title: Irreversible glucuronyl C5-epimerization in the biosynthesis of heparan sulfate.
abstract: Glucuronyl C5-epimerase catalyzes the conversion of d-glucuronic acid to l-iduronic acid units in heparan sulfate biosynthesis. Substrate recognition depends on the N-substituent pattern of the heparan sulfate precursor polysaccharide and requires the adjacent glucosamine residue toward the non-reducing end to be N-sulfated. Epimerization of an appropriately N-sulfated substrate is freely reversible in a soluble system, with equilibrium favoring retention of d-gluco configuration (Hagner-McWhirter, A., Lindahl, U., and Li, J.-P. (2000) Biochem. J. 347, 69-75). We studied the reversibility of the epimerase reaction in a cellular system, by incubating human embryonic kidney 293 cells with d-[5-(3)H]galactose. The label was incorporated with glucuronic acid units into the heparan sulfate precursor polysaccharide and was lost upon subsequent C5-epimerization to iduronic acid. However, analysis of oligosaccharides obtained by deaminative cleavage of the mature heparan sulfate chains indicated that all glucuronic acid units retained their C5-(3)H label, irrespective of whether they had occurred in sequences susceptible or resistant to the epimerase. All (3)H-labels of the final products resisted incubation with epimerase in a soluble system, apparently due to blocking O-sulfate groups. These results indicate that glucuronic acid C5-epimerization is effectively irreversible in vivo and argue for a stringent organization of the biosynthetic machinery.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 582, S-751 23 Uppsala, Sweden.
authors.forename: Asa
authors.initials: A
authors.lastname: Hagner-McWhirter
authors.forename: Jin-Ping
authors.initials: JP
authors.lastname: Li
authors.forename: Stefan
authors.initials: S
authors.lastname: Oscarson
authors.forename: Ulf
authors.initials: U
authors.lastname: Lindahl
mesh_terms: D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005690:Galactose / Q000737:chemistry; D020723:Glucuronic Acid / Q000737:chemistry; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D007067:Iduronic Acid / Q000737:chemistry; D008969:Molecular Sequence Data; D009608:Nitrous Acid / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D013379:Substrate Specificity; D014316:Tritium / Q000737:chemistry
chemical_list: D002238:Carbohydrate Epimerases; D002241:Carbohydrates; D005690:Galactose; D020723:Glucuronic Acid; D006497:Heparan Sulfate; D007067:Iduronic Acid; D009608:Nitrous Acid; D009844:Oligosaccharides; D011134:Polysaccharides; D014316:Tritium; C430881:heparin-heparan sulfate-glucuronic acid C5-epimerase