The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

Casu, Benito; Oreste, Pasqua; Torri, Giangiacomo; Zoppetti, G; Choay, J; Lormeau, J C; Petitou, Maurice; Sînaÿ, Pierre

doi:10.1042/bj1970599

Cited by 333 publications

(127 citation statements)

References 29 publications

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“…The peak indicated at 57.5 ppm is from a trace of ethanol. The assignments for GlcNS-3S were reported by Casu et al [5] and our spectrum is in agreement (Fig. 1C).…”

Section: Preparation Of 3-0-sulphated Monosaccharidessupporting

confidence: 81%

Flavobacterium heparinum 3‐O‐sulphatase for N‐substituted glucosamine 3‐O‐sulphate

Bruce

McLEAN

Long

et al. 1985

European Journal of Biochemistry

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A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-0-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-~-glucose and 2-acetamido-2-deoxy-3-0-sulpho-D-glucose.The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56000. Two novel assays were developed using 24 14C]acetamido-2-deoxy-3-O-sulpho-~-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-~-glucose as respective substrates.The purified 3-0-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K + or Mg2' ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-Ca2+. Inorganic phosphate and sulphate inhibited 3-0-sulphatase activity. The K , value of the N-acetylated substrate was determined to be 42 pmol dm-3. No activity was detected with 2-amino-2-deoxy-3-0-sulpho-~-glucose.The first report of glucosamine-3-0-sulphate in heparin came from permethylation studies by Danishefsky et al. [l]. Subsequently, Leder [2] synthesised l-O-methyl-2-deoxy-2-sulphamido-3-O-sulpho-~-glucopyranoside and detected a 3-0-sulphatase in human urine. The enzyme was partially purified and found to be active against the non-reducing terminal 3-0-sulphate of a pentasaccharide derived from the antithrombin-binding site of heparin [3]. 3C NMR examination of the antithrombin-binding region [4, 51 supported the existence of a 3-0-sulphated glucosamine moiety in this structure.In view of the presence of the mammalian enzyme [2] a similar activity was sought from the Flavobacterium heparinAbbreviutions. GlcN, 2-amino-2-deoxy-~-glucose; GlcNCbz, 2-[(benzyloxycarbonyl)amino]-2-deoxy-~-glucose; GIcN-~S, 2-amino-2-deoxy-3-0-sulpho-~-glucose; GIcNS-~S, 2-deoxy-2-sulphamido-3-O-sulpho-~-g~ucose; GIcNAc-~S, 2-acetamido-2-deoxy-3-O-sulpho-D-glucose; GIcNAc-~S, 2-acetamido-2-deoxy-6-0-sulpho-~-glucose; GIcNS-~S, 2-deoxy-2-sulphamido-6-O-sulpho-~-glucose; GlcNS, 2-deoxy-2-sulphamido-~-glucose ; GalNAc-6S, 2-acetamido-2-deoxy-6-0-sulpho-~-galactose; SDS, sodium dodecylsulphate.Enzymes. Chondroitinase AC, chondroitin sulphate AC lyase (EC 4.2.2.5); chondroitinase B, chondroitin sulphate B lyase (EC 4.2.2.-); heparinase, heparin lyase (EC 4.2.2.7); d4,5glycuronidase, glycuronidase for 4-deoxy-u-~-threo-hex-4-enopyranosyluronic acid-(1 +4)-2-deoxy-2-sulphamido-6-0-sulpho-~-glucose (EC 3.2.1 .-); 2-0-sulphatase for 4-deoxy-2-0-sulpho-a-~-threo-hex-4-enopyranosyl uronic acid-(I +4)-2-deoxy-2-sulphamido-6-0-sulpho-

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“…The peak indicated at 57.5 ppm is from a trace of ethanol. The assignments for GlcNS-3S were reported by Casu et al [5] and our spectrum is in agreement (Fig. 1C).…”

Section: Preparation Of 3-0-sulphated Monosaccharidessupporting

confidence: 81%

Flavobacterium heparinum 3‐O‐sulphatase for N‐substituted glucosamine 3‐O‐sulphate

Bruce

McLEAN

Long

et al. 1985

European Journal of Biochemistry

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“…A unique pentasaccharide sequence in Hep is specifically recognized by AT-III (1,3,15). Since the procedure used to generate Hep oligosaccharides destroys glucosaminyl residues at the reducing terminus, the present studies indicate that HIP peptide also recognizes a motif of five to six saccharide units.…”

Section: Discussionmentioning

confidence: 70%

“…Results of anion exchange chromatography indicate that HA-Hep also has a slightly higher average negative charge density than either LA-Hep or unfractionated Hep (Fig. 1D) (1,3,(13)(14)(15). Therefore, HA-Hep was subjected to both AT-III and bFGF affinity chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…On the other hand, the biosynthesis of a structurally heterogeneous glycosaminoglycan seems to result from a series of incomplete enzymatic reactions, raising concern about the genetic control of glycosaminoglycan fine structure (6). At least one protein, antithrombin III (AT-III), binds to a specific sequence present in some Hep molecules but not in others (1)(2)(3). Other proteins, such as the FGFs bind to Hep with surprisingly high affinity; however, attempts to identify specific binding sites in Hep for different growth factors have given inconclusive and sometimes conflicting results.…”

mentioning

confidence: 99%

“…Because of the microheterogeneity of the sulfate substitutions of the polysaccharides, it has been speculated that specific monosaccharide sequences in Hep bind to selective domains in target proteins (1)(2)(3)(4)(5). A Hep hexasaccharide can theoretically occur in Ͼ10 5 different structural forms, which allows for sufficient structural variations, expected for an information molecule.…”

mentioning

confidence: 99%

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A heparin-binding synthetic peptide of heparin/heparan sulfateinteracting protein modulates blood coagulation activities

Liu¹,

Zhou²,

Höök³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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We have previously identified and characterized a heparin-binding cell surface protein (heparin͞heparan sulfate-interacting protein, or HIP) present on epithelial and endothelial cells. A synthetic peptide mimicking a heparinbinding domain of HIP is now shown to bind a small subset of heparin molecules with high affinity and, therefore, presumably recognizes a specific structural motif in the heparin molecule. Further analyses revealed that the heparin molecules exhibiting a high affinity for the HIP peptide also show an extremely high affinity for antithrombin III (AT-III), a cofactor required for heparin's anticoagulant activity. The HIP peptide was shown to compete with AT-III for binding to heparin and to neutralize the anticoagulant activity of heparin in blood plasma assays. Furthermore, the heparin subfraction that binds to the HIP peptide with high affinity exhibits an extremely high anticoagulant activity. We conclude that although the HIP peptide shows no sequence similarity with AT-III, the two proteins recognize the same or similar structural motifs in heparin.

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Monitoring Heparin Therapy

Johnston

2013

Quality in Laboratory Hemostasis and Thrombosis

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The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

Cited by 333 publications

References 29 publications

Flavobacterium heparinum 3‐O‐sulphatase for N‐substituted glucosamine 3‐O‐sulphate

Flavobacterium heparinum 3‐O‐sulphatase for N‐substituted glucosamine 3‐O‐sulphate

A heparin-binding synthetic peptide of heparin/heparan sulfateinteracting protein modulates blood coagulation activities

Monitoring Heparin Therapy

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