Purification and immunoglobulin E-binding properties of peanut allergen Ara h 6: evidence for cross-reactivity with Ara h 2 (1)
Peanut allergens Ara h 1, Ara h 2, and Ara h 3 have been characterized. Ara h 2 is the most recognized allergen in peanuts. Ara h 6, and to a lesser extent, Ara h 7 are homologous to Arah2. Arah2, Arah6,and Arah7 all belong to the 2S albumin family, and have the characteristically conserved cysteine residues of this group of proteins.
Fifty kilograms of peanuts were extracted three times with hexane. After extraction, the peanuts were dried and ground. The defatted peanut flour (1.3kg) was extracted for 1 h while stirring in 13 L 50 mM Tris/HCl pH 8.2 at 41C. The solution was filtered using a kitchen sieve and cheese cloth. The filtrate is referred to as crude extract.
Ammonium sulphate was added to the crude extract to attain a concentration of 40% saturation at 4C. The solution was centrifuged (45 min, 8000 g, at 4 1C). The cold supernatant was filtered over glass wool to remove fat particles. Ammonium sulphate was then added to a concentration of 80% saturation at 4C. The solution was centrifuged again (45 min, 10 000 g, at 4C). The pellet was then resuspended in 1.3 L 20 mM Tris/HCl pH 8.0 containing 1 mM EDTA. This preparation is referred to as concentrate.
A fraction of 230mL was applied on a Sephadex G75 column (7200 mL column volume, diameter 20cm, height 23cm) and eluted with 20 mM Tris, pH 8.0 at 100 mL/min. The main peak at approximately 3 L is a mixture of Ara h 1 and Ara h 3, both eluting in the void volume of the column.
The enriched fractions were combined, warmed up to 25C and applied to a 3600mL Source 15Q column (diameter 20cm, height 12 cm) previously equilibrated with 20 mM Tris, pH 8.0 (loading buffer). After washing with loading buffer, the column was eluted with a 40 L salt gradient of 0–0.25 M NaCl in loading buffer at a flow of 100mL/min. the arrow indicates where the target protein elutes from the column. The pure fraction contained approximately 800mg protein.
The purity of the target protein (lane 4) is estimated to be >95%. The isolated protein is Ara h 6, and that it has stretches homologous to Ara h 2 in the sequenced N-terminal part of the protein. At cycle 14 and 26 no increase in signal was observed. This is indicative for Cys residues or a glycosylated or phosphorylated amino acid. Ara h 6 only induces basophil degranulation after sensitization of basophils with pooled serum from peanut allergic individuals.
The IgE binding to CPE, purified Ara h 2, and purified Ara h 6 for the tested 23 patients. Some patients (40, 48, and 54) reacted intensely on the IgE-blot, resulting in exhaustion of the colour reaction, visible as a vague, lighter area within protein bands. The majority of the tested patients showed IgE binding to protein bands of approximately 15 kDa in CPE as well as to purified Ara h 6. In fact, only patient 34 and 47 reacted with Ara h 2 without any reaction towards Ara h 6. The serum of patient 47 exclusively reacts with Ara h 2. The serum of patient 34, however, reacts also with Ara h 1 (high molecular weight band in CPE lane), and with proteins of 10–15 kDa, possibly Ara h 5 or Ara h 7, while no reactivity is observed to purified Ara h 6.
10- to 100-fold higher concentrations of Ara h 1 were needed to induce reactions comparable to that of Ara h 2. Ara h 2 is such a potent allergen may be explained by its stability. Ara h 2 belongs to the conglutin and 2S protein family, characterized by a compact structure, and held together by disulphide bonds. Ara h 2 is very stable towards breakdown by digestive proteases pepsin and trypsin, and a stable peptide of 10kDa remains after prolonged incubation with digestive enzymes. Recombinant Ara h 6 was recognized by 38% of sera from the peanut-allergic population of Kleber-Janke et al. IgE-reactivity towards the 15 kDa peanut protein, that is now identified as Ara h 6 in 20 from 32 patients (63%). This indicates that Ara h 6 is an important IgE-binding protein. Differences in percentage recognition may be explained by differences between recombinant vs. peanut-derived allergens, or differences between patient populations. Although recombinant peanut allergens may bind patient IgE, it was shown that post-translational processing of peanut allergens has important consequences for their molecular organization and the IgE-binding properties. IgE epitopes on Ara h 2 have been mapped and these are mainly located at the N-terminal part of the protein (seven out of nine epitopes) while two epitopes are located in the middle part, and one other is found at the C-terminal part of the protein. Interestingly, the primary homology with Ara h 2 is in the middle-and C-terminal of Ara h 6, an area with only three IgE epitopes. The major IgE binding epitope DPYSPS is thus two times present in the heavy iso-form of Ara h 2, only once in the light iso-form of Ara h2,and not at all in Ara h6.