Degeneracy of the Antithrombin Binding Sequence in Heparin: 2‐O‐Sulfated Iduronic Acid Can Replace the Critical Glucuronic Acid

Abstract

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this “canonical” GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation‐transfer‐difference (STD) NMR, NOEs, transferred NOEs (tr‐NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a 1C4‐2S0 equilibrium in the unbound saccharide, shift to full 2S0 and full 1C4 upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.

Building Block Analysis of ATIII Affinity Fractions of Heparins: Application to the ATIII Binding Capacity of Non-conventional 3-O-Sulfated Sequences

“…In the three heparins ( Tables 7 – 9 ), ΔIIs- Is id and ΔIs- Is id were major fragments containing - Is id and their presence, detected essentially in the affinity fractions, was a strong argument for their ATIII binding contribution within a non-conventional pentasaccharide where a 2- O -sulfated iduronic acid is substituted for the canonical central glucuronic acid. These data, compatible with an ATIII binding capacity of sequences including ΔIIs- Is id and ΔIs- Is id , are in line with those recently published ( 5 , 6 ). Table 7 also shows that even in the fraction HA5, the phenomenon of double 3S ATIII pentasaccharides was limited for PMH (5–10% of ΔIIa- IVs glu , and ΔIIa- IIs glu is in ΔIIa- IVs glu - Is id , ΔIIa- IIs glu - Is id sequences) in connection with the low content of Δ Is .…”

“…For the first two disaccharides, Δ Is and Δ IIIs , incompatibility with the structural requirements of the antithrombin III (ATIII) binding sequence suggested that they did not participate in anticoagulant activity and could be classified as non-ATIII-binding units ( 4 ). However, recent studies based on biosynthetic sequences ( 5 , 6 ), showed that the medium glucuronic acid in the pentasaccharide is not mandatory to bind ATIII and could be replaced by a 2- O -sulfated iduronic acid in synthetic octasaccharides, which still have anticoagulant activity. Δ Is and Δ IIIs could then be digested from ATIII binding sequences where their uronic acid is iduronic 2- O -sulfate [-IdoA(2S)-GlcN(NS,3S,6S) and -IdoA(2S)-GlcN(NS,3S)].…”

NMR Characterization of the Interactions Between Glycosaminoglycans and Proteins

“…At the same time, the flexibility of IdoA provided unlimited possibilities for the binding of heparin to protein. A recent study used IdoA2S instead of GlcA in the AT III binding sequence (Elli et al, 2020). The results showed that IdoA2S, which replaced GlcA, was in a pure 1 C 4 conformation when bound, and the affinity was tripled, which provided a basis for the application of bovine heparin.…”

Use of Enoxaparin to Counteract COVID-19 Infection and Reduce Thromboembolic Venous Complications: A Review of the Current Evidence

“…The specific pentasaccharide sequence binds and potentiates the effect of antithrombin a naturally occurring anticoagulant, which can inhibit several serine proteases of the coagulation system, primarily FIIa (thrombin) and FXa. More recently, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which glucuronic acid is replaced with sulfated iduronic acid, was shown to similarly bind to and activate antithrombin, thus paving the way for the development of heparin-like drugs that be obtained by a chemo-enzymatic approach ( Elli et al., 2020 ).…”