Conformational transitions induced in heparin octasaccharides by binding with antithrombin III

Abstract

The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides

“…1c) showed a slightly higher affinity for AT compared with the octasaccharide with AGA*IA positioned at the reducing end (Fig. 1a) (14). Nevertheless, the simultaneous presence of a small amount of AT 2 -dodecasaccharide tertiary complex cannot be excluded by STD data.…”

“…Although an early study suggested that this domain was located prevalently toward the nonreducing end of the molecule (26), other studies suggested a more random distribution (27). In recent studies by our groups, the presence of the pentasaccharide in both sulfated and unsulfated sequences was shown, suggesting a more random distribution of the pentasaccharide along the heparin chain (14). 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).…”

“…The K d value was obtained by monitoring the enhancement of intrinsic fluorescence of the serpin upon its reaction with increasing concentrations of each products, using the procedure previously described (15 (14). Depending on the salt concentration, the dodecasaccharide had 2.1 to 3.2 times higher affinity for AT than fondaparinux.…”

“…These studies demonstrated that the AGA*IA sequence can be flanked by both sulfated and undersulfated disaccharides and that these elongating sequences can have an active role in binding AT (14,15). 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).…”

Antithrombin-binding Octasaccharides and Role of Extensions of the Active Pentasaccharide Sequence in the Specificity and Strength of Interaction

“…Longer AT-binding sequences, such as decasaccharides, were also previously isolated (8). The influence of the position of the pentasaccharide sequence along the oligosaccharide chains together with the knowledge of the role of the residues prolonging the active sequence toward both its reducing and nonreducing side are among the major goals of current heparin research (10). Although the active pentasaccharide AGA*IA is taken as paradigm for a unique heparin sequence targeting a specific protein (i.e.…”