Understanding IgE Glycosylation
Abstract
Evidence favouring the role of glycans in the modulation of immunoglobulin (IG) activities has been mounting. IgE antibodies mostly known for its role in allergic responses and anti-parasitic immune reactions, is the most complex glycosylated IG. Peculiarities include site-specific glycosylation (complex vs. oligomannosidic structures), large fractions of sialylation and significant diversity. Several studies point to the impact of glycosylation on IgE biology. However, researchers lack a broader understanding, mainly due to the current technological limitations on producing complex IgEs with defined glycan structures. Here, we aim to explain and control the processing of IgE N-glycans and investigate their impact on the structure, function and half-life of this antibody class. We aim to understand how complex structures are synthesized, which factors contribute to the generation of glycan diversity on a single protein and how glycosylation can be controlled. IgE will undergo intensive in vivo glycan-engineering processes with the aim of reconstructing single glycan species, an important prerequisite to study glycan functions. Biochemical/biophysical analyses will allow us to determine structural integrity of IgE glycovariants. Importantly, we will carry out functional activity assays, encompassing antigen/receptor binding and the ability of IgE glycovariants to induce effector functions. We expect that the results will significantly contribute to a better understanding of molecular mechanisms driving the synthesis of complex protein glycosylation and will generate novel insights how IgE glycans interact with the immune system. The knowledge gained might be transferred to other immunoglobulins and/or the generation of therapeutic IgE with optimised or novel functions. Methods: Monoclonal IgEs against well-defined antigen targets are chosen as models to enable controlled expression. IgE production will be accomplished by using a plant-based transient expression system, developed by the applicant, allowing rapid and flexible glycan engineering. Structural integrity of the antibodies will be determined by biochemical/biophysical means including MS-based analyses, CD spectroscopy, calorimetry. Functional analyses encompass in vitro binding studies (e.g. antigen-, Fc-receptor) and cell-based assays (e.g. ADCC, ADCP, mast cell degranulation, direct tumour killing); PK values of selected glycan engineered IgE will be determined in a mouse model.
Schlagworte Immunglobulin E Glycosylation
Publikationen
Mitarbeiter*innen
Herta Steinkellner
Ao.Univ.Prof. Mag.rer.nat. Dr.nat.techn. Herta Steinkellner
herta.steinkellner@boku.ac.at
Tel: +43 1 47654-94370
Project Leader
11.04.2016 - 10.12.2019