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Biosynthesis of heparin / heparan sulfate: cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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1997 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 44, 28158-28163 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
1997. Vol. 272, no 44, 28158-28163 p.
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Medical and Health Sciences
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URN: urn:nbn:se:uu:diva-52352DOI: 10.1074/jbc.272.44.28158ISI: A1997YD47300098PubMedID: 9346972OAI: oai:DiVA.org:uu-52352DiVA: diva2:80261
Available from: 2008-10-17 Created: 2008-10-17 Last updated: 2017-12-04Bibliographically approved

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Li, Jin-PingKjellén, Lena

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