From a fundamental perspective, novel NMR-based methodologies have been developed to study the structure, dynamics, presentation, and interactions of intact glycoproteins. A notable early success was the application of paramagnetic solution NMR to highly complex multiantenna N-glycans, elucidating their recognition features with infection-related proteins..
N-glycosylation, the most common post-translational modification in proteins, introduces heterogeneity that adds complexity to biological functions. An NMR-based strategy was developed to structurally characterize the glycan content of the IgE high-affinity receptor (FcεRIα) expressed in human HEK 293 cells. It was applied to study the interaction between the SARS-CoV-2 spike glycoprotein's receptor-binding domain and human immune system lectins, aiming to deepen our understanding of diseases involving glycans (e.g. cancer, influenza, COVID-19).
In parallel, a novel method has been introduced, offering a precise way to identify the specific binding epitopes of glycans, like polyLacNAc, to galectins. Our breakthrough provided a powerful tool for high-resolution studies of glycan-galectin interactions with biomedical relevance.
Another significant finding has demonstrated that the SARS-CoV-2 spike glycoprotein directly binds external sialic acids. Our NMR experiments with isolated spike regions (RBD, NTD) and blocking antibodies confirmed that the spike binds external sialic acids efficiently through its N-terminal domain, offering new potential for developing glycan-based inhibitors to combat COVID-19.
Understanding the role of glycan presentation and multivalency in molecular recognition is a critical focus of the RECGLYCANMR project. To mimic natural membrane environments, liposomes displaying various glycosphingolipids (GSLs) were used, highlighting the critical importance of glycan presentation in achieving efficient recognition by protein receptors and triggering biological responses.
Regarding multivalency, the interaction between galectins and multivalent glycans presented on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers was studied using a combination of NMR, cryo-electron microscopy (cryo-EM), and dynamic light scattering (DLS), underscoring the role of glycan presentation in efficient binding.
From a methodological perspective, RECGLYCANMR also explored the interaction features of L-Sectin, a key lectin in the human immune system, with various N-glycans in solution and surface conditions. NMR studies, coupled with glycan microarray data, revealed contrasting results between the two experimental setups, underscoring the complexity of translating findings across experimental models.
Multivalent glycans on the cell surface, particularly in the glycocalyx, influence their recognition by lectins. In this context, RECGLYCANMR studied multivalent lactose-functionalized glycomacromolecules and their lipid-conjugates, using liposome-based systems to mimic cellular environments.
Galectins are one of the most biologically significant glycan receptors in nature, with functions relevant to both health and disease. RECGLYCANMR particularly focused on galectins’ interactions with histo-blood group antigens.
Other key lectins implicated in many diseases are the human sialic acid-binding immunoglobulin-like lectin receptors (Siglecs), which play a crucial role in regulating the immune response. Our research initially focused on Siglec-8 and Siglec-9 and offers valuable insights for designing the next generation of Siglec inhibitors.
We also developed a NMR method based on a competitive displacement assay using 19F NMR with a T2-filter.
While NMR is widely used to study glycan binding in solution, the weak nature of these interactions under dilute conditions (mM to µM range) in vitro contrasts with the multivalent presentations found in biological environments. To mimic such conditions, we used in-cell and on-cell NMR techniques to study glycan-lectin interactions. For in-cell NMR, galectin-7 was used as a model, marking the first observation of a galectin's NMR spectrum in a cell environment. However, as most lectins function on the cell surface, on-cell STD-NMR experiments were employed to monitor Siglec-10's interaction with a glycomimetic. These experiments successfully identified the binding epitope, aligned with results from isolated lectin experiments under diluted conditions.
In conclusion, we have shown that glycan-mediated molecular recognition events can be studied using NMR that closely replicate the natural cellular environment.
