Biology subjects

Cartmell, A.

Publications and source records attributed to Cartmell, A..

3 recordsLinked to original sources

bioRxiv2020

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current gold standards for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated enzymes. One sentence summarySulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.

biochemistry↗

bioRxiv2020

A single bacterial sulfatase is required for metabolism of colonic mucin O-glycans and intestinal colonization by a symbiotic human gut bacterium

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

microbiology↗

bioRxiv2020

Insights into glycan import by a prominent gut symbiont

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the {beta}2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to [~]2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.

microbiology↗