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

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 kDa and a range of 13–25 kDa. 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 kDa 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 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 >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.