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

Glycobiology, 2017, vol. 27, no. 9, 900–911 doi: 10.1093/glycob/cwx025 Advance Access Publication Date: 13 April 2017 Original Article Structural Biology The β-reducing end in α(2–8)-polysialic acid constitutes a unique structural motif Hugo F Azurmendi2, Marcos D Battistel2, Jasmin Zarb2, Flora Lichaa2, Alejandro Negrete Virgen2, Joseph Shiloach3, and Darón I Freedberg1,2 2 Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20903, USA and 3Biotechnology Unit, MSC 5522, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA To whom correspondence should be addressed: Tel: +1-240-402-9532; e-mail: 1 Received 9 February 2017; Revised 10 March 2017; Editorial decision 12 March 2017; Accepted 14 March 2017 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. Key words: hydrogen bond, molecular dynamics, residual dipolar couplings, sialic acid structure, stereospecific assignments Introduction Sialic acids (Sias) are glycans constituted by nine carbons and a suite of chemical moieties playing diverse and important roles in cell and molecular interactions (Cohen and Varki 2010). Sias are expressed abundantly in vertebrates and higher invertebrates (Angata and Varki 2002); outside these groups, Sias are found only in a small number of species (Vimr et al. 2004). Among the latter, the majority is constituted by disease-causing gram-negative bacteria to human and animal hosts (Varki 2008). Moreover, microbial Sia metabolism is a virulence determinant in a range of infectious diseases (Vimr et al. 2004). Such is the case for the meningitis-causing bacteria N. meningitidis serogroup B and E. coli K1, whose capsular polysaccharides (CPs) are important virulence factors (Schiffer et al. 1976). The N. meningitidis group B (NMB) CP is a linear homopolymer of (2–8)-α-Neu5Ac (N-acetylneuraminic acid) units, also known as α(2–8)-polysialic acid (polySia), and in bacteria can reach a degree Published by Oxford University Press 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US. 900 The β-reducing end in α(2–8)-polysialic acid constitutes a unique structural motif of polymerization of up to 230 Sia units (Pelkonen et al. 1988; Schnaar et al. 2014). Because CPs are less prone to variation among bacterial strains as compared to proteins, they are excellent candidates for vaccine antigens, as has been the case with the development of CP conjugate vaccines against N. meningitidis serogroups A, C, W135, and Y (Cohn et al. 2010). The exception has been the NMB CP that elicits a poor immune response in humans (Wyle et al. 1972). The reason for poor immunogenicity is not known; however, it has been attributed to the fact that humans express α(2–8)-polySia in neural cell-adhesion molecules. Therefore, apprehension that a NMB CP-based vaccine has the potential to induce autoimmune reactions has hampered research and development of a NMB CP-based vaccine (Finne et al. 1983; Saukkonen et al. 1986). However, failure for this concern to materialize among persons carrying anti-CP NMB antibodies has prompted some researchers to call for a NMB CP vaccine development (Robbins et al. 2011). In order to make well-supported decisions in this regard, a detailed understanding of NMB structure and dynamics on varied conditions is among the highest priorities. Structural characterizations of polySia in solution with varying number of units have been advanced mainly using a combination of NMR experimental techniques and molecular modeling tools; however, no clear consensus has been reached, with proposals ranging from a variety of helical conformations to random coils (Michon et al. 1987; Yamasaki and Bacon 1991; Brisson et al. 1992; Henderson et al. 2003; Battistel et al. 2012). With the goal of obtaining greater detail, studies on short oligomers (two and three residues) with the reducing end capped with a methyl group have been reported as well (Yongye et al. 2008). Similar to the internal residues of α(2–8)-polySia, these oligomers have the anomeric carbon of the end residue locked in the α-configuration, with the CO2− axial (Figure 1) making it a good model system to study α(2–8)polySia. However, potential effects arising from the end-cap as compared with the βOH group have not been explored in previous 901 studies. Differences are to be expected, at least in the neighborhood of the reducing end, since the α-configuration represents only about 10% of the population for monomeric Sia in solution. However free α(2–8)-polySia is found mostly (over 99%) with the reducing end in the β configuration (Supplementary data, Figure S1), that is, with the CO2− equatorial and the OH axial (Figure 1). The biological implication or relevance of the β configuration is unknown since the vast majority of Sias are linked in the α configuration. Nevertheless, most in vitro studies utilize free polySia, hence the findings reported herein are relevant primarily to in vitro studies but may have potential in vivo applications. Hints of the potential importance of the different configuration at the anomeric end were noted as early as 1987 by Jennings and co-workers (Michon et al (...truncated)