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The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states. Epithelial cells that line the gut secrete complex glycoproteins that form a mucus layer to protect the gut wall from enteric pathogens. Here, the authors provide a comprehensive characterisation of endo-acting glycoside hydrolases expressed by mucin-degrading members of the microbiome that are able to cleave the O-glycan chains of a range of different animal and human mucins.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-l-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-l-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1–3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3′-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.

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, Rg GH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that Rg GH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed Rg GH98 affinity for blood group A over blood group B and H antigens. The molecular basis of Rg GH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of Rg GH98 in complex with BgA trisaccharide (BgAtri) and of Rg GH98 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 Rg GH98 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 Rg GH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that Rg GH98 releases BgAtri from mucin and that pretreatment of mucin with Rg GH98 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 study of saliva O-glycosylation is receiving increasing attention due to the potential of glycans for disease biomarkers, but also due to easy access and non-invasive collection of saliva as biological fluid. Saliva is rich in glycoproteins which are secreted from the bloodstream or produced by salivary glands. Mucins, which are highly O-glycosylated proteins, are particularly abundant in human saliva. Their glycosylation is associated with blood group and secretor status, and represents a reservoir of potential disease biomarkers. This study aims to analyse and compare O-glycans released from whole human mouth saliva collected 3 times a day from a healthy individual over a 5 days period. O-linked glycans were released by hydrazinolysis, labelled with procainamide and analysed by ultra-high performance liquid chromatography with fluorescence detection (UHPLC-FLR) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). The sample preparation method showed excellent reproducibility and can therefore be used for biomarker discovery. Our data demonstrates that the O-glycosylation in human saliva changes significantly during the day. These changes may be related to changes in the salivary concentrations of specific proteins.

Peptide:N-glycosidases (PNGases) are used by a wide range of organisms to remove N-glycan structures from proteins for use as either nutrients or in glycoprotein processing. PNGaseF is the most well-characterized enzyme of this family and is widely used in glycobiology to allow study of the N-glycome of a specific protein, cell and tissues, for instance. Despite this, PNGaseF has limitations in the types of N-glycan structures it can target. In this study, we explored the specificities of six uncharacterized PNGases selected from diverse parts of the PNGaseF superfamily. One of these enzymes, PNGaseL from Flavobacterium akiainvivens, is the highlight of this study due to its very broad specificity, exemplified by its ability to cleave mammalian-, plant- and invertebrate-type complex N-glycans as well as high-mannose N-glycans. A detailed biochemical and structural characterization was carried out against a variety of substrates to illustrate the advanced capability of PNGaseL in comparison to the canonical PNGaseF and PNGaseA enzymes. To determine the optimal reaction conditions, assess stability and define limitations of PNGaseL, a series of validation studies were performed. The data reveal that PNGaseL has potential utility in a range of glycobiology applications that are superior to the current commercially available options.

Microbial α- l- fucosidases catalyse the hydrolysis of terminal α- l -fucosidic linkages and can perform transglycosylation reactions. Based on sequence identity, α- l- fucosidases are classified in glycoside hydrolases (GHs) families of the carbohydrate-active enzyme database. Here we explored the sequence-function space of GH29 fucosidases. Based on sequence similarity network (SSN) analyses, 15 GH29 α- l- fucosidases were selected for functional characterisation. HPAEC-PAD and LC-FD-MS/MS analyses revealed substrate and linkage specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated oligosaccharides and glycoconjugates, consistent with their SSN clustering. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1,6 linkages and FA2G2 N -glycan was determined by X-ray crystallography and STD NMR. The capacity of GH29 fucosidases to carry out transfucosylation reactions with GlcNAc and 3FN as acceptors was evaluated by TLC combined with ESI–MS and NMR. These experimental data supported the use of SSN to further explore the GH29 sequence-function space through machine-learning models. Our lightweight protein language models could accurately allocate test sequences in their respective SSN clusters and assign 34,258 non-redundant GH29 sequences into SSN clusters. It is expected that the combination of these computational approaches will be used in the future for the identification of novel GHs with desired specificities. Microbial α-L-fucosidases pertain to the glycoside hydrolases (GHs) family and catalyse the hydrolysis of terminal α-L-fucosidic linkages and transglycosylation reactions, however, prediction of their function directly from protein sequences remains challenging. Here, the authors combine a protein sequence similarity network and protein language model to explore the sequence-function space of GH29 fucosidases and substrate specificity of uncharacterised GH29 sequences.

The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insecttype N-glycans, which we discuss from the perspective of insects as a nutrient source.

Glycans are the major carbon sources available to the human colonic microbiota. Numerous N -glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N -glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron ( Bt ) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche. The gut microbiota member Bacteroides thetaiotaomicron can utilize various complex N -glycans, including those from immunoglobulins, via a complex and diverse set of enzymes encoded by multiple non-adjacent loci.

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker. The interlaboratory study showed good correlation between fucosylation levels measured for the 320 cases in the two centers with the correlation coefficient (r) of up to 0.88 for a single trait A3FG3S2. The improved chromatographic separation allowed the identification of six single glycan traits and a derived antennary fucosylation trait that were able to differentiate individuals carrying pathogenic mutations from benign or no HNF1A mutation cases, as determined by the area under the curve (AUC) of up to 0.94. The excellent (r = 0.88) interlaboratory performance of the glycan biomarker for HNF1A-MODY further supports the development of a clinically relevant diagnostic test measuring antennary fucosylation levels.

Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson’s r correlation coefficient of 0.8208 between the two methods.