IDENTIFICATION AND MUTATIONAL ANALYSIS OF THE IMMUNODOMINANT IGE BINDING EPITOPES OF THE MAJOR PEANUT ALLERGEN ARA-H-2

A major peanut allergen, Ara h 2, is recognized by serum IgE from >90% of patients with peanut hypersensitivity. Biochemical characterizatio n of this allergen indicates that it is a glycoprotein of similar to 1 7.5 kDa. Using N-terminal amino acid sequence data from purified Ara h 2, oligonucleotide primers were synthesized and used to identify a cl one (741 bp) from a peanut cDNA library. This clone was capable of enc oding a 17.5-kDa protein with homology to the conglutin family of seed storage proteins. The major linear immunoglobulin E (IgE)-binding epi topes of this allergen were mapped using overlapping peptides synthesi zed on an activated cellulose membrane and pooled serum IgE from 15 pe anut-sensitive patients. Ten IgE-binding epitopes were identified, dis tributed throughout the length of the Ara h 2 protein. Sixty-three per cent of the amino acids represented in the epitopes were either polar uncharged or apolar residues. In an effort to determine which, if any, of the 10 epitopes were recognized by the majority of patients with p eanut hypersensitivity, each set of 10 peptides was probed individuall y with serum IgE from 10 different patients. All of the patient sera t ested recognized multiple epitopes. Three epitopes (aa27-36, aa57-66, and aa65-74) were recognized by all patients tested. In addition, thes e three peptides bound more IgE than all the other epitopes combined, indicating that they are the immunodominant epitopes of the Ara h 2 pr otein. Mutational analysis of the Ara h 2 epitopes indicate that singl e amino acid changes result in loss of IgE binding. Two epitopes in re gion aa57-74 contained the amino acid sequence DPYSP that appears to b e necessary for IgE binding. These results may allow for the design of improved diagnostic and therapeutic approaches to peanut hypersensiti vity. (C) 1997 Academic Press.