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This perspective article is focused on the presentation of the latest advances in NMR methods and applications that are behind the exciting achievements in the understanding of glycan receptors in molecular recognition events. Different NMR-based methodologies are discussed along with their applications to scrutinize the conformation and dynamics of glycans as well as their interactions with protein receptors.

25 p.-7 fig. Carbohydrate Chemistry: Chemical and Biological Approaches.Volume 44. Editors: Amélia Pilar Rauter, Thisbe K Lindhorst, Yves Queneau, This chapter is dedicated to the presentation of different examples of the application of solution NMR to the study of conformation, dynamics of sugar molecules (oligo and polysaccharides, glycopeptides and glycomimetics) including the investigation of glycanrelated molecular recognition events. It is not our intention to be exhaustive, rather to highlight diverse examples since 2016. They are presented depending on the chemical nature of the saccharide molecule, and on the different methodologies that can be employed., We thank MINECO (Spain, Projects) and the European Union (Project RECGLYCANMR and BACTIVAX) for financial support. AF also thanksMINECO for Juan de la Cierva contract.

We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment obtained by depolymerization complexed with a protective mAb. While literature data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a high glycodensity elicited functional antibodies comparably to the full-length conjugated polysaccharide. Here, starting from the X-ray crystallographic structure of the polysaccharide fragment-mAb complex, we designed and synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive Surface Plasmon Resonance, Saturation Transfer Difference NMR and in silico modelling, we demonstrated that the synthetic hexasaccharide recognized the mAb similarly to the dimer and, conjugated to CRM197 (a mutant of diphtheria toxin), elicited a functional immune response non inferior to the conjugated polysaccharide, indicating to suffice as vaccine antigen

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full‐length conjugated polysaccharide. Here, starting from the X‐ray crystallographic structure of the polysaccharide fragment–mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in‐silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM197, a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X‐ray crystallography‐guided design of a synthetic carbohydrate‐based conjugate vaccine., Deciphering the sugar glycocode: Starting from the X‐ray crystallographic structure of a fragment from Group B Streptococcus type III capsular polysaccharide in complex with a protective mAb complex, a hexasaccharide comprising exclusively the relevant positions involved in interactions was obtained. By structural and immunogenicity studies it was shown that the synthetic hexasaccharide recognized the mAb similarly to the polysaccharide fragment and, conjugated to a carrier protein, suffices as vaccine antigen.

The glycan structures of the receptor binding domain of the SARS‐CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS‐based analyses. The interaction of the RBD 13C‐labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15N‐labelled galectins (galectins‐3, ‐7 and ‐8 N‐terminal), Siglecs (Siglec‐8, Siglec‐10), and C‐type lectins (DC‐SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed., Unprecedent structural details of the glycans of the RBD of SARS‐CoV‐2 spike glycoprotein have been revealed by NMR spectroscopy. Unexpected and non‐previously reported glycoepitopes have been detected. The interaction of the RBD glycoprotein with diverse human lectins has been scrutinised by exploiting the NMR signature of the 13C‐glycans. Our analysis permitted to identify the corresponding glycan epitopes responsible for the interaction with each lectin.

Nuclear Magnetic Resonance (NMR) has been widely employed to assess diverse features of glycan–protein molecular recognition events. Different types of qualitative and quantitative information at different degrees of resolution and complexity can be extracted from the proper application of the available NMR-techniques. In fact, affinity, structural, kinetic, conformational, and dynamic characteristics of the binding process are available. Nevertheless, except in particular cases, the affinity of lectin-sugar interactions is weak, mostly at the low mM range. This feature is overcome in biological processes by using multivalency, thus augmenting the strength of the binding. However, the application of NMR methods to monitor multivalent lectin–glycan interactions is intrinsically challenging. It is well known that when large macromolecular complexes are formed, the NMR signals disappear from the NMR spectrum, due to the existence of fast transverse relaxation, related to the large size and exchange features. Indeed, at the heart of the molecular recognition event, the associated free-bound chemical exchange process for both partners takes place in a particular timescale. Thus, these factors have to be considered and overcome. In this review article, we have distinguished, in a subjective manner, the existence of multivalent presentations in the glycan or in the lectin. From the glycan perspective, we have also considered whether multiple epitopes of a given ligand are presented in the same linear chain of a saccharide (i.e., poly-LacNAc oligosaccharides) or decorating different arms of a multiantennae scaffold, either natural (as in multiantennae N-glycans) or synthetic (of dendrimer or polymer nature). From the lectin perspective, the presence of an individual binding site at every monomer of a multimeric lectin may also have key consequences for the binding event at different levels of complexity. We thank generous funding by the European Research Council (RECGLYCANMR, Advanced Grant No. 788143), the Agencia Estatal de Investigación (Spain) for grant PDI2021-1237810B-C21, and CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We also thank Marie-Skłodowska-Curie actions (TN BactiVax, under grant agreement No. 860325).