Pl. Deangelis et Am. Achyuthan, YEAST-DERIVED RECOMBINANT DG42 PROTEIN OF XENOPUS CAN SYNTHESIZE HYALURONAN IN-VITRO, The Journal of biological chemistry, 271(39), 1996, pp. 23657-23660
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.