The Roles of EXT-Like Proteins in Heparan Sulphate Biosynthesis

Abstract

Heparan sulphate (HS) proteoglycans, composed of a core protein and one or more negatively charged HS chains, are a major component of extracellular matrices and are located on the plasma membrane of all animal cells. The HS polysaccharide is synthesized by alternating addition of glucuronic acid (GlcA) and Nacetylglycosamine (GlcNAc) from corresponding UDP-sugar precursors. Due to subsequent modifications, including epimerization of GlcA to iduronic acid and sulphation the resultant polysaccharide is variously sulphate-substituted at different positions. Polymerization of HS chains is believed to be catalyzed by the EXT family of proteins. In humans, the EXT family consists of five members: EXT1, EXT2, EXTL1, EXTL2 and EXTL3. All members of exostoses family harbour in vitro glycosyltransferase activities related to HS chain elongation. EXT1-EXT2 form a heter-oligomeric complex involved in the chain elongation step in HS biosynthesis, but the biological roles of EXTLs are less clearly defined. The aim of the studies presented in this thesis was to investigate the roles of the three EXTLs in heparan sulphate biosynthesis. Down regulation of EXTL2 or EXTL3 expression in a human cell line resulted in increased HS chain length. Also overexpression of EXTL1 or EXTL3 resulted in increased chain length. In contrast, overexpression of EXTL2 resulted in shorter HS chains. The results indicate that these enzymes influence HS synthesis and that their expression levels control HS chain length. In addition, all three EXTL proteins were able to catalyze the addition of a single GlcNAc to [GlcAGlcNAc] n oligosaccharide acceptors, but did not show any detectable transfer of GlcA to GlcNAc[GlcA-GlcNAc]n oligosaccharide acceptors.