Protein Structure Plays a Critical Role in Peanut Allergen Stability and May Determine Immunodominant IgE-Binding Epitopes

Abstract

Hypersensitivity to peanuts is a reaction mediated by IgE Abs in response to several peanut protein allergens. Among these allergenic proteins, Ara h 2 is one of the most commonly recognized allergens. Ara h 2 is a 17-kDa protein that has eight cysteine residues that could form up to four disulfide bonds. Circular dichroism studies showed substantial changes in the secondary and tertiary structures of the reduced Ara h 2 as compared with the native protein. Upon treatment with trypsin, chymotrypsin, or pepsin, a number of relatively large fragments are produced that are resistant to further enzymatic digestion. These resistant Ara h 2 peptide fragments contain intact IgE-binding epitopes and several potential enzyme cut sites that are protected from the enzymes by the compact structure of the protein. The enzyme-treated allergen remains essentially intact despite the action of proteases until the fragments are dissociated when the disulfide linkages are reduced. Amino acid sequence analysis of the resistant protein fragments indicates that they contain most of the immunodominant IgE-binding eptiopes. These results provide a link between allergen structure and the immunodominant IgE-binding epitopes within a population of food-allergic individuals.

Temporal and Spatial Expression of the Major Allergens in Developing and Germinating Peanut Seed

“…The allergenicities of Ara h 1 and Ara h 2 are resistant to heat and several proteinases due to protein structure and modifications to these proteins that occur following these treatments (Koppelman et al, 1999;Maleki et al, 2000aMaleki et al, , 2000bMaleki et al, , 2003. In particular, disulfide bonds contribute significantly to the overall structure and stability of Ara h 2 (Sen et al, 2002).…”

“…Ara h 2 is composed of two glycoproteins containing eight Cys residues that migrate closely on SDS-PAGE (Burks et al, 1992;Sen et al, 2002). An ara h 2 clone of 741 bp was identified from a peanut cDNA library, and this clone hybridizes to an approximately 0.7-kb mRNA .…”

Narrow Leafed Lupin Beta-Conglutin Proteins Epitopes Identification and Molecular Features Analysis Involved in Cross-Allergenicity to Peanut and Other Legumes

“…However, the first one account for the majority of food-induced immune reactions. Generally, storage proteins contained in legume seeds, grains and nuts are the causative of allergy reactions upon ingestion, mainly due to the high stability under extremes of pH and temperature, and variable similarity in their primary sequence among these allergens [38,39].…”

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

“…For instance, a peanut protein extract was fractionated by anion and cation exchange chromatography once or twice (Burks et al, 1992;de Jong et al, 1998), and the precipitate fraction of 40 _ 70% ASF from the extract was further fractionated by anion exchange chromatography, followed by hydrophobic chromatography (Sen et al, 2002). On the SDS-PAGE gel, Ara h 2 can be found as two bands at ~16 kDa and ~18 kDa, representing 2 isoforms containing different numbers of repeated inserted sequence (Chatel et al, 2003).…”

“…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).…”