Major peanut allergen Ara h1 and Ara h 3 epitope post-translational modifications (PTMs)

Abstract

Objective: Peanuts are widely used for the preparation of a variety of foods and are also relied on as a protein extender. Peanut allergies affect a large portion of world population causing reactions ranging from mild to severe that can lead to anaphylaxis and even death. Seed storage proteins Ara h 1 and Ara h 3 are known as major peanut allergens. IgE epitopes of these allergens have been characterized, but little is known about how post-translational modifications (PTMs) affect their allergenicity and digestibility. Our aim was to investigate PTMs present on known epitopes of said proteins using bottom-up proteomcs methods. Material and Methods: Purified 2S albumins (Ara h 1 and Ara h 3) were analysed by a Top5 nLC- MS/MS method by LTQ Orbitrap XL (Thermo Fischer Scientific, Germany). Spectra were compared to Uniprot derived Peanut protein database, hybridized with the Repository of Adventitious Proteins (cRAP), using Peaks 8.5 software package (BSI, Canada). Epitopes were searched for possible PTMs by matching PEAKS PTM results with mapped positions of epitope sequences (found in the Immune Epitope Database – IEDB www.iedb.org). Results: According to IEDB Ara h 1 contains 327 peptide epitopes, within which we detected 8 likely PTMs. Hydroxylation Pro and pyro-glu from Q were found as most common in Ara h 1 epitopes. Ara h 3 has only 110 epitopes, according to IEDB with 10 likely PTMs. Hydroxylation Pro, dehydration and methylation (KR) were found as most frequent in Ara h 3 epitopes. PTMs could be found in the vicinity of trypsin cleavage sites, which could have an impact on digestibility. Conclusions: Peanut allergen epitopes are indeed carriers of PTMs. These results show promise in revealing a possible role PTMs could have on protein allergenicity and digestibility. Further investigation is necessary in order to fully understand the impact protein modifications could have on their allergenic potential