Molecular cloning and epitope analysis of the peanut allergen Ara h 3
Pat Rabjohn, Erica M. Helm, J. Steven Stanley, C. Michael West, Hugh A. Sampson, A. Wesley Burks, Gary A. Bannon
Pat Rabjohn, Erica M. Helm, J. Steven Stanley, C. Michael West, Hugh A. Sampson, A. Wesley Burks, Gary A. Bannon
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Molecular cloning and epitope analysis of the peanut allergen Ara h 3

Abstract

Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from ∼45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3–allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions.

Authors

Pat Rabjohn, Erica M. Helm, J. Steven Stanley, C. Michael West, Hugh A. Sampson, A. Wesley Burks, Gary A. Bannon

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Ara h 3 epitopes can be mutated to non–IgE-binding peptides. Epitope 4 w...
Ara h 3 epitopes can be mutated to non–IgE-binding peptides. Epitope 4 was synthesized with an alanine residue substituted for one of the amino acids at each position in the peptide. The synthesized peptides were probed with a pool of serum IgE from peanut-hypersensitive patients whose IgE has previously been shown to recognize this peptide. The letters to the right of each lane indicate the one-letter amino acid code for the residue normally at that position and the amino acid substituted for that residue. The numbers indicate the position of each residue in the Ara h 3 protein. WT indicates the wild-type peptide with no amino acid substitutions.