Some studies claim that IgE glycosylation is not essential for binding to FcεRI and subsequent Fc receptor-mediated functions ( Basu et al., 1993 Young et al., 1995 Henry et al., 2000), while other investigations have contradicted these results ( Nettletone and Kochan, 1995 Helm et al., 1998 Björklund et al., 1999 Hunt et al., 2005). Attempts to explore IgE glycosylation have concentrated on investigating its interaction with its two Fcε receptors, as they are key players in the allergic cascade: the high affinity Fcε receptor I (FcεRI), present on mast cells, basophils and eosinophils, and the low affinity CD23 (FcεRII) found, amongst others, on B cells and activated macrophages. While N-glycans have been proven to be involved in the modulation of IgG effector functions, relatively little is known about their role on other antibody classes ( Jefferis, 2012). Serum IgE is heavily sialylated, with significant N-glycan diversity between different (patho-) physiological stages ( Arnold et al., 2004 Plomp et al., 2014). The glycosylation of IgE presents peculiarities, like site-specific occupancy (lack of glycans at NGS6) and differential site glycosylation pattern (oligomannose vs. In fact, IgE is the most heavily glycosylated antibody class, with seven N-glycosylation sites (NGS) and ~12% of their molecular weight made up of carbohydrates (see a schematic illustration of human IgE Supplementary Figure 1). Both Fcε receptors and IgE are heavily glycosylated molecules, implying a potential impact of this post-translational modification on their activities. The principal mechanisms of IgE antibodies in allergic diseases are (i) recognizing allergens through their antigen-binding regions (Fab) and (ii) interacting via their Fc regions with their two cell surface receptors to induce the allergic cascade ( Gould and Sutton, 2008). Immunoglobulin E (IgE), normally the least abundant antibody class in human serum, is most commonly known for its role in the allergic response. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities. In contrast, binding to the low affinity receptor CD23 (FcεRII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor FcεRI, and subsequent similar capacity to trigger mast cell degranulation. Recombinant human cell-derived HER2-IgE exhibited large N-glycan heterogeneity. We were able to not only modulate the five NGSs naturally decorated with complex N-glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Plant-derived HER2-IgE exhibited N-glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N-glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Here, we applied an in vivo approach that allows the manipulation of IgE N-glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven N-glycosites (NGS) in its Fab and Fc domains may modulate its functions. 4Breast Cancer Now Research Unit, Guy's Cancer Centre, School of Cancer and Pharmaceutical Sciences, King's College London, London, United Kingdom.John's Institute of Dermatology, Guy's Hospital, London, United Kingdom 3School of Basic and Medical Biosciences, King's College London, St. ![]() 2Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.1Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria. ![]() Karagiannis 3,4, Friedrich Altmann 2 and Herta Steinkellner 1 * Laura Montero-Morales 1, Daniel Maresch 2, Silvia Crescioli 3, Alexandra Castilho 1, Kristina M.
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