title: Biosynthesis of heparin. Use of Escherichia coli K5 capsular polysaccharide as a model substrate in enzymic polymer-modification reactions.
abstract: A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)----(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3'-phosphate 5'-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.
authors.affiliation: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Kusche
authors.forename: H H
authors.initials: HH
authors.lastname: Hannesson
authors.forename: U
authors.initials: U
authors.lastname: Lindahl
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000378:metabolism; D002240:Carbohydrate Sequence; D002845:Chromatography; D004926:Escherichia coli / Q000032:analysis*; D005965:Glucuronates / Q000378:metabolism; D020723:Glucuronic Acid; D006493:Heparin / Q000096:biosynthesis*; D012515:Mast-Cell Sarcoma / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D010724:Phosphoadenosine Phosphosulfate / Q000494:pharmacology; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D019998:Racemases and Epimerases / Q000378:metabolism; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism
chemical_list: D000991:Antithrombins; D005965:Glucuronates; D011135:Polysaccharides, Bacterial; D013431:Sulfates; D010724:Phosphoadenosine Phosphosulfate; D020723:Glucuronic Acid; D006493:Heparin; D015238:Sulfotransferases; D019998:Racemases and Epimerases