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Exploration des enzymes de biosynthèse des protéoglycanes et leurs altérations lors de pathologies articulaires

Abstract : Located at the cell-tissue-organ interface, heparan sulfate proteoglycans (HSPGs) facilitate ligand-receptor interactions crucial to many physiopathological processes. The synthesis of glycosaminoglycans (GAGs) requires the coordinated action of an array of glycosyltransferases (GTs) and sulfotransferases (STs). Although the biosynthetic scheme of HSPGs has been outlined, little is known about the regulation of this complex process. In the first part of our work, we performed structure-function studies of the human [Bêta]1,4-galactosyltransferase 7 ([Bêta]4GalT7) which catalyses a key step of the initiation of GAG synthesis and identified two conserved domains D163VD165 and 221FWGWGREDDE230 in the [Bêta]4GalT family. In vitro studies combined to ex vivo analysis of PG synthesis by 35S incorporation allowed us to determine the impact of site-directed mutations on the catalytic and functional properties of [Bêta]4GalT7. We identified key residues for donor UDP-galactose/Mn2+ and acceptor substrate binding, and catalysis. Secondarily, to investigate more in depth the mechanisms involved in the regulation of PG biosynthetic pathway, we inspected the 5' region of the genes coding for GT and ST enzymes and identified the presence of typical CpG islands with the most striking hypermethylation pattern for the 3-OST gene subfamily in the chondrosarcoma cell line H-EMC-SS (HEMC). Aberrant methylation was associated with downregulation of these genes and altered HS-GAG chains, as demonstrated both at biochemical and cellular levels. Treatment of cells with an inhibitor of DNA methyltransferases (5-aza-2'deoxycytidine) or reintroduction of 3-OST cDNA expression into HEMC cells resulted in a decrease in the proliferative and migration capacities of HEMC cells, and augmented adhesion ability of the cells. These findings underline the significance of HS alterations in the invasive phenotype of HEMC and identify 3-O-sulfation as a contributing factor to this process. We show for the first time that a specific set of HS-O-sulfotransferases is regulated via an epigenetic mechanism in HEMC cells, may be of particular relevance in understanding the process of cartilage tumor pathogenesis, towards the development of therapies targeting DNA methylation.
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Catherine Bui. Exploration des enzymes de biosynthèse des protéoglycanes et leurs altérations lors de pathologies articulaires. Médecine humaine et pathologie. Université Henri Poincaré - Nancy 1, 2009. Français. ⟨NNT : 2009NAN10100⟩. ⟨tel-01748292⟩



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