Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation (1)
Ara h 1 (63 kD), Ara h 2(17–19 kD doublet) and Ara h 3 (60 kD) have been designated major peanut allergens based on the frequency of patients whose IgE binds to these proteins. Ara h 6, another important peanut allergen, is 59% homologous with Ara h 2. Ara h 2 and Ara h 6 belong to the conglutin family of seed storage proteins, and are part of the 2S albumin family. There are two main isoforms, Ara h 2.01 and Ara h 2.02, containing 10 independent IgE-binding epitopes stretching throughout the linear structure.
Crude peanut extract was prepared with the following procedures. Twenty grams of fresh raw Georgia green peanuts were frozen in liquid nitrogen and finely ground with a mortar and pestle. Following extensive defatting with diethyl ether, the dried peanut flour was resuspended in 100 mL of ice-cold buffer [150 mM NaCl, 50 mM Tris, pH 7.4 with protease inhibitors and mixed overnight at 41C. The extract [25 mg/mL] was clarified by centrifugation (10,000 g, 30 min), sterile filtered, and frozen in aliquots at -70C.
Two peptide epitopes (13–14 amino acids each) of Ara h 2 [GenBank sequence AAN77576 (gi:26245477)] were chosen based on uniqueness, antigenicity, surface probability, flexibility, and an antigenic index. The antibody that was most useful was directed against the C-terminal sequence, CDLEVESGGRDRY, which consists of amino acids 160–172 of Ara h 2.02 (GenBank sequence AAN77576 and SwissProt accession number, Q8GV20) and is also the C-terminus of Ara h 2.01. The second peptide was DRRDPYSPSPYDR, which consists of amino acids 79–91 of Ara h 2.02 (above), and includes the immunodominant IgE-binding epitope DPYSPS that is also present in Ara h 2.01. Two rabbits were immunized with peptide-KLH conjugates of both peptides. Anti-peptide antibodies against Ara h 1 (amino acid #599–612; KE- SPEKEDQEEE NQ; Swiss-Prot, Q547W5), Ara h 3 (amino acid # 90–103; EEPHTQGRRSQSQR; Swiss-Prot, O82580), and Ara h 6 (amino acid # 42–59; EQEQYD- SYNFGSTRSSDQ; Swiss-Prot, Q9SQG5) were raised and used for immunoblots.
Incubation of CPE with the anti-Ara h 2 agarose column successfully removed 99 +/- 1% (mean +/- SD, n = 3 comparisons) of Ara h 2 without depleting Ara h 1, Ara h 3 or Ara h 6. The sham-treated extract contained 0.18 +/- 0.070 (mean +/- range of two measurements) ug of Ara h 2 per 100 ug of CPE and estimated that the Ara h 2- depleted extract contained 0.005 ug of Ara h 2 per 100 ug of CPE (> 97% depleted).
Depletion of 99% of Ara h 2 from the CPE has only a small effect on the ability of the CPE to cross-link IgE effectively from peanut-allergic patients as measured in the RBL SX-38 cell assay. The dose response of sham-treated CPE is very similar to that of Ara h 2-depleted CPE either for cells sensitized with sera pooled from 10 donors.
Experiments with basophils (naturally sensitized with IgE) from three of the peanut-allergic donors showed no consistent effect of removal of 99% of the Ara h 2 from the CPE.
A control blot was probed with serum from a peanut-tolerant patient with allergic rhinitis (total IgE = 960 IU/mL; IgE = o0.35 IU/mL). Ara h 2 is clearly visible although the density of binding of IgE to Ara h 2 is dwarfed by that of other proteins especially in the blots using nitrocellulose. Previous work has shown that purified Ara h 2 binds IgE less well on immunoblots than does Ara h 1 and this is recapitulated here. The 2D blot probed with the control serum showed no specific spots after a 5-min exposure. The 2D gel transferred onto a PVDF membrane instead of nitrocellulose showed a somewhat different pattern with decreased detection of higher molecular weight allergens and enhanced detection of lower molecular weight allergens.
To use extracts of raw peanuts were for two reasons. First, peanut extracts used clinically for skin testing and peanut flour used for food challenges are made with raw peanuts. Second, roasted peanuts have been reported to bind more IgE and the Ara h 2 trypsin inhibition activity is increased.