Biosynthesis of heparin/heparan sulfate. cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung.
Glucuronyl C5-epimerases catalyze the conversion of D-glucuronic acid (GlcUA) to L-iduronic acid (IdceA) units during the biosynthesis of glycosaminoglycans. An epimerase implicated in the generation of heparin/heparan sulfate was previously purified to homogeneity from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-p. (1994) J. Biol. Chem. 269, 26953-26958). The present report describes the molecular cloning and functional expression of the lung enzyme. The cloned enzyme contains 444 amino acid residues and has a molecular mass of 49,905 Da. N-terminal sequence analysis of the isolated liver enzyme showed this species to be a truncated form lacking a 73-residue N-terminal domain of the deduced amino acid sequence. The coding cDNA insert was cloned into a baculovirus expression vector and expressed in Sf9 insect cells. Cells infected with recombinant epimerase showed a 20-30-fold increase in enzyme activity, measured as release of 3H2O from a polysaccharide substrate containing C5-3H-labeled hexuronic acid units. Furthermore, incubation of the expressed protein with the appropriate (GlcUA-GlcNSO3)n substrate resulted in conversion of approximately 20% of the GlcUA units into IdceA residues. Northern analysis implicated two epimerase transcripts in both bovine lung and liver tissues, a dominant approximately 9-kilobase (kb) mRNA and a minor approximately 5-kb species. Mouse mastocytoma cells showed only the approximately 5-kb transcript. A comparison of the cloned epimerase with the enzymes catalyzing an analogous reaction in alginate biosynthesis revealed no apparent amino acid sequence similarity.
Li
J
J
Department of Medical and Physiological Chemistry, University of Uppsala, S-751 23 Uppsala, Sweden. Jin-Ping.Li@medkem.uu.se
Hagner-McWhirter
A
A
Kjellén
L
L
Palgi
J
J
Jalkanen
M
M
Lindahl
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics*; D002417:Cattle; D003001:Cloning, Molecular; D015342:DNA Probes; D018076:DNA, Complementary; D006497:Heparan Sulfate / Q000096:biosynthesis*; D008168:Lung / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data
D002238:Carbohydrate Epimerases; D015342:DNA Probes; D018076:DNA, Complementary; D006497:Heparan Sulfate; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase