Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2

J. Steven Stanley, Nina King, A. Wesley Burks, Shau K. Huang, Hugh Sampson, Gael Cockrell, Ricki M. Helm, C. Michael West, Gary A. Bannon

Research output: Contribution to journalArticle

302 Citations (Scopus)

Abstract

A major peanut allergen, Ara h 2, is recognized by serum IgE from >90% of patients with peanut hypersensitivity. Biochemical characterization of this allergen indicates that it is a glycoprotein of ~17.5 kDa. Using N- terminal amino acid sequence data from purified Ara h 2, oligonucleotide primers were synthesized and used to identify a clone (741 bp) from a peanut cDNA library. This clone was capable of encoding a 17.5-kDa protein with homology to the conglutin family of seed storage proteins. The major linear immunoglobulin E (IgE)-binding epitopes of this allergen were mapped using overlapping peptides synthesized on an activated cellulose membrane and pooled serum IgE from 15 peanut-sensitive patients. Ten IgE-binding epitopes were identified, distributed throughout the length of the Ara h 2 protein. Sixty-three percent 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 peanut hypersensitivity, each set of 10 peptides was probed individually with serum IgE from 10 different patients. All of the patient sera tested recognized multiple epitopes. Three epitopes (aa27-36, aa57-66, and aa65-74) were recognized by all patients tested. In addition, these three peptides bound more IgE than all the other epitopes combined, indicating that they are the immunodominant epitopes of the Ara h 2 protein. Mutational analysis of the Ara h 2 epitopes indicate that single amino acid changes result in loss of IgE binding. Two epitopes in region aa57-74 contained the amino acid sequence DPYSP that appears to be necessary for IgE binding. These results may allow for the design of improved diagnostic and therapeutic approaches to peanut hypersensitivity.

Original languageEnglish
Pages (from-to)244-253
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume342
Issue number2
DOIs
Publication statusPublished - Jun 15 1997
Externally publishedYes

Fingerprint

Immunoglobulin E
Epitopes
Peanut Hypersensitivity
Amino Acids
Serum
Allergens
Peptides
Amino Acid Sequence
Clone Cells
Seed Storage Proteins
Arachis hypogaea Ara h 2 allergen
Immunodominant Epitopes
Proteins
DNA Primers
Gene Library
Cellulose
Glycoproteins
Membranes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. / Stanley, J. Steven; King, Nina; Burks, A. Wesley; Huang, Shau K.; Sampson, Hugh; Cockrell, Gael; Helm, Ricki M.; West, C. Michael; Bannon, Gary A.

In: Archives of Biochemistry and Biophysics, Vol. 342, No. 2, 15.06.1997, p. 244-253.

Research output: Contribution to journalArticle

Stanley, J. Steven ; King, Nina ; Burks, A. Wesley ; Huang, Shau K. ; Sampson, Hugh ; Cockrell, Gael ; Helm, Ricki M. ; West, C. Michael ; Bannon, Gary A. / Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. In: Archives of Biochemistry and Biophysics. 1997 ; Vol. 342, No. 2. pp. 244-253.
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