Stornaiuolo, Alessia (2025) Lectins expression and purification for biochemical and physico-chemical studies of their interaction with eukaryotic glycans. [Tesi di dottorato]

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Abstract

The immune system’s extraordinary capacity to discriminate between "self" and "non-self" and respond accordingly is fundamental to health. This intricate balance, crucial for inducing acquired immune responses, regulating inflammation, and establishing tolerance, is increasingly understood to be profoundly influenced by glycans. This post-translational process, found on host cell and pathogen proteins (glycoproteins) and lipids (glycolipids), exhibit natural heterogeneity, acting as crucial carriers of biological information. This information is precisely decoded by lectins, diverse protein families including C-type lectin receptors (CLRs), galectins, and Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs), which trigger specific tolerogenic or immunogenic signaling pathways upon glycan recognition. Siglecs are cell surface receptors that recognize sialic acids. They are known modulators of immune responses, modulating immune cell response upon binding with sialylated ligands. These interactions mediate events such as cell adhesion and the inhibition or regulation of immune cell activation. Galectins are soluble proteins binding β-galactoside-containing glycans. They play critical roles in a myriad of physiological and pathological processes. Glycans and their complementary glycan-binding proteins are essential components in controlling both innate and adaptive immunity. Indeed, Sialic acid-Siglec interactions are associated with a broad spectrum of diseases, from autoimmunity to neurodegeneration and cancer. Similarly, Galectin-3 modulates a range of cellular interactions, such as immune response, cell adhesion, migration, and angiogenesis. Consequently, it is a key player in fibrosis and carcinogenesis, as its upregulation often correlates with poor prognosis. Given this context, strategies for rationally modulating lectin-glycan interactions in pathological processes offer significant therapeutic potential. Recent decades have seen the development of various inhibitors, including small molecules, multivalent saccharide ligands, peptides, and peptidomimetics, offering alternatives against neoplastic and other diseases. This thesis project aimed to elucidate the molecular details of the binding between Siglec-8 and Galectin-3 with specific glycomimetics. The research involved the expression and purification of these proteins, followed by binding studies with sialic acid-containing glycans (for Siglec-8) and β-galactoside-containing glycans (for Galectin-3). Structural insights into these molecular recognition processes were provided by Nuclear Magnetic Resonance (NMR) spectroscopy, employing both ligand-based and protein-based approaches, complemented by biophysical and computational methods, such as docking and molecular dynamics simulations. This comprehensive approach facilitated a detailed examination of the three-dimensional complexes, which enabled the thorough characterization of their recognition and binding processes and provided a deeper understanding of the molecular mechanisms underlying their inhibitory or modulatory activities.

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