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: M Kusche, H H Hannesson, U Lindahl
• Journal: The Biochemical journal (Biochem J) Vol. 275 ( Pt 1) Pg. 151-8 (Apr 01 1991) ISSN: 0264-6021 [Print] England
• PMID: 1902083 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antithrombins
• Glucuronates
• Polysaccharides, Bacterial
• Sulfates
• Phosphoadenosine Phosphosulfate
• Glucuronic Acid
• Heparin
• Sulfotransferases
• Racemases and Epimerases

Topics

• Animals
• Antithrombins (metabolism)
• Carbohydrate Sequence
• Chromatography
• Escherichia coli (analysis)
• Glucuronates (metabolism)
• Glucuronic Acid
• Heparin (biosynthesis)
• Mast-Cell Sarcoma (enzymology)
• Mice
• Molecular Sequence Data
• Phosphoadenosine Phosphosulfate (pharmacology)
• Polysaccharides, Bacterial (metabolism)
• Racemases and Epimerases (metabolism)
• Substrate Specificity
• Sulfates (metabolism)
• Sulfotransferases (metabolism)