Sequencing of 3-O-Sulfate Containing Heparin Decasaccharides with a Partial Antithrombin III Binding Site

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Shriver, Zachary
Raman, Rahul
Venkataraman, Ganesh
Drummond, Katherine
Turnbull, Jeremy
Toida, Toshihiko
Linhardt, Robert
Biemann, Klaus
Sasisekharan, Ram
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is DeltaU(2S)H(NS,6S)I(2S)H(NS, 6S)I(2S)H(NS,6S)IH(NAc,6S)GH(NS,3S,6S) (+/-DDD4-7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.
Proceedings of the National Academy of Sciences (USA), 97, 10359-10364
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Sequencing of 3-O-Sulfate Containing Heparin Decasaccharides with a Partial Antithrombin III Binding Site, Z. Shriver, R. Raman, G. Venkataraman, K. Drummond, J. Turnbull, T. Toida, R. Linhardt, K. Biemann, R. Sasisekharan, Proceedings of the National Academy of Sciences (USA), 97, 10359-10364, 2000.
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