Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants

Abstract

Rationale-An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized.Methods-Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut allergic donors.Results-Following gel filtration, 75±7% of the applied protein and 76±16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85±2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of ~20 kD and a range of 13-25 kD. When all the individual fractions were recombined, the measured activity was similar to that of the original extract (140±43% when measured with a pool of serum (n=2) and 66±7% when measured with individual sera (n=4)); when all individual fractions excluding the 20 kD fraction were recombined, the measured activity was only 8±2% (n=2) of the original extract when assayed with the serum pool and 10±4% (n=3) when assayed with the individual sera. Two dimensional gel electrophoresis of this biologically active fraction by revealed >60 protein spots . Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that greater than 97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins.Conclusions-Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.

Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

“…A previous report found that Ara h 2 treated with trypsin/chymotrypsin displayed minimal reduction in IgE binding capacity, and mediator release from rat basophilic leukemia cells was not reduced by protease treatment, which was attributed to the proteolytic resistant core of Ara h 2 [23]. Additionally, studies have examined the effector activity of the peanut 2S albumins, Ara h 2 and 6, and have demonstrated that these proteins are the most potent peanut allergens in terms of degranulating basophils [7] and inducing anaphylaxis in a mouse model of peanut allergy [25]. Our current data show Ara h 2 fragments are present following any of the enzymatic hydrolyses, and thus may account for the persistent basophil reactivity we measured in response to the hydrolysates.…”

“…Ara h 3/4 has been recognized by serum IgE from 44 to 54% of patients with a history of peanut hypersensitivity [4,5]. Importantly, studies investigating the capacity of peanut proteins to cross-link IgE on effector cells have demonstrated that Ara h 2, together with its homolog Ara h 6, account for more than 80% of effector activity in peanut extracts [6,7]. …”

Evaluation of Bar, Barnase, and Barstar recombinant proteins expressed in genetically engineered Brassica juncea (Indian mustard) for potential risks of food allergy using bioinformatics and literature searches

“…Among the various immune mechanisms that are known to cause allergy, IgE mediated immune responses are responsible for most food allergy including severe and life threatening reactions to foods (Burks, 2002). All allergens are proteins but only a small number of proteins, for example the seed storage proteins 2S albumins (Ara h 2, Ara h 6), glycinin (Ara h 3) and beta-conglycinin (Ara h 1) and possibly four low-abundance proteins out of the hundreds of proteins present in peanuts are responsible for peanut food allergy (Porterfield et al, 2009;Peeters et al, 2007). The prevalence of food allergy is relatively low in the general population, ranging from 1 to 5% in adults to 6e8% in children (Thomas et al, 2005;Sicherer and Sampson, 2014).…”

Identification of peanut seed prolamins with an antifungal role by 2D-GE and drought treatment

“…34) Despite water solubility of the seed albumins, a large amount of the peanut albumins could be extracted by NaCl buffer. 35) In a soybean 2D-GE model, little contamination of globulins and albumins was evident together with other hydrophilic proteins after one-step extraction by relatively high alcohol strength using 40% isopropanol.…”