoxin like protein, as a target of both Lf and ΔLf transcriptional activity and suggested that secreted Lf acts as a transcription factor.