Molecular cloning and epitope analysis of the peanut allergen Ara h 3 (1)
The peanut (Arachis hypogaea) is a member of the legume (Leguminosae) family, which includes the soybean, jack bean, lima bean, and pea. Two of these fractions, Ara h 1 (63.5 kDa) and Ara h 2 (17 kDa), have been extensively characterized in our laboratory. Both proteins are recognized by serum IgE from >90% of peanut-allergic patients, establishing them as major peanut allergens. Sequencing of the Ara h 3 cDNA identified this allergen as a legumin-like seed-storage protein. The recombinant form of this protein is recognized by serum IgE from 44% (8/18) of our peanut-hypersensitive patient population.
The Ara h 3 cDNA is an open-reading frame of 1,530 nucleotides, coding for 510 amino acids. Ara h 3 showed 62%–72% sequence identity to glycinins from Glycine max (soybean) and legumins from Pisum satvium (pea). There was no homology noted between this allergen and the other major peanut allergens already identified (Ara h 1 or Ara h 2).
Forty-four percent (8/18) of the patients tested had IgE that recognized the recombinant protein.
The four IgE-binding regions and their corresponding location within the Ara h 3 primary amino acid sequence. These IgE-binding regions were represented by amino acid residues 21–55, 134–154, 231–269, and 271–328.
An immunoblot of six synthetic peptides and the amino acid sequence representing this region and the amino acid sequences represented by each individual peptide were shown.
The four IgE-binding epitopes identified in this manner are shown. Epitope 3 was recognized by serum IgE from 100% (8/8) of the Ara h 3–allergic patients, classifying it as an immunodominant epitope within the Ara h 3–allergic population.
The pool of serum IgE did not recognize the peptide, or a decrease in binding was observed when alanine was substituted for the wild-type amino acid at positions 308, 309, 310, 311, 312, and 314. An alanine substitution increases IgE binding at positions 304 and 305.
Central amino acids within each epitope are favored for mutation. All mutations that led to a significant decrease in IgE binding were located at residues found within each core epitope.
Epitopes 3 and 4 are located within the hypervariable region of the acidic chain, a stretch of amino acids that is highly variable in length among 11S storage proteins. This region contains a high proportion of glutamate, aspartate, and arginine residues and will tolerate large, naturally occurring insertions or deletions. Soybean glycinins have long been the target of genetic engineering studies aimed at improving their nutritional value. Initial experiments revealed that mutations within the 35-kDa acidic subunit had little effect on the ability of glycinins to oligomerize into higher-order structures.