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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 insect-type N -glycans, which we discuss from the perspective of insects as a nutrient source.

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

Circulating mannose-binding lectin (MBL) plays an important role in the progression of tissue damage caused by ischemic events, an account of its high-affinity, multivalent binding to carbohydrate arrays exposed on damaged endothelium (damage-associated molecular patterns, DAMPS). MBL inhibitors have therefore been proposed as a novel protective therapeutic strategy to prevent secondary injury. We developed a new, convenient, robust surface plasmon resonance (SPR) assay for in vitro screening of compounds interfering with the binding of native human MBL to an appropriate chip surface functionalized with a pattern of sugar moieties mimicking DAMPS. We also characterized the procedure to regenerate the chip surface after each experimental session, accomplished by sequential cleaning with piranha solution followed by UV exposure. The SPR assay detects the specific binding of human recombinant MBL and native MBL present in human serum, and can identify inhibitors of this binding. We observed inhibitory effects of mannose (IC50 5 mM), of a nine mannose residues carrying glycan (IC50 0.33 mg/mL, corresponding to ~ 175 mu M), and mainly mannose-coated gold nanoparticles (IC50 1.1 mu g /mL). These in vitro results serve as a basis for testing the protective properties of these molecules/nanoparticles later inthe more expensive and time-consuming studies in cells and animal models of MBL mediated-injuries. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 814236.