Redefining the major peanut allergens

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Redefining the major peanut allergens (1)

The peanut, Arachis hypogaea, is a member of the legume family and is related to beans and peas, but not to tree nuts, although up to 50 % of peanut-allergic patients are also sensitized to tree nuts. Eleven potentially important allergens of peanut have been described and, of these, Ara h 1, Ara h 2, and Ara h 3 have been designated as the major peanut allergens. Two highly related 2S albumins, Ara h 2 and Ara h 6, are in fact the major peanut allergens. Food allergens belong to a select group of protein super- families [33–35]. These are (1) the cupin superfamily (7S and 11S seed storage proteins) including viculins and legumins (glycinins); (2) the prolaminin superfamily char- acterized by 2S albumins, nsLTPs (nonspecific lipid- transfer proteins), a-amylase, and some protease inhibitors; and (3) the plant defense proteins including Prs (patho- genesis-related proteins), proteases, and protease inhibitors.

Ara h 1 belongs to the vicilin (7S) family of seed storage proteins. It is a glycoprotein and contains 23 independent IgE-binding epitopes. The relative weak activity of native (trimer) Ara h 1 in cross-linking IgE and the strong binding of IgE to denatured monomers (63 kD on immunoblots)

Ara h 2 belongs to the conglutin family of seed storage proteins that is related to the 2S albumin family. There are two isoforms, Ara h 2.01 and Ara h 2.02, and two alleles of each. The larger isoform contains 12 extra amino acids, including a duplication of a strong IgE- binding sequence, DPYSPS and binds more IgE.

Ara h 3, a peanut glycinin, belongs to the legumin (11S) family of seed storage proteins. Native glycinin is a 360–380-kD protein constructed from 60-kD monomers. After unfolding in 6M urea and reduction of disulfide bonds by DTT, peptides composed of 14, 16, 25, 28, 42, and 45 kD. IgE-binding epitopes are found in the 14-, 42-, and 45-kD fragments.

Arah6is59%homologoustoArah2butis2–4- kD smaller. It is a heat and digestion stable protein with a molecular weight of *14.5 kD, a protease- stable core, and allergenic potency similar to that of Ara h 2.

Ara h 8 is homologous to Bet v 1, an important allergen in birch pollen and may account for peanut sensitivity in patients who are allergic to birch pollen.

Ara h 9 belongs to the nonspecific lipid-transfer proteins (nsLTPs) allergen family and seems to play an important part in peanut allergy for patients from the Mediterranean region.

It is important to point out that all in vitro assays of binding of IgE to allergens are measuring some aspects of the immunochemical association of IgE and allergen, but none of these assays measures the ability of the allergen to cross-link IgE/FceRI complexes on the surface of mast cells or basophils leading to cell activation. De Groot et al. depleted an extract of cat dander of Fel d I (by 95 %) with monoclonal and with polyclonal antibodies. In basophil histamine release (BHR) tests, the depleted extracts were 30–300 times less potent than the original extracts, demonstrating that Fel d I is a major allergen of cat dander. Of note, similar experiments were performed to remove Der p 1 from a dust mite extract (whole body extract), and there was no effect on the potency of the extract.

RBL SX-38 cells assay

RBL SX-38 cells are rat basophilic leukemia cells that stably express approximately 70,000 copies per cell of a, b, and c chains of the human high-affinity receptor for IgE, FceRI. Ara h 1, Ara h 2, and Ara h 6, to degranulate RBL SX-38 cells following sensitization of the cells with IgE from either a pool of subjects or from individual subjects.

Oral challenges

Oral challenges commonly are used in murine model using the peanut-allergic female C3H/HeJ mouse that is sensitized with freshly ground whole peanut with cholera toxin as an adjuvant.

20-kD fraction from crude peanut extracts revealed more than 60 protein spots, and, unexpectedly, mass spectrometry revealed that >97 % of this biologically active fraction consisted of Ara h 2, Ara h 6, and variants of these proteins.

Anti-peptide anti-Ara h 2 antibodies and found that one of the rabbit anti-peptide antibodies (against the C-terminal peptide of Ara h 2, CDLEVESGGRDRY) was able to bind to Ara h 2 in solution and specifically and quantitatively ([99 %) remove it from the CPE. The rabbit anti- peptide antibodies (against the unique peptide of Ara h 6, EQEQYDSYDIRSTRSSDQ) was consistently capable of removing Ara h 6 without removing Ara h 2. Ara h 2 and Ara h 6 together are the most potent peanut allergens in vivo and when used in immunotherapy are sufficient to prevent allergic reactions to a complete CPE.

Ara h 2 is an ‘‘important’’ peanut allergen in that it accounts for 0–40 % of the effector activity of a CPE depending upon patients, but it does not account for the majority of the effector activity in a CPE for any patient examined to date.

1. Y. Zhuang, S. C. Dreskin, Redefining the major peanut allergens. Immunol. Res. 55, 125–134 (2013).

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