Enzymes involved in heparan sulfate chain elongation : Function of a novel family of tumor suppressors?
Abstract: Heparan sulfate (HS) is a glycosaminoglycan associated with many physiological functions such as regulation of growth factor action and cell adhesion. The HS polysaccharide is synthesized by alternating addition of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) units from the corresponding UDP-sugar precursors. Due to subsequent modifications including epimerization and sulfation the resultant polymer has a highly heterogeneous structure.The aims of this work were to isolate the enzymes involved in HS chain elongation and to also characterize a functionally similar bacterial enzyme, KfiC. An additional aim was to clone and express UDP-glucose dehydrogenase, i.e. the enzyme generating UDP-GlcA, one of the precursors in HS chain elongation.A single protein associated with the two glycosyltransferase activities (HS-POL) was purified from bovine serum. Cloning and expression of the protein confirmed its activities. HS-POL was identified as EXT2, a gene associated with hereditary multiple exostoses. This human skeletal disorder, characterized by multiple cartilaginous tumors, is ascribed to mutations in three putative tumor suppressor genes, denoted EXT1-3. EXT1 also showed HS-POL activities, implying that both EXT1 and EXT2 are glycosyltransferases required for HS biosynthesis.Bovine UDP-glucose dehydrogenase was cloned and expressed. Both a full length and a truncated form of the protein yielded UDP-glucose dehydrogenase activity.In the bacterial protein KfiC two aspartic acid residues suggested to be critical for the β-glycosyltransferase reaction were found. By site directed mutagenesis these residues were shown to be essential for GlcA transfer.
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