Molecular recognition of tumour-associated O-glycans and mimetics by imune lectins

• Autor(a) principal: Travecedo, Maria Alejandra De Castro
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/160974
• topic: Molecular recognition; NMR; MGL; Siglec-7; Glycomimetics; cancer; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Cancer cells overexpress tumour-associated O-glycans that interact with immune lectins leading to antitumour immune suppression. In this context, the rational design of molecules to block these aberrant interactions is an attractive strategy for the development of novel immunotherapies. Herein, the molecular recognition process of different tumour associated O-glycans (Tn, STn and TF), analogues, and mimetics, including those with trivalent presentation, by two immune lectins, Siglec-7 and MGL, was scrutinized. Ligand- and protein-based Nuclear Magnetic Resonance (NMR) binding methods assisted with molecular dynamics (MD) simulations were integrated to investigate this molecular recognition event. STn-Thr and STn-analogue similarly interact with the carbohydrate recognition domain (CRD) of Siglec-7, the Siglec-7d1, in the same binding site and mainly through the Neu5Ac moiety. The tail of STn-analogue does not interfere with the binding, being oriented towards the solvent, and therefore suitable for derivatization. Our NMR integrative approach (1H,15N-HSQC based titration, Tr-ROESY, STD-NMR) indicate that both Tn-analogue and STn-analogue bind to MGL-CRD and that the binding mode orientation of GalNAc towards MGL is similar in both structures. Additionally, the tail does not interact significantly with MGL-CRD, as determined in the Tr-ROESY and STD-NMR, and thus suitable for derivatization and dendrimerization since the tail is oriented towards the solvent, as revealed in the MD simulation. The binding of a trimer of Tn-antigen was also investigated. Our NMR data shows that Tn-Trimer competes with α-MeGalNAc for the same binding site in MGL-CRD in the competition titration assay. DOSY-NMR experiments suggests that Tn-Trimer can bind to more than one MGL-CRD simultaneously. With the obtained results it was not possible to infer if the affinity/avidity was improved with the multivalent presentation of Tn-antigen. Finally, 1H,15N-HSQC-based titration with a TF-mimetic and its corresponding trimer shows an increased binding affinity due to multivalent presentation, highlighting the importance of multivalency when creating mimetics.