Structure of the Major Peanut Allergen Ara h 1 May Protect IgE-Binding Epitopes from Degradation

Abstract

In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.

Structural and Immunologic Characterization of Ara h 1, a Major Peanut Allergen

“…Cloning, Expression, and Purification-Natural Ara h 1 (nAra h 1) was purified as described previously (16). The DNA expression construct for recombinant full-length Ara h 1 (rAra h 1) was synthesized by EZbiolab (Carmel, IN), and the protein was purified using the same protocol as for nAra h 1.…”

Simplified Methods for Purification of Peanut Allergenic Proteins: Ara h 1, Ara h 2, and Ara h 3

“…The fractionation condition employed was at 0 _ 70% AS and 70 _ 100% AS saturation (Maleki et al, 2000). In the 70 _ 100% AS fraction, Ara h 1 was recovered at ~ 64 kDa, as well as proteins at 11 _ 15 kDa, ~ 21 kDa, and ~ 37 kDa (Fig.…”

“…For instance, Ara h 1 is purified as follows: the peanut protein extract is subjected to ammonium sulfate fractionation (ASF), and after removal of the precipitate fraction at 0 _ 70% AS, the precipitate fraction at 70 _ 100% AS is collected. The collected fraction is then purified by cation exchange chromatography (Maleki et al, 2000). In another procedure, the peanut protein extract is fractionated by gel permeation chromatography twice (van Boxtel et al, 2006).…”

“…For instance, 3.5% of the total population in France (Kanny et al, 2001), 3.6% in Germany (Zuberbier et al, 2004), and 3.5 _ 4.0% in the United 2001). It is utilized as a nitrogen source during peanut seedling growth (Maleki et al, 2000). Ara h 1 is recognized by more than 90% of serum IgE from peanut allergic patients, indicating its importance in the etiology of peanut allergy (Burks et al, 1995).…”

“…In previous reports, target allergens have been purified as follows: protein extraction from defatted peanut by using a weakly basic buffer solution, ammonium sulfate fractionation (ASF) once or twice, and final purification steps using a combination of several column chromatography techniques, such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography (Maleki et al, 2000;van Boxtel et al, 2006;Burks et al, 1992;de Jong et al, 1998;Sen et al, 2002;Koppelman et al, 2003).…”