Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins

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 that 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 monoclonal antibodies (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.

Critical structural elements for the antigenicity of wheat allergen LTP1 (Tri a 14) revealed by site-directed mutagenesis

“…Recently, conformational epitopes were mapped on the mugwort pollen allergen Art v 3 using directed mutagenesis in combination with a novel approach, H/D (hydrogen/deuterium) exchange memory NMR. This method measures differences in signals between the allergen–antibody complex and free allergen 41 .…”

Critical structural elements for the antigenicity of wheat allergen LTP1 (Tri a 14) revealed by site-directed mutagenesis

“…Very interestingly, these mAbs react similarly to patient sera towards the set of tested LTP1 variants, indicating that they surely bind the dominant IgE epitope regions containing the cluster C13, C27, C28, C73 and K72. This was also the case of mAbs generated to Art v 3, which showed good overlap with patient IgE 41 . This subgroup of anti-Nat-LTP mAbs could enable generation of recombinant monoclonal IgE as substitutes mimicking patients reactivity 49 .…”

“…Residue K72 is well conserved among fruit and cereal LTP1s. Together with cysteine C73, it belongs to epitope zones already revealed on Tri a 14 (66-80) and on other allergenic LTPs, Pru p 3 (71-80) 35 and Art v 3 (conformational epitope comprising K73 and C74) 41 . Its position between a proline that reduces the trypsin proteolytic activity and a cysteine forming a disul de bridge may assure its protection from enzymatic digestion.…”

“…simultaneous mutations of 39-40-43-44-76-77-78-79) 38 . Similarly, N77 appeared in a conformational epitope area of Art v 3 in combination with other residues (24, 33, 34, 36 and 39) 41 .…”

“…The structural integrity and preservation of disul de bonds is crucial for IgE-binding to wheat LTP1 34 and has also been observed to be important for other LTPs 41,48 . Among the four LTP1 disul de bonds that are characteristic of the prolamin superfamily, we identi ed two (C13-C27 and C28-C73) as critical elements for wheat LTP1 antigenicity.…”