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Les enzymes de biosynthèse des glycosaminoglycanes : étude structurale et fonctionnelle de la [bêta]4GalT7 humaine et caractérisation moléculaire des mutations responsables du syndrome progéroide d'Ehlers-Danlos

Abstract : Proteoglycans (PGs) and their glycosaminoglycan chains (GAGs), play a major role in the architecture of extracellular matrices and are implicated in numerous cell events. The impairment of GAG synthesis and sulfation is involved in degenerative, tumor and genetic diseases, such as the progeroid form of Ehlers-Danlos (ED) syndrome. This inherited disorder is due to mutations of human [bêta]4GalT7 ([bêta]4GalT7) causing a defect in GAG synthesis, associated with severe musculo-skeletal alterations. Indeed, this enzyme catalyzes a key step in GAG synthesis linked to the core protein of PGs and from exogenous xylosides. Our work has been focused on the structural and functional characterization of human recombinant [bêta]4GalT7 enzyme. We combined in vitro and ex vivo approaches to explore the role of amino acids located in 163DVD165, 221FWGWGREDDD230 and 257HLH259 motifs, which are highly conserved within [bêta]4GalTs. The study of the consequences of site-directed mutations on kinetic and functional properties of the [bêta]4GalT7 enzyme allowed us to identify key active site amino acids. Our results indicate that D165 and H257 residues form coordination bonds with Mn2+ divalent cations. Furthermore, we suggested a catalytic role for D228 residue and highlighted a central role of W224 residue via interactions with the donor and acceptor substrates. We also determined the molecular basis of [bêta]4GalT7 mutations associated with ED syndrome. Finally, the study of epigenetic regulation mechanisms by DNA methylation of GAG biosynthesis in human chondrosarcoma cells (H-EMC-SS) revealed the specific hypermethylation of the 3-O-sulfotransferase gene family, associated with the invasive phenotype of these cells. Together, this work paves the way towards innovative strategies in the treatment of arthropathies
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Ibtissam Talhaoui. Les enzymes de biosynthèse des glycosaminoglycanes : étude structurale et fonctionnelle de la [bêta]4GalT7 humaine et caractérisation moléculaire des mutations responsables du syndrome progéroide d'Ehlers-Danlos. Médecine humaine et pathologie. Université Henri Poincaré - Nancy 1, 2010. Français. ⟨NNT : 2010NAN10126⟩. ⟨tel-01746345⟩

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