Contribution of chemical modifications and conformational epitopes to IgE binding by Ara h 3

Authors: SCOTT, DYER - Maine Medical Center Research Institute (MMCRI); Nesbit, Jacqueline; CABANILLAS, BEATRIZ - University Of Bonn; Cheng, Hsiaopo; Hurlburt, Barry; Maleki, Soheila

Submitted to: Foods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/2018
Publication Date: 11/14/2018
Citation: Scott, D., Nesbit, J.B., Cabanillas, B., Cheng, H., Hurlburt, B.K., Maleki, S.J. 2018. Contribution of chemical modifications and conformational epitopes to IgE binding by Ara h 3. Foods. 7(11):189. https://doi.org/10.3390/foods7110189.
DOI: https://doi.org/10.3390/foods7110189

Interpretive Summary: We have previously shown that roasting can alter allergenic properties of peanut proteins. Here we purify a major peanut allergen, Ara h 3 from raw, light roast (LR) and dark roast peanuts (DR) and show that the altered allergenic properties, due to roasting are more likely due to the chemical modifications that are induced by heat treatment than the changes in the structure of the protein. We then ask what role the structure plays in allergenic properties. We assess the immunoglobulin E (IgE) antibody binding to Ara h 3 that has been partially unfolded and find that it is significantly reduced compared to the folded Ara h 3. Therefore we conclude that the structure of the protein is important for maintaining the IgE binding properties and can also influence the allergenic properties of peanuts. This may allow us to determine methods to reduce the allergenic potential of peanuts.

Technical Abstract: We have previously shown that processes, such as roasting, can alter the allergenic properties of peanuts. To understand these observations at a molecular level, the IgE binding and structural characteristics of Ara h 3 from raw and roasted peanuts were assessed. Ara h 3 (A3) was purified from raw (R), light roast (LR) and Dark Roast (DR) peanuts, the purity was assessed using SDS-PAGE and the secondary structures compared with circular dichroism (CD) spectroscopy. In order to understand the contribution of structure to IgE binding, the N Ara h 3 was partially denatured (PD) by heat treatment (65oC for 2hrs), subjected to CD spectroscopy and IgE spot blot analysis with sera from peanut allergic individuals. While we observed that the secondary structure of purified Ara h 3 from N and LR peanut, in solution, is affected with reduction of disulfide bonds and heat treatment, when purified from the peanut following the roasting process, only small alterations are seen in the secondary structure. The purified LR Ara h 3 binds higher levels of IgE than the N Ara h 3. CD spectroscopy of PD Ara h 3 reveals a reduction in the percentage of alpha helixes, and serum IgE binding. So, while Ara h 3 purified from roasted peanuts does not show significant changes in secondary structure it shows higher IgE binding than RAra h 3. Therefore, the higher IgE binding to LR Ara h 3 is more likely due to the chemical modifications than structural changes. Howerver, we found that if R Ara h 3 is deliberately unfolded, a decrease is seen in the IgE binding indicating that the structure plays an important role in IgE binding to Ara h 3.