Proteomic Analysis of Peanut Seed Storage Proteins and Genetic Variation in a Potential Peanut Allergen

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, lacking several seed proteins, which were identified as Ara h 3 isoforms by peptide sequencing and named iso-Ara h 3. Total seed proteins were analyzed by one-dimensional (SDS-PAGE) and two-dimensional gel electrophoreses (2-D PAGE). The total protein extracts were also 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. Peanut genotypes of GT-C9 and GT-C20 exhibited significantly lower levels of AGE adducts and of IgE binding. This 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. Anti-Ara h 3 antibodies reacted with two groups of protein peptides, one with strong reactions and another with weak reactions. These peptide spots with weak reaction on 2-D PAGE to anti-Ara h 3 antibodies are subunits or isoallergens of this potential peanut allergen iso-Ara h 3. A recent study suggested that Ara h 3 basic subunits may be more significant allergenicity than the acidic subunits.

AllerML: Markup language for allergens

“…Finally, the ontology is flexible enough to permit data exchange between other databases that are not specifically for allergenic proteins, such as the Immune Epitope Database and Analysis Resource (IEDB; http://immuneepitope.org), which contains an extensive list of T- and B-cell epitopes assembled from large scale screening (Zhang et al, 2008). Other extensions of AllerML could include data on allergenic medical symptoms or biomarkers from new technologies, such as proteomics (Chapman et al, 2007; Guo et al, 2008; Reisdorph et al, 2009) or microarray approaches (Bublin et al, 2011; Hiller et al, 2002; Hochwallner et al, 2010; Mari et al, 2010; Shreffler et al, 2005). …”

The Molecular Basis of Peanut Allergy

“…The two cupin domains are known as the acidic and basic subunit and can be readily separated by isolectric focusing [19]. Certain cultivars of peanuts lacking the basic subunit of Ara h 3 have been studied as potentially less allergenic [20]. …”

Strategies in Prevention of Preharvest Aflatoxin Contamination in Peanuts: Aflatoxin Biosynthesis, Genetics and Genomics

“…Two submissions were 7454 and 2184 sequences from seed libraries of the Chinese accessions Luhua 14 and Shanyou 523, respectively. The majority represented the 21,777 ESTs (accession numbers ES702769 to ES724546) from developing seeds (Guo et al, 2008a) of the U.S. runner type cultivar Tifrunner and the Spanish breeding line GT-C20 (Guo et al, 2008b). The seed and leaf EST originated from 10 non-normalized peanut cDNA libraries (Guo et al, 2007b;Guo et al, 2008a), resulting in over 13,824 unigene ESTs, which will be used for the production of a long-oligo microarray for peanut.…”