Cloning, Golgi Localization, and Enzyme Activity of the Full-length Heparin/Heparan Sulfate-Glucuronic Acid C5-epimerase

• Published in: The Journal of biological chemistry 2001-06, Vol.276 (24), p.21538-21543
• Main Authors: Crawford, Brett E., Olson, Sara K., Esko, Jeffrey D., Pinhal, MariaA.S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Caenorhabditis elegans - enzymology; Carbohydrate Epimerases - chemistry; Carbohydrate Epimerases - genetics; Carbohydrate Epimerases - metabolism; Cattle; Cell Membrane - enzymology; CHO Cells; Cloning, Molecular; Cricetinae; Drosophila melanogaster - enzymology; Gene Library; Genes, Reporter; glucuronic acid C5-epimerase; Golgi Apparatus - enzymology; heparan sulfate; Humans; Kinetics; Mast-Cell Sarcoma; Mice; Molecular Sequence Data; Recombinant Proteins - analysis; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Transfection; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M100880200

While studying the cellular localization and activity of enzymes involved in heparan sulfate biosynthesis, we discovered that the published sequence for the glucuronic acid C5-epimerase responsible for the interconversion ofd-glucuronic acid and l-iduronic acid residues encodes a truncated protein. Genome analysis and 5′-rapid amplification of cDNA ends was used to clone the full-length cDNA from a mouse mastocytoma cell line. The extended cDNA encodes for an additional 174 amino acids at the amino terminus of the protein. The murine sequence is 95% identical to the human epimerase identified from genomic sequences and fits with the general size and structure of the gene from Drosophila melanogasterand Caenorhabditis elegans. Full-length epimerase is predicted to have a type II transmembrane topology with a 17-amino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An assay with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike other enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myctag or green fluorescent protein to the highly conserved COOH-terminal portion of the protein inhibits its activity. The amino-terminally truncated epimerase does not localize to any cellular compartment, whereas the full-length enzyme is in the Golgi, where heparan sulfate synthesis is thought to occur.