Exploring glycosylation patterns driving antibody and pathogen recognitions

Glycosylation is a ubiquitous post-translational modification. Its existence is mostly evident e.g. in the thick layer of glycans, glycoproteins, and glycolipids that covers the surface of all cells in the human body. At the cell surface, glycans regulate multiple biological events. Among these, glycosylated molecules can serve the role of receptors for glycan binding agents (GBAs) such as monoclonal antibodies (mAbs) and pathogenic surface/secreted proteins, also referred to as adhesins. Considering the intrinsically weak interactions between individual glycans and their protein binding partner, multivalence of glycan presentation is postulated to be an essential factor to achieve a biologically significant response. Consequently, recognitions at the cell surface are determined not only by the structure of glycans but also by their nanoscale configuration and spatial distribution on the lipid/protein backbone and in the natural context of the cell membrane. Due to the high level of complexity of these glycan-protein interactions, a complete understanding of the interplay between the spatial organization of glycans and the components of the cell surface is still missing (Chapter 1).

To deepen this understanding, I aimed to gain insight into the structural attributes responsible for driving the specificity of a particular GBA through examples of interactions where multiple factors play a critical role.

One such example is the recognition of glycopeptide epitopes by mAbs, where both glycan and peptide backbone determine the binding specificity. In an attempt to advance the comprehension of the molecular elements essential for the specific affinity of glycoprotein-specific mAbs, two mAbs against a Muc21 TR reporter domain bearing the cancer-associated Tn antigen were produced via Hybridoma Technology and further characterized (Chapter 2).

Within this framework, though shifting the focus from glycan-specific mAbs to pathogen adhesins, this thesis also examines the intricate pattern of recognition between the red blood cell glycoprotein glycophorin A (GYPA) and the Plasmodium falciparum EBA175 surface adhesin. In an attempt to resolve this interaction, a genetic approach relying on a recently developed cell-based glycan array allowed the display and production of GYPA reporter protein with defined glycosylation identities, called glycoforms, for further characterization (Chapter 3).

Lastly, I implemented an engineering strategy relying on synthetic biology to modify biological membranes by means of chemical tools. Specifically, a cholesteryl-peptide-based platform was designed as a flexible chemical module for the presentation of the carbohydrate GM1 on probiotic bacteria and used as a mimic receptor decoy for cholera toxin neutralization (Chapter 4, manuscript in preparation).

In summary, this thesis contributes to the understanding of the principles driving glycan recognition at the cell surface. To provide a comprehensive overview of the main elements involved, each chapter contains an example of a specific pattern of recognition in the context of pathogen adhesins (P. falciparum EBA175 and cholera toxin) or mAbs targeting glycosylated epitopes (anti-Tn-Muc21 mAbs). Specifically, I showed that the use of a genetically engineered cell platform (Chapters 2, 3) represents an invaluable tool to shed light on intricate and poorly understood patterns of glycan presentation. The chemical modification described in Chapter 4 serves as an alternative approach, providing a means by which to both modify membranes for a better understanding of glycans’ presentation and provide an applicable toolbox for therapeutic development.