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Deciphering Human-microbiome glyco-code: An integrative approach to uncover protein-glycan interactions in the gut environment
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O microbioma humano é uma comunidade complexa de microorganismos que estão envolvidos em vários processos biológicos do hospedeiro. A bactéria Bacteroides thetaiotaomicron é altamente adaptável, alterando as preferências metabólicas de acordo com os glicanos complexos que estão disponíveis no intestino. O genoma desta espécie contém sistemas complexos, com genes co-regulados, designados Polysaccharide Utilization Loci (PUL) que codificam proteínas, tais como módulos de ligação a hidratos de carbono (CBMs), SusDs e enzimas modulares, com especificidade para um determinado glicano. Esta bactéria, tem a particularidade de expressar uma diversidade de PULs que previsívelmente degradam glicanos do hospedeiro, tais como O-glicanos de mucinas, glicoproteínas constituintes da mucosa intestinal que proteje o epitélio. A sua degradação excessiva pode levar à danificação da mucosa intestinal, facilitando a entrada de patógenos e doenças inflamatórias. O trabalho desenvolvido nesta Tese pretendeu contribuir para o estudo do reconhecimento de O-glicanos do hospedeiro por estes PULs a nível molecular, que é ainda vago, e focou para este fim em proteínas do PUL que direccionam o sistema para o substrato (CBMs anotados na família 32, CBM32), ou para importe no periplasma (proteínas do tipo SusD). Utilizando a tecnologia dos microarrays de glicanos, foram identificadas especificidades de ligação únicas para os CBMs a motivos presentes em O-glicanos, sendo o terminal não redutor galactose o monossacárido comum reconhecido: sequências do tipo LacNAc (Galβ1-4/3GlcNAc-) e LacDiNAc (GalNAcβ1-4GlcNAc-) para o BT0865-CBM, core 1 (Galβ1-3GalNAcαSer/Thr) e core 2 (GlcNAcβ1-6(Galβ1-3)GalNAcαSer/Thr) para o BT3015C-CBM e Lewisa (Galβ1-3(Fucα1-4)GlcNAc-) para o BT4040-CBM. Tendo em conta a novidade dos glicanos reconhecidos, os CBMs BT3015C e BT4040 foram selecionados para caracterização estrutural por Cristalografia de raios-X e posterior análise da interação com o ligando. A estrutura 3D do BT3015C-CBM em complexo com core-1 e core-2 O-glicanos revelou um local de ligação canónico para a galactose e os amino ácidos Asn59, Asn89 and Asp147 em loops variáveis adjacentes a conferir especifidade para o ligando. A estrutura 3D da proteína SusD BT3013 do mesmo PUL, permitiu identificar uma região variável de loops com vários amino ácidos aromáticos, que podem constituir um sítio de ligação putativo. A caracterização estrutural do BT4040-CBM e de mutantes funcionais, identificou o trissacárido de Lewisa como motivo mínimo necessário para o reconhecimento e o amino ácido Glu73 como determinante da especificidade para o ligando fucosilado. A caracterização destes complexos de proteínas do B. thetaiotaomicron com o glicano alvo é essencial para perceber o metabolismo de glicanos por esta bactéria e seu o impacto no hospedeiro, conhecimento que pode ser usado no melhoramento estratégico da saúde do trato gastro-intestinal.
The human microbiome is a complex microbial community, involved in biological processes such as nutrition, protection against pathogens and immune system modulation. Bacteroides thetaiotaomicron is a highly adaptable gut commensal that can switch its metabolic preferences according to the complex glycans available (dietary carbohydrates and highly glycosylated host mucins) and is considered as a model microorganism of glycan utilization. To orchestrate the breakdown of complex glycans, B. thetaiotaomicron holds highly efficient polysaccharide utilization loci (PUL) systems, comprising sets of co-regulated genes. These genes encode proteins, such as carbohydrate-binding modules (CBMs), SusD-like proteins and modular enzymes with appended CBMs, that target a specific glycan substrate. A high number of PULs are predicted to target host glycoconjugates, e.g., mucin O-glycoproteins. Targeting mucin O-glycosylation will disturb the intestinal mucosal layer, opening the way to pathogens and inflammatory diseases. But glycan recognition by these PULs remains largely uncharacterized, making this human-microbiome recognition an uncharted territory. This work aimed to fill this gap by studying the molecular recognition of host glycans by family 32 CBMs and SusD-like proteins from B. thetaiotaomicron PULs. To this end, we applied an integrative strategy, combining as main approaches glycan microarrays for ligand discovery, and X-ray crystallography for characterization of protein and protein-glycan interactions, complemented with O-glycopeptide microarrays, NMR and ITC or MST biophysical methods. Overall, unique CBM specificities were identified towards the sequences LacNAc (Galβ1-4/3GlcNAc-) and LacDiNAc (GalNAcβ1-4GlcNAc-) for BT0865, core 1 (Galβ1-3GalNAcαSer/Thr) and core 2 (GlcNAcβ1-6(Galβ1-3)GalNAcαSer/Thr) O-glycans for BT3015C and Lewisa (Galβ1-3(Fucα1-4)GlcNAc-) for BT4040, with the non-reducing end galactose as a common recognition motif. Due to the novelty of unique specificities, BT3015C and BT4040 CBMs were selected for structural characterization to detail the molecular recognition. The 3D structure of BT3015C-CBM in complex with core 1 and core 2 O-glycans revealed a typical canonical galactose-binding site with amino acids Asn59, Asn89 and Asp147 in adjacent loops conferring the ligand specificity. For BT3013 SusD of the same PUL, the 3D structure was also determined, which revealed the variable loop-rich region with several aromatic residues that possibly constitute the putative binding site. Structural and functional studies of BT4040 WT and mutants, identified Lewisa trisaccharide as the minimum binding epitope and Glu73 as a key for the ligand specificity. The assignment of binding specificity and structural characterization of these protein-glycan complexes helps to understand the role of specific proteins in B. thetaiotaomicron, which is required to understand the impact of this microorganism on the host. Also, as human gut diseases are increasing, this knowledge can be used to design new strategies to improve gut health.
The human microbiome is a complex microbial community, involved in biological processes such as nutrition, protection against pathogens and immune system modulation. Bacteroides thetaiotaomicron is a highly adaptable gut commensal that can switch its metabolic preferences according to the complex glycans available (dietary carbohydrates and highly glycosylated host mucins) and is considered as a model microorganism of glycan utilization. To orchestrate the breakdown of complex glycans, B. thetaiotaomicron holds highly efficient polysaccharide utilization loci (PUL) systems, comprising sets of co-regulated genes. These genes encode proteins, such as carbohydrate-binding modules (CBMs), SusD-like proteins and modular enzymes with appended CBMs, that target a specific glycan substrate. A high number of PULs are predicted to target host glycoconjugates, e.g., mucin O-glycoproteins. Targeting mucin O-glycosylation will disturb the intestinal mucosal layer, opening the way to pathogens and inflammatory diseases. But glycan recognition by these PULs remains largely uncharacterized, making this human-microbiome recognition an uncharted territory. This work aimed to fill this gap by studying the molecular recognition of host glycans by family 32 CBMs and SusD-like proteins from B. thetaiotaomicron PULs. To this end, we applied an integrative strategy, combining as main approaches glycan microarrays for ligand discovery, and X-ray crystallography for characterization of protein and protein-glycan interactions, complemented with O-glycopeptide microarrays, NMR and ITC or MST biophysical methods. Overall, unique CBM specificities were identified towards the sequences LacNAc (Galβ1-4/3GlcNAc-) and LacDiNAc (GalNAcβ1-4GlcNAc-) for BT0865, core 1 (Galβ1-3GalNAcαSer/Thr) and core 2 (GlcNAcβ1-6(Galβ1-3)GalNAcαSer/Thr) O-glycans for BT3015C and Lewisa (Galβ1-3(Fucα1-4)GlcNAc-) for BT4040, with the non-reducing end galactose as a common recognition motif. Due to the novelty of unique specificities, BT3015C and BT4040 CBMs were selected for structural characterization to detail the molecular recognition. The 3D structure of BT3015C-CBM in complex with core 1 and core 2 O-glycans revealed a typical canonical galactose-binding site with amino acids Asn59, Asn89 and Asp147 in adjacent loops conferring the ligand specificity. For BT3013 SusD of the same PUL, the 3D structure was also determined, which revealed the variable loop-rich region with several aromatic residues that possibly constitute the putative binding site. Structural and functional studies of BT4040 WT and mutants, identified Lewisa trisaccharide as the minimum binding epitope and Glu73 as a key for the ligand specificity. The assignment of binding specificity and structural characterization of these protein-glycan complexes helps to understand the role of specific proteins in B. thetaiotaomicron, which is required to understand the impact of this microorganism on the host. Also, as human gut diseases are increasing, this knowledge can be used to design new strategies to improve gut health.
Descrição
Palavras-chave
Bacteroides thetaiotaomicron Carbohydrate-Binding Modules SusD-like proteins Protein-glycan recognition Glycan microarrays X-ray crystallography