Exploration des glycosyltransférases impliquées dans la synthèse et l'assemblage des glycosaminoglycanes. Impact de la phosphorylation/sulfatation de la chaîne polysaccharidique – GAGnetwork

Hypothesis - Understanding the biosynthetic pathway of glycosaminoglycans (GAGs) is a major issue in cell biology since this process determines the composition of GAG chains that govern the signalling functions of these complex polysaccharides in health and disease. Of special interest is their crucial role in the oncogenic process, including cell transformation, tumour growth, invasion and metastasis. However, the underlying mechanisms of GAG chains assembly remains elusive although most of the heparan-sulfates (HS)- and chondroitin-sulfates (CS)-synthesizing glycosyltransferases have now been identified. Our current knowledge of the mechanisms governing CS- and HS-sorting is limited and the contribution of each particular HS-synthase (EXT/EXTL) and CS-synthase is still an enigma. Objectives - The main goal of our proposal is to elucidate the molecular mechanisms underlying the three major steps of the GAG biosynthetic pathways ie initiation / orientation and polymerization of the polysaccharide chains by combining in vitro and ex vivo approaches. Specifically, we aim to assess (1) the impact of xylose phosphorylation on the maturation of the linkage region (2) the influence of sulfation of the linkage sequence in the sorting of CS and HS chains and (2) the role of sulfatation of the growing nascent GAG chains in the elongation and possibly termination of the synthetic pathway. Towards this goal, we will generate series of substituted (phosphorylated/sulfated) and unsubstituted GAG-derivatives that will be tested as potential substrates of the recombinant human glycosyltransferases involved HS and CS chains synthesis. We next will determine the molecular basis governing the substrate specificity of these enzymes with regard to phosphorylation and sulfation. For this purpose, we will investigate the structural determinants that confer acceptor selectivity and govern the recognition of modified or unmodified oligosaccharide structures using bio-informatic approaches, protein-engineering strategies and surface plasmon resonance (SPR, BIAcore) analysis of substrate-enzyme interactions. Finally, we wish to complement these in vitro approaches by the investigation of GAG chains processing and assembly in cellular models (chondrocytes and chondrosarcoma cell lines). We aim to define the individual contribution of the glycosyltransferases in HS and CS chain assembly in these cells. For this purpose we will develop (1) gene transfer overexpression (2) siRNA and DNA methylation silencing strategies and evaluate the consequences of these manipulations on the composition and structure of cellular ad extracellular GAG chains. Expected results – Two main advances are expected from this project : (1) Understanding of how the network of glycosyltransferases contribute to the mechanism of assembly of the different types of GAG chains. Particularly, the introduction of sulfate and phosphate substitutents on analogs of acceptor substrate should allow to identify key-regulatory mechanisms of GAG synthesis. This project should lead to the design of molecules that are able to initiate GAG formation or to direct the biosynthesis towards a selected GAG type (2) Taking into account the key role played by GAGs in tumorigenesis, the knowledge gain from these studies is expected to open avenues in the development of gAG-based therapeutics especially in the field of musculosqueletal oncology.