Submitted to: Protein and Peptide Letters
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/11/2008
Publication Date: 6/20/2008
Citation: Guo, B., Liang, X., Chung, S., Holbrook, C.C., Maleki, S.J. 2008. Proteomic analysis of peanut seed storage proteins and genetic variation in a potential peanut allergen. Protein & Peptide Letters. 15:567-577. Interpretive Summary: Plant seeds contain a number of storage proteins which serve as nitrogen sources for germination and growth. Most allergens identified in peanut, are seed storage proteins. Unfortunately, the incidence of allergy to plant foods has increased over the years. There is currently no treatment for peanut allergy. The most effective strategy is to avoid peanut and peanut-containing products by those allergic individuals. Therefore, new approaches to reduce the food allergy associated with peanut should be identified, such as developing peanut cultivars that are less allergenic. One effort to alleviate the allergenicity problem is to identify peanut germplasm with lower levels of allergens which could be used in traditional cross-breeding to produce a less allergenic peanut cultivar. In this study, we identified one peanut line, GT-C9, with a different 2-D protein profile with reduced, or absent, levels of several protein peptides resulting in significantly lower levels of AGE and IgE binding. By measuring levels of protein-bound end products or adducts and IgE binding along with peptide sequencing and Western blot analysis, we confirmed that this group of peptides with a weak reaction to monoclonal anti-Ara h 3 antibodies are subunits or isoallergens and would be a novel potential peanut allergen named iso-Ara h 3. These results suggest that iso-ara h 3 is a newly identified gene encoding a unique Ara h 3-like protein, iso-Ara h 3. Future studies could be designed to test the allergenicity of GT-C9 and to develop peanut cultivars with reduced allergens, particularly in light of the importance of the basic subunit of Ara h 3.
Technical Abstract: Peanut allergy is one of the most severe food allergies. One effort to alleviate this problem is to identify peanut germplasm with lower levels of allergens which could be used in conventional breeding to produce a less allergenic peanut cultivar. In this study, we identified one peanut line, GT-C9, which was lacking several seed protein peptides, which were identified as Ara h 3 isoforms by peptide sequencing and therefore named iso-Ara h 3, a novel potential allergen. The total seed proteins were analyzed by one-dimensional (SDS-PAGE) and two-dimensional gel electrophoreses (2-D PAGE) and tested for levels of protein-bound end products or adducts such as advanced glycation end products (AGE) and N-(carboxymethyl) lysine (CML), and IgE binding. CML and AGE adducts were present in all tested peanut lines. GT-C9 exhibited a significantly lower levels of IgE binding and of AGE adducts. The identification of a novel potential peanut allergen iso-Ara h 3 was confirmed by peptide sequences and Western blot analysis using specific anti-Ara h 1, Ara h 2, and Ara h 3 antibodies. A full-length sequence of iso-ara h 3 (GenBank number DQ855115) was obtained. The deduced amino acid sequence iso-Ara h 3 (ABI17154) has the first three of four IgE-binding epitopes of Ara h 3. All peptide fragments had significant sequence homology to peanut allergen Ara h 3 but had 100% identity to iso-Ara h 3 for most of the peptide fragments. Nevertheless, anti-Ara h 3 antibodies reacted with two groups of protein peptides, one with strong reactions and another with weak reactions. These weak reactive protein peptide spots on 2-D PAGE to anti-Ara h 3 antibodies are subunits or isoallergens of a novel potential peanut allergen iso-Ara h 3.