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Projeto de investigação
Picturing pathogen infection: A structure-functional approach to interrogate host-glycans/Salmonella crosstalk
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Decoding the Molecular Basis of the Specificity of an Anti-sTn Antibody
Publication . Soares, Cátia O.; Laugieri, Maria Elena; Grosso, Ana Sofia; Natale, Mariangela; Coelho, Helena; Behren, Sandra; Yu, Jin; Cai, Hui; Franconetti, Antonio; Oyenarte, Iker; Magnasco, Maria; Gimeno, Ana; Ramos, Nuno; Chai, Wengang; Corzana, Francisco; Westerlind, Ulrika; Jiménez-Barbero, Jesús; Palma, Angelina S.; Videira, Paula A.; Ereño-Orbea, June; Marcelo, Filipa; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; Faculdade de Ciências e Tecnologia (FCT); ACS - American Chemical Society
The mucin O-glycan sialyl Tn antigen (sTn, Neu5Acα2-6GalNAcα1-O-Ser/Thr) is an antigen associated with different types of cancers, often linked with a higher risk of metastasis and poor prognosis. Despite efforts to develop anti-sTn antibodies with high specificity for diagnostics and immunotherapy, challenges in eliciting high-affinity antibodies for glycan structures have limited their effectiveness, leading to low titers and short protection durations. Experimental structural insights into anti-sTn antibody specificity are lacking, hindering their optimization for cancer cell recognition. In this study, we used a comprehensive structural approach, combining X-ray crystallography, NMR spectroscopy, computational methods, glycan/glycopeptide microarrays, and biophysical techniques, to thoroughly investigate the molecular basis of sTn recognition by L2A5, a novel preclinical anti-sTn monoclonal antibody (mAb). Our data unequivocally show that the L2A5 fragment antigen-binding (Fab) specifically binds to core sTn moieties. NMR and X-ray structural data suggest a similar binding mode for the complexes formed by the sTn moiety linked to Ser or Thr and the L2A5 Fab. The sugar moieties are similarly oriented in the paratope of mAb, with the Neu5Ac moiety establishing key interactions with the receptor and the GalNAc moiety providing additional contacts. Furthermore, L2A5 exhibits fine specificity toward cancer-related MUC1 and MUC4 mucin-derived sTn glycopeptides, which might contribute to its selective targeting against tumor cells. This newfound knowledge holds promise for the rational improvement and potential application of this anti-sTn antibody in diagnosis and targeted therapy against sTn expressing cancers such as breast, colorectal, and bladder cancer, improving patient care.
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.
Revisiting the immunopathology of congenital disorders of glycosylation
Publication . Pascoal, Carlota; Francisco, Rita; Mexia, Patrícia; Pereira, Beatriz Luís; Granjo, Pedro; Coelho, Helena; Barbosa, Mariana; dos Reis Ferreira, Vanessa; Videira, Paula Alexandra; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; DQ - Departamento de Química; Frontiers
Glycosylation is a critical post-translational modification that plays a pivotal role in several biological processes, such as the immune response. Alterations in glycosylation can modulate the course of various pathologies, such as the case of congenital disorders of glycosylation (CDG), a group of more than 160 rare and complex genetic diseases. Although the link between glycosylation and immune dysfunction has already been recognized, the immune involvement in most CDG remains largely unexplored and poorly understood. In this study, we provide an update on the immune dysfunction and clinical manifestations of the 12 CDG with major immune involvement, organized into 6 categories of inborn errors of immunity according to the International Union of Immunological Societies (IUIS). The immune involvement in phosphomannomutase 2 (PMM2)-CDG - the most frequent CDG - was comprehensively reviewed, highlighting a higher prevalence of immune issues during infancy and childhood and in R141H-bearing genotypes. Finally, using PMM2-CDG as a model, we point to links between abnormal glycosylation patterns in host cells and possibly favored interactions with microorganisms that may explain the higher susceptibility to infection. Further characterizing immunopathology and unusual host-pathogen adhesion in CDG can not only improve immunological standards of care but also pave the way for innovative preventive measures and targeted glycan-based therapies that may improve quality of life for people living with CDG.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
CEEC IND 3ed
Número da atribuição
2020.03261.CEECIND/CP1586/CT0012