Enzymes involved in heparan sulfate chain elongation: Function of a novel family of tumor suppressors?
1999 (English)Doctoral thesis, comprehensive summary (Other academic)
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.
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 1999. , 46 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 805
Biochemistry, heparan sulfate, biosynthesis, glycosyltransferase, EXT1, EXT2, UDP-glucose
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject Medical and Physiological Chemistry
IdentifiersURN: urn:nbn:se:uu:diva-263ISBN: 91-554-4347-8OAI: oai:DiVA.org:uu-263DiVA: diva2:161947
1999-03-05, lecture hall B21, Uppsala Biomedical Centre, Uppsala University, Uppsala, 09:15