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 molecular weight hyaluronan (hyaluronic acid; HA) in vitro. Saccharomyces are eukaryotes that do not naturally produce HA or any other molecules 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 [14C]glucuronic acid and N-[3H]acetylglucosamine from exogenously supplied UDP-sugar nucleotides into a high molecular mass (10(6) to 10(7) Da) polymer in the presence of magnesium ions. Both sugar precursors were simultaneously required for elongation. Control extracts prepared from cells with the vector plasmid alone or the DG42 cDNA in the antisense orientation 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 by protease or chitinase treatments. The fragments generated by HA lyase were identical to fragments derived from authentic vertebrate HA as compared by high performance liquid chromatography. We conclude that DG42 is a membrane-associated HA synthase capable of transferring both glucuronic acid and N-acetylglucosamine groups.
DeAngelis
P L
PL
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Achyuthan
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D016695:Glycosyltransferases; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029867:Xenopus Proteins; D014982:Xenopus laevis
D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D006820:Hyaluronic Acid; D016695:Glycosyltransferases; C056944:HAS1 protein, Xenopus