The β-reducing end in α(2–8)-polysialic acid constitutes a unique structural motif

Abstract

Over the years, structural characterizations of α(2-8)-polysialic acid (polySia) in solution have produced inconclusive results. Efforts for obtaining detailed information in this important antigen have focused primarily on the α-linked residues and not on the distinctive characteristics of the terminal ones. The thermodynamically preferred anomeric configuration for the reducing end of sialic acids is β, which has the [I]CO2- group equatorial and the OH ([I]OH2) axial, while for all other residues the CO2- group is axial. We show that this purportedly minor difference has distinct consequences for the structure of α(2-8)-polySia near the reducing end, as the β configuration places the [I]OH2 in a favorable position for the formation of a hydrogen bond with the carboxylate group of the following residue ([II]CO2-). Molecular dynamics (MD) simulations predicted the hydrogen bond, which we subsequently directly detected by NMR. The combination of MD and residual dipolar couplings shows that the net result for the structure of Sia2-βOH is a stable conformation with well-defined hydration and charge patterns, and consistent with experimental NOE-based hydroxyl and aliphatic inter-proton distances. Moreover, we provide evidence that this distinct conformation is preserved on Sia oligosaccharides, thus constituting a motif that determines the structure and dynamics of α(2-8)-polySia for at least the first two residues of the polymer. We suggest the hypothesis that this structural motif sheds light on a longtime puzzling observation for the requirement of 10 residues of α(2-8)-polySia in order to bind effectively to specific antibodies, about four units more than for analogous cases.

Molecular Conformations of Di-, Tri-, and Tetra-α-(2→8)-Linked Sialic Acid from NMR Spectroscopy and MD Simulations

“…Dimer2 βH showed a more prominent intermolecular hydrogen-bonding pattern, as was reported by Azurmendi et al as well [21].…”

Molecular Recognition Insights of Sialic Acid Glycans by Distinct Receptors Unveiled by NMR and Molecular Modeling

“…The authors observed an H-bond between OH 2 of Sia I and O 1 of Sia II, which dictates a well-defined conformation on the reducing end residues of (Sia) 4 ( Figure 2B ). This structural motif was previously observed in shorter (Sia) 2 homologue, and it was suggested to be a common feature of PolySia structures ( Azurmendi et al, 2017 ). For the nonreducing end (Sia III-IV residues), the authors reported high potential flexibility together with the typical structural motif of the glycerol side chain of the terminal end Sia IV, characterized by the stable intra-residue H-bond between OH 8 and O1 ( Novakovic et al, 2021 ).…”

“…Antibodies recognize PolySia polymers with 10 or more Neu5Ac residues as minimal epitope ( Jennings et al, 1985 ; Turupcu et al, 2020 ). Therefore, it was hypothesized that PolySia structures with a certain degree of polymerization adopt a specific conformation in solution that would promote the binding of antibodies ( Azurmendi et al, 2017 ). In this regard, several conformational studies were carried out on PolySia with different degrees of polymerization ( Henderson et al, 2003 ; Battistel et al, 2012 ; Hanashima et al, 2013 ; Turupcu et al, 2020 ; Novakovic et al, 2021 ).…”

The capsule ofCryptococcus neoformans

“…While research has identified the molecular composition and primary structure of both GXM and GXMGal, how these monosaccharides come together to form three-dimensional structure of >1 mDa PS capsule remains to be solved [5] (Figure 3). Recent work in the bacterial PS field indicates that with the use of specific HPLC isolation techniques and new 2D NMR techniques the three-dimensional structure of capsular PSs can be determined [46,47], and these methods may be applicable to C. neoformans PS.…”