YEAST-DERIVED RECOMBINANT DG42 PROTEIN OF XENOPUS CAN SYNTHESIZE HYALURONAN IN-VITRO

We demonstrate in this report that the Xenopus DG42 gene product made in the yeast Saccharomyces cerevisiae can synthesize authentic high mo lecular weight hyaluronan (hyaluronic acid; HA) in vitro. Saccharomyce s are eukaryotes that do not naturally produce HA or any other molecul es known to contain glucuronic acid, Therefore bakers' yeast is a good model system to determine the enzymatic activity of the DG42 protein, which is the topic of recent debate. Membrane extracts prepared from cells expressing DG42 encoded on a plasmid incorporated [C-14]glucuron ic acid and N-[H-3]acetylglucosamine from exogenously supplied UDP-sug ar nucleotides into a high molecular mass (10(6) to 10(7) Da) polymer in the presence of magnesium ions. Both sugar precursors were simultan eously required for elongation. Control extracts prepared from cells w ith the vector plasmid alone or the DG42 cDNA in the antisense orienta tion did not display this activity. The double-labeled polysaccharide product synthesized in vitro was deemed to be HA by enzymatic analyses ; specific HA lyase could degrade the polymer, but it was unaffected b y protease or chitinase treatments. The fragments generated by HA lyas e were identical to fragments derived from authentic vertebrate HA as compared by high performance liquid chromatography. We conclude that D G42 is a membrane-associated HA synthase capable of transferring both glucuronic acid and N-acetylglucosamine groups.