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.

Erythropoietin (EPO) is one of the main therapeutics used to treat anemic patients, greatly improving their quality of life. In this study, biosimilars Binocrit and a development product, called here CIGB-EPO, were compared to the originator product, Eprex. All three are epoetin alpha products, reputed to have similar glycosylation profiles. The quality, safety, and efficacy of this biotherapeutic depend on the following glycosylation critical quality attributes (GCQAs): sialylation, N-glycolyl-neuraminic acid (Neu5Gc) content, branching, N-acetyl-lactosamine (LacNAc) extensions, and O-acetylation pattern. Reverse-phase ultra-high-pressure liquid chromatography (RP-UHPLC) analysis of acid-released, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labeled sialic acid derivatives and hydrophilic interaction liquid chromatography (HILIC) in combination with mass spectrometry (HILIC-UHPLC-MS) of procainamide (PROC) labeled N-glycans were the analytical tools used. An automated method for enzymatic release and PROC labeling was applied for the first time to the erythropoiesis stimulating agent (ESA) products, which facilitated novel, in-depth characterization, and allowed identification of precise structural features including the location of O-acetyl groups on sialic acid (SA) moieties. Samples were digested by a sialate-O-acetylesterase (NanS) to confirm the presence of O-acetyl groups. It was found that Eprex contained the greatest relative abundance of O-acetylated derivatives, Binocrit expressed the least Neu5Gc, and CIGB-EPO showed the greatest variety of high-mannose-phosphate structures. The sialylation and LacNAc extension patterns of the three ESAs were similar, with a maximum of four N-acetyl-neuraminic acid (Neu5Ac) moieties detected per glycan. Such differences in SA derivatization, particularly O-acetylation, could have consequences for the quality and safety of a biotherapeutic, as well as its efficacy.

Monoclonal antibodies (mAbs) are one of the cornerstones of modern medicine, across an increasing range of therapeutic areas. All therapeutic mAbs are glycoproteins, i.e., their polypeptide chain is decorated with glycans, oligosaccharides of extraordinary structural diversity. The presence, absence, and composition of these glycans can have a profound effect on the pharmacodynamic and pharmacokinetic profile of individual mAbs. Approaches for the glycoengineering of therapeutic mAbs—the manipulation and optimisation of mAb glycan structures—are therefore of great interest from a technological, therapeutic, and regulatory perspective. In this review, we provide a brief introduction to the effects of glycosylation on the biological and pharmacological functions of the five classes of immunoglobulins (IgG, IgE, IgA, IgM and IgD) that form the backbone of all current clinical and experimental mAbs, including an overview of common mAb expression systems. We review selected examples for the use of small molecule inhibitors of glycan biosynthesis for mAb glycoengineering, we discuss the potential advantages and challenges of this approach, and we outline potential future applications. The main aim of the review is to showcase the expanding chemical toolbox that is becoming available for mAb glycoengineering to the biology and biotechnology community.

Naturally occurring asymmetric and triantennay N -glycans can be isolated from hen egg yolk glycopeptides.

Cardiovascular disease (CVD), diabetes and cancer pose increasing global healthcare burdens. New biomarkers could enable earlier diagnosis of these diseases, leading to more effective treatment and...

Biomarkers to guide clinical decision-making at diagnosis of inflammatory bowel disease (IBD) are urgently needed. We investigated a composite serum N-glycomic biomarker to predict future disease course in a discovery cohort of 244 newly diagnosed IBD patients. Forty-seven individual glycan peaks were analysed using ultra-high performance liquid chromatography identifying 105 glycoforms from which 24 derived glycan traits were calculated. Multivariable logistic regression was performed to determine associations of derived glycan traits with disease. Cox proportional hazard models were used to predict treatment escalation from first-line treatment to biologics or surgery (hazard ratio (HR) 25.9, p=1.1×10^-12; 95% confidence interval (CI), 8.52–78.78). Application to an independent replication cohort of 54 IBD patients yielded a HR of 5.1 (p=1.1×10^-5; 95% CI, 2.54–10.1). These data demonstrate the prognostic capacity of serum N-glycan biomarkers and represent a step towards personalized medicine in IBD.