profiling-permethylated-mucin-o-glycans-using-matrix-assisted-laser

Profiling of Permethylated Mucin <em>O</em>-glycans Using Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the profiling of mucin glycans that ...

Quadram Institute Bioscience

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives.

Membrane-enclosed multienzyme (MEME) synthesis of 2,7-anhydro-sialic acid derivatives.

Protein glycosylation is emerging as an important feature in bacteria. Protein glycosylation systems have been reported and studied in many pathogenic bacteria, revealing an important diversity of glycan structures and pathways within and between bacterial species. These systems play key roles in virulence and pathogenicity. More recently, a large number of bacterial proteins have been found to be glycosylated in gut commensal bacteria. We present an overview of bacterial protein glycosylation systems (- and -glycosylation) in bacteria, with a focus on glycoproteins from gut commensal bacteria, particularly Lactobacilli. These emerging studies underscore the importance of bacterial protein glycosylation in the interaction of the gut microbiota with the host.

How Sweet Are Our Gut Beneficial Bacteria? A Focus on Protein Glycosylation in Lactobacillus.

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein-carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly- N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule-grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.-Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcβ(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.

Serine-rich repeat protein adhesins from Lactobacillus reuteri display strain specific glycosylation profiles.

Serine-rich repeat proteins (SRRPs) have emerged as an important group of cell surface adhesins found in a growing number of Gram-positive bacteria. Studies focused on SRRPs from streptococci and staphylococci demonstrated that these proteins are -glycosylated on serine or threonine residues and exported via an accessory secretion (aSec) system. In pathogens, these adhesins contribute to disease pathogenesis and represent therapeutic targets. Recently, the non-canonical aSec system has been identified in the genomes of gut microbes and characterization of their associated SRRPs is beginning to unfold, showing their role in mediating attachment and biofilm formation. Here we provide an update of the occurrence, structure, and function of SRRPs across bacteria, with emphasis on the molecular and biochemical properties of SRRPs from gut symbionts, particularly Lactobacilli. These emerging studies underscore the range of ligands recognized by these adhesins and the importance of SRRP glycosylation in the interaction of gut microbes with the host.

Serine-rich repeat proteins from gut microbes.

Sialic acid (N-acetylneuraminic acid (Neu5Ac)) is commonly found in the terminal location of colonic mucin glycans where it is a much-coveted nutrient for gut bacteria, including Ruminococcus gnavus. R. gnavus is part of the healthy gut microbiota in humans, but it is disproportionately represented in diseases. There is therefore a need to understand the molecular mechanisms that underpin the adaptation of R. gnavus to the gut. Previous in vitro research has demonstrated that the mucin-glycan-foraging strategy of R. gnavus is strain dependent and is associated with the expression of an intramolecular trans-sialidase, which releases 2,7-anhydro-Neu5Ac, rather than Neu5Ac, from mucins. Here, we unravelled the metabolism pathway of 2,7-anhydro-Neu5Ac in R. gnavus that is underpinned by the exquisite specificity of the sialic transporter for 2,7-anhydro-Neu5Ac and by the action of an oxidoreductase that converts 2,7-anhydro-Neu5Ac into Neu5Ac, which then becomes a substrate of a Neu5Ac-specific aldolase. Having generated an R. gnavus nan-cluster deletion mutant that lost the ability to grow on sialylated substrates, we showed that-in gnotobiotic mice colonized with R. gnavus wild-type (WT) and mutant strains-the fitness of the nan mutant was significantly impaired, with a reduced ability to colonize the mucus layer. Overall, we revealed a unique sialic acid pathway in bacteria that has important implications for the spatial adaptation of mucin-foraging gut symbionts in health and disease.

Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut.

The human gut symbiont  scavenges host-derived -acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into  ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (NanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro--acetylneuraminic acid intermediate and NAD regeneration. The crystal structure of NanOx in complex with the NAD cofactor showed a protein dimer with a Rossman fold. Guided by the NanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of NanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the  homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that  could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using  mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in  depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in .

Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.

Polysaccharides such as β-2,1-linked fructans including inulin or fructose oligosaccharides are well-known prebiotics with recognised immunomodulatory properties. In recent years, other fructan types covering β-2,6-linked fructans, particularly microbial levans, have gained increasing interest in the field. β-2,6-linked fructans of different degrees of polymerisation can be synthesised by plants or microbes including those that reside in the gastrointestinal tract. Accumulating evidence suggests a role for these β-2,6 fructans in modulating immune function. Here, we provide an overview of the sources and structures of β-2,6 fructans from plants and microbes and describe their ability to modulate immune function in vitro and in vivo along with the suggested mechanisms underpinning their immunomodulatory properties. Further, we discuss the limitations and perspectives pertinent to current studies and the potential applications of β-2,6 fructans including in gut health.

The Immunomodulatory Properties of β-2,6 Fructans: A Comprehensive Review.

Levan, a β-2,6 fructofuranose polymer produced by microbial species, has been reported for its immunomodulatory properties via interaction with toll-like receptor 4 (TLR4) which recognises lipopolysaccharide (LPS). However, the molecular mechanisms underlying these interactions remain elusive. Here, we investigated the immunomodulatory properties of levan using thoroughly-purified and characterised samples from Erwinia herbicola and other sources. E. herbicola levan was purified by gel-permeation chromatography and LPS was removed from the levan following a novel alkali treatment developed in this study. E. herbicola levan was then characterised by gas chromatography-mass spectrometry and NMR. We found that levan containing LPS, but not LPS-depleted levan, induced TLR4-mediated cytokine production by bone marrow-derived dendritic cells and/or activated TLR4 reporter cells. These data indicated that the immunomodulatory properties of the levan toward TLR4-expressing immune cells were mediated by the LPS. This work also demonstrates the importance of LPS removal when assessing the immunomodulatory activity of polysaccharides.

Lipopolysaccharide associated with β-2,6 fructan mediates TLR4-dependent immunomodulatory activity in vitro.

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.

The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract.

Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of  and  strains to utilise xylan derivatives. We showed that  ATCC 53608 and  ATCC 27340 produced β-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial (-nitrophenyl β-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in  but not in  strains, and quantitative proteomics identified other enzymes and transporters involved in  XOS utilisation. Based on these findings, we proposed a model for an XOS metabolism pathway in  and  strains. Together with phylogenetic analyses, the data also revealed the extended xylanolytic potential of the gut microbiota.

Biochemical Basis of Xylooligosaccharide Utilisation by Gut Bacteria.

 is a prevalent member of the human gut microbiota, which is over-represented in inflammatory bowel disease and neurological disorders. We previously showed that the ability of  to forage on mucins is strain-dependent and associated with sialic acid metabolism. Here, we showed that mice monocolonized with  ATCC 29149 (-mice) display changes in major sialic acid derivatives in their cecum content, blood, and brain, which is accompanied by a significant decrease in the percentage of sialylated residues in intestinal mucins relative to germ-free (GF) mice. Changes in metabolites associated with brain function such as tryptamine, indolacetate, and trimethylamine -oxide were also detected in the cecal content of -mice when compared to GF mice. Next, we investigated the effect of  monocolonization on hippocampus cell proliferation and behavior. We observed a significant decrease of PSA-NCAM immunoreactive granule cells in the dentate gyrus (DG) of -mice as compared to GF mice and recruitment of phagocytic microglia in the vicinity. Behavioral assessments suggested an improvement of the spatial working memory in -mice but no change in other cognitive functions. These results were also supported by a significant upregulation of genes involved in proliferation and neuroplasticity. Collectively, these data provide first insights into how  metabolites may influence brain regulation and function through modulation of granule cell development and synaptic plasticity in the adult hippocampus. This work has implications for further understanding the mechanisms underpinning the role of  in neurological disorders.

The role of the mucin-glycan foraging  in the communication between the gut and the brain.

The human gastrointestinal (GI) tract harbors diverse microbial communities collectively known as the gut microbiota that exert a profound impact on human health and disease. The repartition and availability of sialic acid derivatives in the gut have a significant impact on the modulation of gut microbes and host susceptibility to infection and inflammation. Although N-acetylneuraminic acid (Neu5Ac) is the main form of sialic acids in humans, the sialic acid family regroups more than 50 structurally and chemically distinct modified derivatives. In the GI tract, sialic acids are found in the terminal location of mucin glycan chains constituting the mucus layer and also come from human milk oligosaccharides in the infant gut or from meat-based foods in adults. The repartition of sialic acid in the GI tract influences the gut microbiota composition and pathogen colonization. In this review, we provide an update on the mechanisms underpinning sialic acid utilization by gut microbes, focusing on sialidases, transporters, and metabolic enzymes.

Biochemical and structural basis of sialic acid utilization by gut microbes.

Alterations in intestinal mucin glycosylation have been associated with increased intestinal permeability and sensitivity to inflammation and infection. Here, we used mice lacking core 3-derived O-glycans (C3GnT) to investigate the effect of impaired mucin glycosylation in the gut-brain axis. C3GnT mice showed altered microbial metabolites in the caecum associated with brain function such as dimethylglycine and N-acetyl-L-tyrosine profiles as compared to C3GnT littermates. In the brain, polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive granule cells showed an aberrant phenotype in the dentate gyrus of C3GnT mice. This was accompanied by a trend towards decreased expression levels of PSA as well as ZO-1 and occludin as compared to C3GnT. Behavioural studies showed a decrease in the recognition memory of C3GnT mice as compared to C3GnT mice. Combined, these results support the role of mucin O-glycosylation in the gut in potentially influencing brain function which may be facilitated by the passage of microbial metabolites through an impaired gut barrier.

Role of mucin glycosylation in the gut microbiota-brain axis of core 3 O-glycan deficient mice.

Bacterial extracellular vesicles (BEVs) produced by members of the intestinal microbiota can not only contribute to digestion but also mediate microbe-host cell communication via the transfer of functional biomolecules to mammalian host cells. An unresolved question is which host factors and conditions influence BEV cargo and how they impact host cell function. To address this question, we analysed and compared the proteomes of BEVs released by the major human gastrointestinal tract (GIT) symbiont  (Bt) in vivo in fed versus fasted animals using nano-liquid chromatography with tandem mass spectrometry (LC-MSMS). Among the proteins whose abundance was negatively affected by fasting, nine of ten proteins of the serine protease family, including the regulatory protein dipeptidyl peptidase-4 (DPP-4), were significantly decreased in BEVs produced in the GITs of fasted animals. Strikingly, in extracellular vesicles produced by the intestinal epithelia of the same fasted mice, the proteins with the most increased abundance were serine protease inhibitors (serpins). Together, these findings suggest a dynamic interaction between GI bacteria and the host. Additionally, they indicate a regulatory role for the host in determining the balance between bacterial serine proteases and host serpins exported in bacterial and host extracellular vesicles.

Proteomics of Bacterial and Mouse Extracellular Vesicles Released in the Gastrointestinal Tracts of Nutrient-Stressed Animals Reveals an Interplay Between Microbial Serine Proteases and Mammalian Serine Protease Inhibitors.

Article	Year
Membrane-enclosed multienzyme (MEME) synthesis of 2,7-anhydro-sialic acid derivatives. Carbohydrate research\| PubMed ID: 28851488	2017
How Sweet Are Our Gut Beneficial Bacteria? A Focus on Protein Glycosylation in Lactobacillus. International journal of molecular sciences\| PubMed ID: 29301365	2018
Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 29401627	2018
Serine-rich repeat protein adhesins from Lactobacillus reuteri display strain specific glycosylation profiles. Glycobiology\| PubMed ID: 30371779	2019
Serine-rich repeat proteins from gut microbes. Gut microbes\| PubMed ID: 31035824	2020
Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut. Nature microbiology\| PubMed ID: 31636419	2019
Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria. The Journal of biological chemistry\| PubMed ID: 32669363	2020
The Immunomodulatory Properties of β-2,6 Fructans: A Comprehensive Review. Nutrients\| PubMed ID: 33921025	2021
Lipopolysaccharide associated with β-2,6 fructan mediates TLR4-dependent immunomodulatory activity in vitro. Carbohydrate polymers\| PubMed ID: 34893207	2022
The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract. PLoS biology\| PubMed ID: 34936658	2021
Biochemical Basis of Xylooligosaccharide Utilisation by Gut Bacteria. International journal of molecular sciences\| PubMed ID: 35328413	2022
The role of the mucin-glycan foraging in the communication between the gut and the brain. Gut microbes\| PubMed ID: 35579971	2022
Biochemical and structural basis of sialic acid utilization by gut microbes. The Journal of biological chemistry\| PubMed ID: 36758803	2023
Role of mucin glycosylation in the gut microbiota-brain axis of core 3 O-glycan deficient mice. Scientific reports\| PubMed ID: 37634035	2023
Proteomics of Bacterial and Mouse Extracellular Vesicles Released in the Gastrointestinal Tracts of Nutrient-Stressed Animals Reveals an Interplay Between Microbial Serine Proteases and Mamm... International journal of molecular sciences\| PubMed ID: 40362319	2025

Dimitrios Latousakis

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