Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate.
The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, A., Kjellén, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997) J. Biol. Chem. 272, 28158-28163) as probe. The approximately 11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N-terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96-99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The approximately 52-kDa, similarly highly active, enzyme originally purified 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) was found to be associated with an approximately 22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein.
Li
J P
JP
Department of Medical Biochemistry and Microbiology, the University of Uppsala, the Biomedical Center, Box 582, SE-751 23 Uppsala, Sweden.
Gong
F
F
El Darwish
K
K
Jalkanen
M
M
Lindahl
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D006493:Heparin / Q000096:biosynthesis*; D006497:Heparan Sulfate / Q000096:biosynthesis*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002238:Carbohydrate Epimerases; D018076:DNA, Complementary; D006493:Heparin; D006497:Heparan Sulfate; D011994:Recombinant Proteins; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase