Biology subjects

Crouch, L. I.

Publications and source records attributed to Crouch, L. I..

5 recordsLinked to original sources

bioRxiv2025

PNGaseA-mediated N-glycan stripping from peptides by infant-derived Bifidobacterium bifidum

N-glycans are highly common sources of nutrition for human colonic-dwelling bacteria. These microbes have evolved a several methods to remove N-glycans from proteins; herein we describe the biochemical and structural characterisation of one such enzyme, a PNGaseA superfamily member produced by the infant-associated Bifidobacterium bifidum LMG13195. This PNGase was demonstrated to elicit activity against a wide variety of N-glycan structures yet exhibited a high preference for N-glycans attached to a peptide rather than to a denatured or native protein. This unusual specificity highlights how bacterial species tune their enzymology to different types of substrates. The structural characterisation of this PNGase reveals how its structure determines this specificity while being the first structure presented from the PNGaseA superfamily, revealing a unique ten-stand {beta}-sheet cradling a canonical PNGase catalytic module.

biochemistry↗

bioRxiv2025

PNGaseL from Flavobacterium akiainvivens targets a diverse range of N-glycan structures

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-characterised 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 uncharacterised 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 characterisation 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 was performed. The data reveal that PNGaseL has potential utility in a range of glycobiology applications that are superior to the current commercially available options.

biochemistry↗

bioRxiv2024

Carbohydrate-active enzymes from Akkermansia muciniphila breakdown mucin O-glycans to completion

Akkermansia muciniphila is a human microbial symbiont residing in the mucosal layer of the large intestine. Its main carbon source is the highly heterogeneous mucin glycoprotein and A. muciniphila uses an array of Carbohydrate-active enzymes and sulfatases to access this complex energy source. Here we describe the biochemical characterisation of fifty-four glycoside hydrolases, eleven sulfatases, and one polysaccharide lyase from A. muciniphila to provide a holistic understanding of the carbohydrate-degrading activities. The results provide an extensive insight into the sequence of O-glycan degradation and how A. muciniphila can access this structurally variable substrate. One of the most outstanding elements of this work was the demonstration that these enzymes can act synergistically to degrade the O-glycans on the mucin polypeptide to completion, down to the core GalNAc. Additionally, human breast milk oligosaccharide, ganglioside, and globoside glycan structures were included in the study to understand the full degradative capability of A. muciniphila.

biochemistry↗

bioRxiv2022

Plant N-glycan breakdown by human gut Bacteroides

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 characterised 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 characterise five CAZymes involved, including structures of the PNGase and GH92 -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 insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.

biochemistry↗

bioRxiv2019

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown

The human gut microbiota (HGM) are closely associated with health, development and disease. The thick intestinal mucus layer, especially in the colon, is the key barrier between the contents of the lumen and the epithelial cells, providing protection against infiltration by the microbiota as well potential pathogens. The upper layer of the colonic mucus is a niche for a subset of the microbiota which utilise the mucin glycoproteins as a nutrient source and mucin grazing by the microbiota appears to play a key role in maintaining barrier function as well as community stability. Despite the importance of mucin breakdown for gut health, the mechanisms by which gut bacteria access this complex glycoprotein are not well understood. The current model for mucin degradation involves exclusively exo-acting glycosidases that sequentially trim monosaccharides from the termini of the glycan chains to eventually allow access to the mucin peptide backbone by proteases. However, this model is in direct contrast to the Sus paradigm of glycan breakdown used by the Bacteroidetes which involves extracellular cleavage of glycans by surface located endo-acting enzymes prior to import of the oligosaccharide products. Here we describe the discovery and characterisation of endo-acting family 16 glycoside hydrolases (GH16s) from prominent mucin degrading gut bacteria that specifically target the oligosaccharide side chains of intestinal mucins from both animals and humans. These endo-acting O-glycanases display {beta}1,4-glactosidase activity and in several cases are surface located indicating they are involved in the initial step in mucin breakdown. The data suggest a new paradigm for mucin breakdown by the microbiota and the endo-mucinases provide a potential tool to explore changes that occur in mucin structure in intestinal disorders such as inflammatory bowel disease and colon cancer.

biochemistry↗