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Projeto de investigação
Advances in MUC1 Glycan Cancer Antigens: From structure to function in the fight against cancer
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Translating the human-microbiome molecular cross-talk using glycan microarray and structural biology strategies
Publication . Trovão, Filipa; Palma, Maria Angelina; Carvalho, Ana Luísa; Liu, Yan
O microbioma intestinal humano apresenta uma elevada capacidade de utilizar como nutrientes glicanos derivados do hospedeiro e da dieta. Isto promove uma comunicação complexa com o hospedeiro humano tendo impacto na nutrição, na regulação do sistema imunológico e em mecanismos de patologia. Estirpes proeminentes da microbiota, nomeadamente como as do filo Bacteroidetes, possuem múltiplos genes codificadores de enzimas em clusters, designados polysaccharide-utilization loci (PULs). Cada PUL codifica todos os genes necessários para o reconhecimento e degradação de glicanos, incluindo enzimas ativas em hidratos de carbono (CAZYmes) ou peptidases (mucinases) com módulos auxiliares de ligação a hidratos de carbono (CBMs). No entanto, como as bactérias exploram a diversidade estrutural dos glicanos derivados da dieta e do hospedeiro e como isso influencia as interações benéficas versus patogénicas é amplamente desconhecido a nível molecular. O trabalho desenvolvido na presente Tese teve como objetivo responder a estas questões, elucidando e caracterizando o reconhecimento de glicanos por proteínas de superfície bacteriana, particularmente os CBMs, de duas bactérias modelos de intervenientes comensais do intestino, o Bacteroides thetaiotaomicron e o B. caccae, envolvidos na degradação de glicanos derivados da dieta e do hospedeiro.
Para uma melhor compreensão da comunicação molecular complexa dos comensais B. thetaiotaomicron e B. caccae com o hospedeiro humano, foi seguida uma estratégia integrativa, incluindo análise bioinformática de sequências anotadas no genoma e produção de CBMs recombinantes; descoberta de ligandos usando microarrays de glicanos com estruturas diversas de polissacáridos, glicoproteínas tipo mucina e de oligossacáridos de sequência definida; e caracterização estrutural das interações proteína-glicano identificadas usando cristalografia de raios X e outros métodos biofísicos e bioquímicos complementares. A estratégia adotada permitiu a caracterização de 2 novos CBMs: o BT0996-C, o membro fundador dos CBMs de família 97, que reconhece cadeias de ácido poligalacturónico ligadas em α1-4, um glicano derivado da dieta de polissacáridos pécticos; e o BC16100-C, um CBM da família 32 que reconhece o antigénio Tn (GalNAcα-Ser/Thr), um O-glicano do tipo mucina derivado do hospedeiro. Além disso, um novo microarray de glicoproteínas tipo mucina foi desenvolvido para high-throughput screening de novos CBMs da família 32, com o objetivo de descobrir estruturas de glicano biologicamente relevantes, seguindo a abordagem de beam search microarray. Esta abordagem contribuirá para desvendar estruturas de glicanos como mediadores específicos de interações comensais/patogénicas com impacto na saúde humana. As descobertas relatadas nesta Tese avançaram o conhecimento da interface hospedeiro-microbioma a nível molecular e podem ser exploradas para o estudo de outras estirpes microbianas e para desenvolver novas soluções em relação à saúde intestinal.
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.
Deciphering the structural features of glycan-lectin interactions in the immune system
Publication . Lima, Carlos David Lourenço; Marcelo, Filipa Margarida Barradas de Morais
This thesis is focused on the study of the molecular recognition of two cancer associated
glycan epitopes, the LacdiNAc (GalNAcβ1-4GlcNAc) and the LacNAc (Galβ1-4GlcNAc)
structures by two immune-related lectins, the human macrophage galactose-type lectin (MGL)
and human galectin-3 (Gal-3) through a multidisciplinary approach that combines NMR
spectroscopy, isothermal titration calorimetry (ITC), molecular dynamics (MD) and X-ray
crystallography.
In the case of the MGL, the KD of MGL/LacdiNAc complex is at low µM range (5.3 µM)
and similar that obtain for the MGL/α-Me-GalNAc complex, while MGL/LacNAc complex is at the
mM range (higher than 1 mM). STD-NMR binding studies demonstrate that MGL preferentially
binds LacdiNAc and LacNAc structures, through the non-reducing end of the disaccharides. The
1H,15N-HSQC based titrations show that both ligands perturb the same region of MGL, however,
the magnitude of this perturbation is much higher in the case of LacdiNAc than LacNAc.
Furthermore, significant differences in the chemical shift perturbations of Y236, H286 and D294
residues were observed. MD models show that these differences are intimately related with the
specific engagement of the NHAc group of the GalNAc unit of LacdiNAc.
With respect of Gal-3 complexes both ligands bind Gal-3 with similar affinity (30-40 µM).
Nevertheless, the 1H,15N-HSQC based titration shows that additional residues are perturbed in βstrand S3 in the case of Gal-3/LacdiNAc complex. X-Ray structure of the complex Gal-3/LacdiNAc
confirms modifications at this region of the protein showing that the LacdiNAc induces an alternate
conformation for the side chain of R144. The R144B conformation, absent in the case of the
complex with LacNAc, mediates a hydrogen-bond with the N-acetyl group of GalNAc of LacdiNAc,
and increase the residence of a water molecule (W2) that in turn establish a hydrogen-bond
network involving the N-acetyl group of GalNAc, the glycosidic bond of LacdiNAc and the R144B.
Decoding the molecular recognition of sialic acid-containing glycans by Siglecs
Publication . Soares, Cátia Alexandra Oliveira; Marcelo, Filipa; Coelho, Helena
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.
Structural basis for the synthesis of the core 1 structure by C1GalT1
Publication . González-Ramírez, Andrés Manuel; Grosso, Ana Sofia; Yang, Zhang; Compañón, Ismael; Coelho, Helena; Narimatsu, Yoshiki; Clausen, Henrik; Marcelo, Filipa; Corzana, Francisco; Hurtado-Guerrero, Ramon; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Nature Portfolio
C1GalT1 is an essential inverting glycosyltransferase responsible for synthesizing the core 1 structure, a common precursor for mucin-type O-glycans found in many glycoproteins. To date, the structure of C1GalT1 and the details of substrate recognition and catalysis remain unknown. Through biophysical and cellular studies, including X-ray crystallography of C1GalT1 complexed to a glycopeptide, we report that C1GalT1 is an obligate GT-A fold dimer that follows a SN2 mechanism. The binding of the glycopeptides to the enzyme is mainly driven by the GalNAc moiety while the peptide sequence provides optimal kinetic and binding parameters. Interestingly, to achieve glycosylation, C1GalT1 recognizes a high-energy conformation of the α-GalNAc-Thr linkage, negligibly populated in solution. By imposing this 3D-arrangement on that fragment, characteristic of α-GalNAc-Ser peptides, C1GalT1 ensures broad glycosylation of both acceptor substrates. These findings illustrate a structural and mechanistic blueprint to explain glycosylation of multiple acceptor substrates, extending the repertoire of mechanisms adopted by glycosyltransferases.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-MIB/31028/2017