Cloning, Golgi localization, and enzyme activity of the full-length heparin/heparan sulfate-glucuronic acid C5-epimerase.
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 of d-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 melanogaster and 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-myc tag 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.
Department of Cellular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687, USA.
B E
BE
Crawford
S K
SK
Olson
J D
JD
Esko
M A
MA
Pinhal
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D017173:Caenorhabditis elegans / Q000201:enzymology; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D006224:Cricetinae; D004331:Drosophila melanogaster / Q000201:enzymology; D015723:Gene Library; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D012515:Mast-Cell Sarcoma; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C430881:heparin-heparan sulfate-glucuronic acid C5-epimerase