Investigation of the role of xylosyltransferases I and II and of the kinase Fam20B in the regulation of proteoglycan synthesis

Abstract : Heparan- (HS) and chondroitin-sulfate (CS) proteoglycans (PGs) are essential regulators of many biological processes including cell differentiation, signalization, proliferation and morphogenesis. Indeed, PGs act through their glycosaminoglycan (GAG) chains as receptors for growth factors, enzymes and cell adhesion proteins, thereby modulating their bioavailability, gradient formation and biological activity. The assembly of HS and CS GAG chains is initiated by the transfer of xylose to serine residues of PG core protein by the xylosyltransferases (XT) enzymes, XT-I and XT-II. These enzymes catalyze a rate-limiting step in the biosynthesis pathway and therefore considered as a regulating factors in the GAG biosynthesis process. Beside the regulation by XT enzymes, GAG chain synthesis may also be regulated by phosphorylation of the xylose residue at 2-0 position by the kinase Fam20B. Ithas been shown that XT-I and XT-II are able to restore GAG-attached PG synthesis in xylosyltransferase-deficient cells, suggesting that they are functionally redundant. However, nothing is known of the specific roles of XT-I and XT-II ifany and of the impact of XT-I and XT-II mutations on the synthesis of CS- and HS-PG. Here, we showed that XT-I initiates PGs with large size CS- and HS-GAG chains compared to XT-II and demonstrated that this was linked to their subcellular localisation. In addition, we have addressed the question of whether genetic mutations of XT-I and XT-II associated with various diseases impact CS- and HS-PG synthesis and found that mutations in XT-I strongly reduced the capacity of the enzyme to initiate the synthesis of both CS and HS GAG chains. However, two mutations in XT-II abrogated the capacity of the enzyme to initiate CS and HS GAGs and led to the mislocalisation of the enzyme. Furthermore, we demonstrated using variouse cell lines and dead mutants that Fam20B negatively regulates GAG synthesis process and that phosphorylation of xylose residue by this kinase resulted in a blokage of the polymerisation procees of the GAG chain
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Irfan Shaukat. Investigation of the role of xylosyltransferases I and II and of the kinase Fam20B in the regulation of proteoglycan synthesis. Biochemistry, Molecular Biology. Université de Lorraine, 2015. English. ⟨NNT : 2015LORR0267⟩. ⟨tel-01754553⟩

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