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Projeto de investigação
The Glycan Toolbox: From structure to function through the development of glycan-based therapies
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Unraveling Molecular Recognition of Glycan Ligands by Siglec-9 via NMR Spectroscopy and Molecular Dynamics Modeling
Publication . Atxabal, Unai; Nycholat, Corwin; Pröpster, Johannes M.; Fernández , Andrea; Oyenarte, Iker; Lenza, Maria Pia; Franconetti, Antonio; Soares, Cátia O.; Coelho, Helena; Marcelo, Filipa; Schubert, Mario; Paulson, James C.; Jiménez-Barbero, Jesús; Ereño-Orbea, June; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; ACS - American Chemical Society
Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G β-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G′ loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B′-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.
Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b
Publication . Lenza, Maria Pia; Egia-Mendikute, Leire; Antoñana-Vildosola, Asier; Soares, Cátia O.; Coelho, Helena; Corzana, Francisco; Bosch, Alexandre; Manisha, Prodhi; Quintana, Jon Imanol; Oyenarte, Iker; Unione, Luca; Moure, María Jesús; Azkargorta, Mikel; Atxabal, Unai; Sobczak, Klaudia; Elortza, Felix; Sutherland, James D.; Barrio, Rosa; Marcelo, Filipa; Jiménez-Barbero, Jesús; Palazon, Asis; Ereño-Orbea, June; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Nature Portfolio
Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.
Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses
Publication . Bermejo, Iris A.; Guerreiro, Ana; Eguskiza, Ander; Martínez-Sáez, Nuria; Lazaris, Foivos S.; Asín, Alicia; Somovilla, Víctor J.; Compañón, Ismael; Raju, Tom K.; Tadic, Srdan; Garrido, Pablo; García-Sanmartín , Josune; Mangini, Vincenzo; Grosso, Ana S.; Marcelo, Filipa; Avenoza, Alberto; Busto, Jesús H.; García-Martín, Fayna; Hurtado-Guerrero, Ramón; Peregrina, Jesús M.; Bernardes, Gonçalo J. L.; Martínez, Alfredo; Fiammengo, Roberto; Corzana, Francisco; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; ACS - American Chemical Society
Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
CEEC IND 3ed
Número da atribuição
2020.00233.CEECIND/CP1586/CT0011