Abstract
Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.
Highlights
Interaction of antibodies with their antigen-binding sites is vital to maintain health but may contribute to immunological diseases
More than 100 tertiary structures of allergens have been solved, but information on their antibody-binding epitopes is very limited [23]. This applies to nonspecific lipid transfer proteins (LTPs) that were identified as allergens in food, pollen, and latex [6, 24]
We developed hydrogen/deuterium exchange memory (HDXMEM), a novel NMR method, and elucidated three clinically relevant IgE-binding epitopes of the mugwort pollen allergen Art v 3
Summary
To generate mAbs, mice were immunized with recombinant Art v 3.0201 (for simplicity termed Art v 3). To map the functional epitopes of Art v 3 with the three intact mAbs in situ despite the tight binding and extremely slow koff rates, we developed an innovative approach based on H/D exchange This overcomes the limitations of conventional H/D exchange measurements because such tight interactions between antigens and intact mAbs that are not directly detectable by NMR spectroscopy due to their large size, or rely on a tedious protocol of quenching the exchange and separating the antigen from the mAb for each time point [21]. H/D exchange times could be extracted for 36% of all amino acids at 278 K for the free protein and for 33–36% for the samples containing mAbs. To map the binding epitope, the H/D exchange times of the allergen were compared between the measurements with and without mAb H2O D2O mAb mAb k(H/Dex, bound) koff allergen kon Lyophilization D2O kon k(H/Dex, free) detectable differences koff mAb kon detectable differences detectable by NMR undetectable by NMR detectable by NMR
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
