title: Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide.
abstract: Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)n, was chemically modified through N-deacetylation, N-sulphation and O-sulphation [Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr. Res. 263, 271-284]. Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT). A high-affinity subfraction, M(r) approx. 36,000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin. Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a Kd of approx. 200 nM. The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues. Another preparation, M(r) approx. 13,000, of higher overall O-sulphate content, exhibited a single binding site per chain, with Kd approx. 1 microM, and an anti-(Factor Xa) activity of 70 units/mg. Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO3(3,6-di-OSO3)- disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin. The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36,000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide. These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin.
authors.affiliation: Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
authors.forename: N
authors.initials: N
authors.lastname: Razi
authors.forename: E
authors.initials: E
authors.lastname: Feyzi
authors.forename: I
authors.initials: I
authors.lastname: Björk
authors.forename: A
authors.initials: A
authors.lastname: Naggi
authors.forename: B
authors.initials: B
authors.lastname: Casu
authors.forename: U
authors.initials: U
authors.lastname: Lindahl
mesh_terms: D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology; D016667:Bacterial Capsules; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000737:chemistry*; D006493:Heparin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013464:Sulfuric Acids / Q000737:chemistry
chemical_list: D000925:Anticoagulants; D011135:Polysaccharides, Bacterial; D013464:Sulfuric Acids; C050673:capsular polysaccharide K5; D006493:Heparin