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Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus
Publication . Correia, Viviana G.; Trovão, Filipa; Pinheiro, Benedita A.; Brás, Joana L. A.; Silva, Lisete M.; Nunes, Cláudia; Coimbra, Manuel A.; Liu, Yan; Feizi, Ten; Fontes, Carlos M. G. A.; Mulloy, Barbara; Chai, Wengang; Carvalho, Ana Luísa; Palma, Angelina S.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; American Society for Microbiology
A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.
The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome
Publication . Trovão, Filipa; Correia, Viviana G.; Lourenço, Frederico M.; Ribeiro, Diana O.; Carvalho, Ana Luísa; Palma, Angelina S.; Pinheiro, Benedita A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Academic Press Inc.
The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.
Protein-carbohydrate recognition in the biodegradation of the plant cell wall: Functional and structural studies using carbohydrate microarrays and X-ray crystallography
Publication . Ribeiro, Diana de Oliveira; Palma, Angelina; Carvalho, Ana; Feizi, Ten
The plant cell wall is, in its majority, constituted by complex and structurally diverse polysaccharides that are valuable resources for industrial and biotechnological applications. Anaerobic microbial organisms are highly efficient for plant cell wall polysaccharide biodegradation and have evolved a multi-enzyme complex system, the Cellulosome, where catalytic enzymes have non-catalytic Carbohydrate Binding Modules (CBMs) appended that highly potentiate the enzymes’ catalytic efficiency. Deciphering at molecular level the mechanisms underlying plant cell wall carbohydrate recognition and deconstruction by different cellulolytic bacteria is crucial to elucidate these complex biological systems, as well as to further promote novel potential applications. The work developed in this Thesis focused on the unique approach of combining carbohydrate microarrays with X-ray crystallography, to uncover carbohydrate ligands for CBMs and to structurally characterize novel CBM-carbohydrate interactions of two anaerobic bacteria that reside in different ecological niches: Clostridium thermocellum, found in soils, and Ruminococcus flavefaciens FD-1, present in the rumen of herbivorous. To this end, microarrays featuring carbohydrate probes with polysaccharide and oligosaccharide sequences representative of the structural diversity found on plant cell walls, but also in fungal and bacterial cell walls, were developed and then used to screen the carbohydrate-binding and ligand-specificity of 150 CBMs of C. thermocellum and R. flavefaciens CBMomes. The groups of polysaccharides that are differentially recognised were revealed for 59 CBMs and novel CBM-ligand specificities were identified for 23 modules from C. thermocellum and 21 from R. flavefaciens. Overall, the two bacteria differentially expressed CBM families with different carbohydrate-binding specificities, which may reflect adaptation to substrate availability in their specific ecological niche or the complexity of their Cellulosome. Using the information derived from the high-throughput microarray analysis, and according to their biotechnological relevance or novelty, CBMs and the respective ligands were selected for further structural studies. The novel CBM structures solved, complemented with biochemical and biophysical data, enabled the characterization of the molecular determinants for the recognition of mixed-linked β1,3-1,4-glucans by C. thermocellum family 11 CBM, chitin and peptidoglycan-derived sequences by a novel LysM domain from C. thermocellum family 50 CBMs, and pectic arabinans by R. flavefaciens family 13 CBM. The results reported here allow to assign a functional role for these CBMs and CBM families and contribute to the classification of the novel CBMs identified in the genome of the two bacteria, particularly those from R. flavefaciens FD1. Furthermore, the information derived from this integrative study, can promote a better understanding of cellulolytic capabilities of these bacteria, as well as to potentiate biotechnological applications of CBMs.
Feasibility of Brewer’s Spent Yeast Microcapsules as Targeted Oral Carriers
Publication . Reis, Sofia F.; Martins, Vítor J.; Bastos, Rita; Lima, Tânia; Correia, Viviana G.; Pinheiro, Benedita A.; Silva, Lisete M.; Palma, Angelina S.; Ferreira, Paula; Vilanova, Manuel; Coimbra, Manuel A.; Coelho, Elisabete; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
Brewer’s spent yeast (BSY) microcapsules have a complex network of cell-wall polysaccharides that are induced by brewing when compared to the baker’s yeast (Saccharomyces cerevisiae) microcapsules. These are rich in (β1→3)-glucans and covalently linked to (α1→4)- and (β1→4)-glucans in addition to residual mannoproteins. S. cerevisiae is often used as a drug delivery system due to its immunostimulatory potential conferred by the presence of (β1→3)-glucans. Similarly, BSY microcapsules could also be used in the encapsulation of compounds or drug delivery systems with the advantage of resisting digestion conferred by (β1→4)-glucans and promoting a broader immunomodulatory response. This work aims to study the feasibility of BSY microcapsules that are the result of alkali and subcritical water extraction processes, as oral carriers for food and biomedical applications by (1) evaluating the resistance of BSY microcapsules to in vitro digestion (IVD), (2) their recognition by the human Dectin-1 immune receptor after IVD, and (3) the recognition of IVD-solubilized material by different mammalian immune receptors. IVD digested 44–63% of the material, depending on the extraction process. The non-digested material, despite some visible agglutination and deformation of the microcapsules, preserved their spherical shape and was enriched in (β1→3)-glucans. These microcapsules were all recognized by the human Dectin-1 immune receptor. The digested material was differentially recognized by a variety of lectins of the immune system related to (β1→3)-glucans, glycogen, and mannans. These results show the potential of BSY microcapsules to be used as oral carriers for food and biomedical applications.
Helicobacter pylori lipopolysaccharide structural domains and their recognition by immune proteins revealed with carbohydrate microarrays
Publication . Silva, Lisete M.; Correia, Viviana G.; Moreira, Ana S. P.; Domingues, Maria Rosário M.; Ferreira, Rui Manuel; Figueiredo, Céu; Azevedo, Nuno F.; Marcos-Pinto, Ricardo; Carneiro, Fátima; Magalhães, Ana; Reis, Celso A.; Feizi, Ten; Ferreira, José A.; Coimbra, Manuel A.; Palma, Angelina S.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Elsevier
The structural diversity of the lipopolysaccharides (LPSs) from Helicobacter pylori poses a challenge to establish accurate and strain-specific structure-function relationships in interactions with the host. Here, LPS structural domains from five clinical isolates were obtained and compared with the reference strain 26695. This was achieved combining information from structural analysis (GC-MS and ESI-MSn) with binding data after interrogation of a LPS-derived carbohydrate microarray with sequence-specific proteins. All LPSs expressed Lewisx/y and N-acetyllactosamine determinants. Ribans were also detected in LPSs from all clinical isolates, allowing their distinction from the 26695 LPS. There was evidence for 1,3-D-galactans and blood group H-type 2 sequences in two of the clinical isolates, the latter not yet described for H. pylori LPS. Furthermore, carbohydrate microarray analyses showed a strain-associated LPS recognition by the immune lectins DC-SIGN and galectin-3 and revealed distinctive LPS binding patterns by IgG antibodies in the serum from H. pylori-infected patients.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-MIB/31730/2017