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 i n heparan sulfate biosynthesis, we discovered that the published sequence f or 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 prot ein. 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-a mino acid transmembrane domain and an 11-amino acid cytoplasmic tail. An as say with increased sensitivity was devised that detects enzyme activity in extracts prepared from cultured cells and in recombinant proteins. Unlike o ther enzymes involved in glycosaminoglycan biosynthesis, the addition of a c-myc tag or green fluorescent protein to the highly conserved COOH-termina l portion of the protein inhibits its activity. The amino-terminally trunca ted epimerase does not localize to any cellular compartment, whereas the fu ll-length enzyme is in the Golgi, where heparan sulfate synthesis is though t to occur.