Decoding the molecular recognition of sialic acid-containing glycans by Siglecs

• Autor(a) principal: Soares, Cátia Alexandra Oliveira
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/132001
• topic: Siglecs; Cancer; Carbohydrate-lectin interactions; NMR Spectroscopy; Epitope Map; Conformational analysis; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Siglecs are a family of cell surface lectins majorly expressed in immune cells, which through the recognition of sialic acids, present at the terminals of glycans in glycolipids and glycoproteins, modulate immune responses. Hypersialylation is a common phenomenon in cancer cells, which exploits aberrant interactions between cancer-associated sialoglycans and siglecs (namely Siglec-7, -9 and -15) in a strategy to dampen the anti-tumour immune activity. Therefore, understanding the molecular details of the recognition of cancer-associated sialoglycans by siglecs is essential for the development of anticancer therapies. In this work, the molecular recognition of ubiquitous sialoglycans (3’SL and 6’SL) by Siglec-7, -9 and -15, as well the specific recognition of the cancer-associated STn-antigens by Siglec-15, were characterized, using ligand-based Nuclear Magnetic Resonance (NMR) binding experiments such as saturation transfer difference (STD) NMR and tr-NOESY. Overall Siglec-7, -9 and -15 recognize 3’SL and 6’SL similarly. The major determinant for the binding is the Neu5Ac unit, especially its N-acetyl group and glycerol side chain. Subtle differences between siglecs were deduced by STD-derived epitope analysis. Tr-NOESY suggests for 3’SL a conformational selection of the -g conformer (φ=-60°) upon binding. In the case of 6’SL the -g conformer (φ=-60°) is predominant in both free and bound states. Around ω angle, the major conformation in solution is gt but additional studies should be performed to ascertain the conformation in the bound state. Concerning STn antigens (STn-Ser/Thr and STn-PDTRP), the binding towards Siglec-15 is majorly dependent on the Neu5Ac residue, however, GalNAc seems also relevant for the binding. The bioactive conformation around φ dihedral angle for STn-antigens is stabilized around -60° (-g conformer). In the bound state, the determination of ω angle is not conclusive and more studies should be performed. Finally, the effectiveness of the 5G12 antibody in blocking Siglec-15/STn-Ser interactions was also proved by STD-NMR competition experiment.