Effector Activity of Peanut Allergens: A critical role for Ara h 2, Ara h 6, and their variants(1).
The term “major allergen” is defined as the following criteria.
1) a prominent band seen with sera from most patients on IgE immunoblots of 1D gels, 2) activity in basophil histamine release (BHR) assays, 3) activity in competitive ELISA assays, and 4) activity in animal models]. Finally, 5) the contribution of the allergen to the total potency of the extract should be demonstrated by depletion studies in which the putatively important allergen is specifically removed from the whole extract and the residual is demonstrated to have reduced activity. Ara h 1, Ara h 2, and Ara h 3 were identified as major peanut allergens based on the first four criteria but not the fifth.
When crude peanut extract (CPE) was passed over a gel filtration column, the recovery of both protein (75±7%) and effector activity (76±16%) were similar.
With a theoretical average molecular weight of 20 kDa, which from this point on we will refer to as the 20 kD fraction (open circles), is more potent than either the CPE (closed squares), or any other fraction (other open symbols).
Proportional recombination of all fractions (closed circles) reconstituted the activity found in the CPE (closed squares). Two separate assays with the pool of 11 sera demonstrated that 140±43% of the activity of the original CPE was found in the reconstituted material. However, if all fractions except the 20 kD fraction were reconstituted, the 20 kD-depleted reconstituted material (open circles), had 8±2% when assayed with the serum pool and 10±4% when assayed with individual sera of the activity compared to the CPE.
Fraction D shows faint bands of 17 and 19 kD, presumably Ara h 2. Fraction E, the second most potent fraction, contains a similar distribution of molecular masses to those seen in fraction D but the components are less abundant. Fractions F and G contained a variety of very faint bands (difficult to visualize in this exposure) that are likely the result of non-specific absorption of peanut proteins to the column.
Proteins in the 20 kD fraction were identified by two independent analytical strategies. First, the intact proteins (800 μg) were separated by two dimensional gel-electrophoresis (2D-GE) and stained. The protein spots were excised and digested with trypsin. Mass fingerprinting was performed by MALDI-TOF mass spectrometry. Almost all of the protein spots were identified as variants of Ara h 2 and h 6. The various forms of Ara h 2 were grouped into 3 categories: variants of 2.0101 (spots #1–10), variants of 2.0201 (spots #11–23) and those that were indistinguishable (i.e. forms of Ara h 2 where only shared peptides were identified; spots #24–27).
The majority (80–90%) of the effector activity of a CPE recovered from a gel filtration column resides in a single fraction comprised of components in the 13–25 kD range that are refered to as the 20 kD fraction. Selective removal of ~99 % of Ara h 2 as assessed by 2D gel and immunoblots resulted in a very small decrement in effector activity. The RBL SX-38 cells used in this study are an excellent model system for evaluating the effector activity of allergens. This is a stable cell line that effectively binds human IgE and is easy to activate. Data with this cell line are similar to, but more reproducible than those obtained with basophil histamine release assays.