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Title: Allosteric communication in immunoglobulin E and the mechanisms of its interactions with allergens
Author: Bucaite, Gintare
ISNI:       0000 0004 8505 4947
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2020
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The incidence and severity of allergic disorders is increasing worldwide and there is an urgent need for better treatments. Immunoglobulin E (IgE) antibody is the central triggering molecule in allergic reactions, and an allergic reaction is rapidly generated when allergens bind and crosslink IgE-FceRI complexes on mast cells and basophils, resulting in cell degranulation and release of proinflammatory mediators. In this project, we have established and characterised a human patient-derived IgE/pollen-specific allergen interaction system. This model system was employed to study (i) the role of allergen affinity, oligomeric state, valency and spacing of IgE binding epitopes in the effector cell activation, and (ii) the long-range allosteric communication between the functional domains of IgE. We have biophysically and functionally characterised the interactions between two monoclonal timothy-grass pollen-specific IgE antibodies and four recombinant pollen-specific polcalcin allergens (from alder, birch, olive tree and timothy grass pollen). Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to determine the affinity, kinetics and thermodynamics between the antibodies and allergens. We have found that despite their high homology, polcalcins display different binding characteristics towards human patient-derived antibodies. Nuclear magnetic resonance (NMR) spectroscopy was used to map the IgE epitope on the timothy grass pollen allergen Phl p 7. Native and engineered polcalcin multimers were used in functional effector cell degranulation assays and we have determined that polcalcin multimers are re- 1 quired to stimulate high levels of mast cell degranulation and that an allergen's low affinity to the antibody may be overcome with an increased number of binding sites per molecule. IgE, as all antibody classes, contains two antigen-binding regions (Fab) and an Fc region, responsible for interactions with its receptors. Historically, the antigen recognition by Fabs and receptor binding by the Fc region in antibodies have been thought to be functionally and structurally independent. We have used anti-IgE Fc Fabs to capture IgE antibodies in different conformations for SPR experiments and measured the effect this had on the allergen binding to the IgE. We have identified a Fab (aeFab3), which when bound to the IgE Fc region may induce large-scale conformational changes between the Fab and Fc regions. We have characterised the interaction between Ce2 domain of IgE-Fc and aeFab3 using SPR and NMR methods. Future studies will be aimed to further investigate the allosteric mechanisms of antibody Fab and Fc cross-talk. We expect that the allergen-antibody model system we have established and the results presented here will contribute to a better understanding of the factors affecting allergen-IgE interactions and enable further elucidation of the mechanisms of allergic immune responses.
Supervisor: McDonnell, James Michael ; Sutton, Brian John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available