background: Contribution of conformational epitopes to the IgE reactivity of peanut allergens Ara h 2 and Ara h 6 is at least as important as that of the linear epitopes. However, little is known about these conformational IgE-binding epitopes.
objective: We investigated the distribution of conformational epitopes on chimeric 2S-albumins.
methods: Recombinant chimeras were generated by exchanging structural segments between Ara h 2 and Ara h 6. Well-refolded chimeras, as verified by circular dichroism analysis, were then used to determine the epitope specificity of mAbs by performing competitive inhibition of IgG binding. Furthermore, we delineated the contribution of each segment to the overall IgE reactivity ... More
background: Contribution of conformational epitopes to the IgE reactivity of peanut allergens Ara h 2 and Ara h 6 is at least as important as that of the linear epitopes. However, little is known about these conformational IgE-binding epitopes.
objective: We investigated the distribution of conformational epitopes on chimeric 2S-albumins.
methods: Recombinant chimeras were generated by exchanging structural segments between Ara h 2 and Ara h 6. Well-refolded chimeras, as verified by circular dichroism analysis, were then used to determine the epitope specificity of mAbs by performing competitive inhibition of IgG binding. Furthermore, we delineated the contribution of each segment to the overall IgE reactivity of both 2S-albumins by measuring the chimeras' IgE-binding capacity with sera from 21 patients allergic to peanut. We finally assessed chimeras' capacity to trigger mast cell degranulation.
results: Configuration of the conformational epitopes was preserved in the chimeras. Mouse IgG mAbs, raised against natural Ara h 6, and polyclonal human IgE antibodies recognized different conformational epitopes distributed all along Ara h 6. In contrast, we identified human IgG mAbs specific to different Ara h 2 linear or conformational epitopes located in all segments except the C-terminal one. The major conformational IgE-binding epitope of Ara h 2 was located in a segment located between residues 33 and 81 that also contains the major linear hydroxyproline-containing epitope. Accordingly, this segment is critical for the capacity of Ara h 2 to induce mast cell degranulation.
conclusions: Chimeric 2S-albumins provide new insights on the conformational IgE-binding epitopes of Ara h 2 and Ara h 6. Proximity of the immunodominant linear and conformational IgE-binding epitopes probably contributes to the high allergenic potency of Ara h 2.
