Protein Structure Plays a Critical Role in Peanut Allergen Stability and May Determine Immunodominant IgE-Binding Epitopes (1)
Prospective studies have indicated that ∼5% of children less than 4 years of age experience IgE-mediated food-allergic reactions, with ∼1.5% of young children reacting to cow’s milk, ∼1.3% to hen’s egg, and 0.5% to peanut. One of the more significant food allergen characteristics is that they are stable to the proteolytic and acidic conditions of the digestive tract, which imparts an increased probability of reaching the intestinal mucosa, where absorption can occur. The Ara h 2 peanut allergen is recognized by serum IgE from >90% of peanut-allergic patients, thus establishing the importance of this protein in the etiology of the disease. Ara h 2 has been shown to be resistant to acidic conditions and digestion with gastrointestinal (GI) tract enzymes. Ara h 2 contains 10 IgE-binding epitopes detected with linear peptides representing the major epitopes (mainly epitopes 3, 6, and 7) recognized by serum from a peanut-sensitive patient population. Ara h 2 protein structure. However, there are eight cysteine residues that could form up to four disulfide bonds in Ara h 2, disulfide bonds having been shown in other allergens to contribute to the overall allergenicity of the molecule. Native Ara h 2 are 18.2% of the molecule in α-helices, 54% in β-pleated sheet, and 27.7% in a random coil configuration. Reduced Ara h 2 exhibits a secondary structure predominated by β-pleated sheet (82.3%), with the remainder of the molecule mostly in a random coil configuration.
The native, nonreduced Ara h 2 protein migrated as a doublet protein with an average molecular mass of 12 kDa. In contrast, the reduced Ara h 2 migrated as a slightly larger doublet with an average molecular mass of 17 kDa.
Digestion of Ara h 2 with either chymotrypsin or pepsin gave essentially similar results. The nonreduced Ara h 2 protein showed little change in its migration on polyacrylamide gels even after 40 min of enzyme digestion. However, when the disulfide bonds of the digested protein are reduced, the characteristic protein doublet disappears and a prominent 10-kDa protein fragment is obvious after only a short digestion time. The 10-kDa protein fragment was resistant to digestion for the length of the experiment. The native Ara h 2 protein digested with trypsin produced the 10-kDa protein fragment previously observed that was stable for the length of the experiment. In contrast, the Ara h 2 protein that was first reduced and then digested with trypsin did not produce any significant enzyme-resistant protein fragment and appeared to be much more susceptible to the action of the protease when compared with the native protein.
The 10-kDa Ara h 2 fragment is resistant to proteolysis by the three proteases when they are allowed to digest the protein one after the other.
1M urea had no effect on the stability of the 10-kDa protease-resistant fragment. Only after the disulfide bonds were reduced with DTT did the Ara h 2 protein become susceptible to degradation.
The 10-kDa protease-resistant peptide contained intact binding sites that could be recognized by IgE. Knowing that Ara h 2 contains 10 IgE binding sites that are evenly distributed along the linear sequence of the molecule. The amino acid sequence indicated that the 10-kDa fragment begins at aa position 23 and contains ∼90 aa. This portion of the Ara h 2 protein contains IgE-binding epitopes 2–7 and 6 of 8 of the cysteine residues.
Many of the food allergens are proteins containing intramolecular disulfide bonds that may be important to their allergenicity has led to the assumption that protein structure may be an important factor in an allergen’s ability to resist denaturation. Ara h 2 structure is not completely randomized when the disulfide bonds are reduced, but instead is predominated by a β-pleated sheet and β-turn configuration. Ara h 1 can form a stable trimer complex that may afford the molecule some protection from protease digestion and denaturation, allowing passage of Ara h 1 containing several intact IgE-binding epitopes across the small intestine, contributing to its overall allergenicity.