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.

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...

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLe^X) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLe^X in CRC. The SW620FUT6 cell line expressed high levels of sLe^X antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.

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.

The impact of human IgE glycosylation on structure, function and disease mechanisms is not fully elucidated, and heterogeneity in different studies renders drawing conclusions challenging. Previous reviews discussed IgE glycosylation focusing on specific topics such as health versus disease, FcεR binding or impact on function. We present the first systematic review of human IgE glycosylation conducted utilizing the PRISMA guidelines. We sought to define the current consensus concerning the roles of glycosylation on structure, biology and disease. Despite diverse analytical methodologies, source, expression systems and the sparsity of data on IgE antibodies from non‐allergic individuals, collectively evidence suggests differential glycosylation profiles, particularly in allergic diseases compared with healthy states, and indicates functional impact, and contributions to IgE‐mediated hypersensitivities and atopic diseases. Beyond allergic diseases, dysregulated terminal glycan structures, including sialic acid, may regulate IgE metabolism. Glycan sites such as N394 may contribute to stabilizing IgE structure, with alterations in these glycans likely influencing both structure and IgE‐FcεR interactions. This systematic review therefore highlights critical IgE glycosylation attributes in health and disease that may be exploitable for therapeutic intervention, and the need for novel analytics to explore pertinent research avenues.