An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence

Abstract

The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide-antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.

Current structural biology of the heparin interactome

“…This interaction induces a change in the 1 C 4 / 2 S 0 equilibrium of the IdoA2S ring conformation to pure 2 S 0 skew-boat. Some recent studies have identified multiple copies of this motif in heparin-derived oligosaccharides [34,35 ]. For instance, a heparin dodecasaccharide containing two AT-binding motifs intercalated by a non-sulfated IdoA unit was characterized and studied by NMR in interactions with AT.…”

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides

“…The different order of elution on the AT affinity column of an octasaccharide mixture suggested the active role of the flanking residue in the binding of the pentasaccharide sequence with AT (15). Moreover, structural modifications within the AGA*IA sequence, originating with the process used to depolymerize the polysaccharide, such as the presence of the 1,6-anhydrohexosamine residue at the reducing end or naturally present in the parent heparin, such as a pentasaccharide with two A* residues, have a strong influence in the AT binding properties of these oligosaccharides (16,17). The possibility of heparin chains with high AT-binding site density was suggested for high molecular weight fractions of porcine mucosal heparins (10), clam heparins (27), "macromolecular" rat skin heparins (28), and high affinity low molecular weight heparins (11).…”

“…Notably, the presence of glucuronic acid instead of iduronic acid before the pentasaccharide moiety increases the affinity to AT by 1 order of magnitude, whereas the presence of 1,6-anhydrohexosamine at the reducing side of the pentasaccharide significantly reduces the affinity (15,16). Recently, an octasaccharide having a pentasaccharide sequence at the nonreducing end with two 3-O-sulfated glucosamine residues has been characterized (17). Fluorescence titration experiments indicated that the AT affinity of this octasaccharide was characterized by an equilibrium dissociation constant 20-fold lower than that measured for the AGA*IA-AT complex.…”

Heparan sulfate and heparin interactions with proteins

“…In fact, other types of carbohydrate structures, distinct from GAGs, including those with low or no activity, have also been identified that can fulfil the structural requirements of AT binding [72] and a proposal has been made that the stabilization of AT is the key determinant of its activity [73]. It has also been shown that N-acetylation or N-sulfation is permitted in the substitution pattern of residues adjacent to and within, the first glucosamine residue, the pentasaccharide and that specific residues outside of the pentasaccharide region active for AT can influence AT activity [74][75][76][77][78]. This reinforces the idea that binding, even with low affinity, does not necessarily equate to activity.…”