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Ara h 2 and Ara h 6 Have Similar Allergenic Activity and Are Substantially RedundantChen X. · Wang Q. · El-Mezayen R. · Zhuang Y. · Dreskin S.C.
Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado Denver, Aurora, Colo., USA Corresponding Author
Correspondence to: Dr. Stephen C. Dreskin
Division of Allergy and Clinical Immunology
Department of Medicine, University of Colorado Denver
Aurora, CO 80045 (USA)
Tel. +1 303 724 7190, E-Mail email@example.com
Background: The moderately homologous (approx. 60%) proteins Ara h 2 and Ara h 6 are the most potent peanut allergens. This study was designed to define the relative individual contributions of Ara h 2 and Ara h 6 to the overall allergenic activity of a crude peanut extract (CPE). Methods: Ara h 2 and Ara h 6 were removed from CPE by gel filtration chromatography. Ara h 2.01, Ara h 2.02 and Ara h 6 were further purified (>99%). The potency of each allergen and the ability of these allergens to reconstitute the allergenic activity of CPE depleted of Ara h 2 and Ara h 6 was measured with RBL SX-38 cells sensitized with IgE from sensitized peanut allergic patients. Results: The potency of the native proteins were significantly different (p < 0.0001) although not dramatically so, with a rank order of Ara h 2.01 > Ara h 2.02 > Ara h 6. The addition of either purified Ara h 2 or Ara h 6 independently at their original concentration to CPE depleted of both Ara h 2 and Ara h 6 restored 80–100% of the original CPE allergenic activity. Addition of both Ara h 2 and Ara h 6 consistently completely restored the allergenic activity of CPE. Conclusions: These studies indicate that either Ara h 2 or Ara h 6 independently can account for most of the allergenic activity in a CPE and demonstrate important redundancy in the allergenic activity of these related molecules.
© 2012 S. Karger AG, Basel
The prevalence of peanut allergy in the Western world has increased over the last decade, representing a significant health concern affecting more than 1% of children [1,2,3]. Eleven peanut allergens have been identified [4,5]. Of these allergens, the 2S albumins, Ara h 2 and Ara h 6, are the most important in peanut-allergic patients, as determined by a combination of immunoblots, skin prick tests, ex vivo basophil histamine release assays and in vitro experiments with RBL cells expressing human FcεRI [6,7,8,9,10]. Furthermore, Ara h 2 and Ara h 6 are the major elicitors of anaphylaxis in a murine model of peanut allergy and can effectively desensitize peanut-allergic mice .
Ara h 2 has two isoforms, Ara h 2.01 and Ara h 2.02, coded by homologous genes with molecular sizes of 17 and 19 kDa, respectively. The latter has a duplication of 12 amino acids in the middle of the sequence that contains a linear epitope that strongly binds IgE [12,13,14,15,16]. Similarly, multiple genes for Ara h 6 with minor differences in primary structure are expressed in a seed-specific fashion and during seed development [17,18,19,20]. Both Ara h 2 and Ara h 6 contain multiple disulfide-bridged cysteine residues, resulting in a tightly coiled, heat-stable, protease resistant core structure that may be important for allergenicity. Overall, Ara h 2 and Ara h 6 have 59% amino acid homology and this is higher (75%) in the alpha-helical regions accounting for their immunologic cross-reactivity [6,18].
In order to elucidate the most clinically important peanut allergens we have worked to define the relative contributions of specific peanut allergens towards the overall allergenic activity of crude peanut extract using RBL SX-38 cells, an in vitro model of mast cell activation. We have defined the ‘allergenic activity’ of an allergen as its ability to cross-link complexes of IgE and the high-affinity receptor for IgE, FcεRI (IgE/FcεRI), which is likely a more precise measure of clinical relevance than the ability of the allergen to simply bind IgE (immunoreactivity). In our previous studies, we have demonstrated that specifically removing Ara h 2 and Ara h 6 together from a crude peanut extract (CPE) either by size exclusion chromatography or by immunodepletion with specific rabbit antibodies results in a significant decrease in the allergenic activity of a CPE [9,10]. Importantly, removing either Ara h 2 or Ara h 6 alone has little effect . This suggests that these two moderately homologous allergens could act either synergistically or in a redundant fashion.
In this study, we further investigated the individual contributions of Ara h 2 and Ara h 6 to the potency of a crude peanut extract. To do this, we first purified native Ara h 2.01, Ara h 2.02 and Ara h 6 from CPE, determined the concentrations of these allergens in our CPE and compared their potencies in the RBL SX-38 cell assay. Then, we removed >99% of Ara h 2 and Ara h 6 from CPE by gel filtration chromatography and assessed quantitatively the effect of adding back purified Ara h 2 and Ara h 6 separately and together. The data demonstrate that Ara h 2.01, Ara h 2.02 and Ara h 6 have similar potencies and that there is significant functional redundancy between Ara h 2 and Ara h 6.
Individuals were selected on the basis of a strong history of systemic allergic reactions to peanuts and having high concentrations of peanut-specific IgE (>14 kU/l) to peanuts, a level associated with a 95% confidence of a positive double-blind placebo-controlled challenge . A pool of 10 sera was prepared in which each serum was combined proportionally based on the concentration of anti-peanut IgE. This serum pool is different from that described in Chen et al. . In addition, we studied 6 individual sera, 2 of which had slightly higher levels of anti-Ara h 2 IgE than anti-Ara h 6 IgE (the anti-Ara h 2 IgE:anti-Ara h 6 IgE ratio was 1.8:1.0), 2 of which had slightly higher levels of anti-Ara h 6 IgE than that of anti-Ara h 2 (anti-Ara h 6 IgE: anti-Ara h 2 IgE was 1.7:1.0) and 2 of which had equal levels of anti-Ara h 2 and anti-Ara h 6. Clinical characteristics, the total IgE, the peanut-specific IgE of the individual sera and the sera in the serum pool are shown in online suppl. table 1 (for all online suppl. material, see www.karger.com?doi=10.1159/000341642). This study was approved by the Institutional Review Board of the University of Colorado Denver. All subjects signed informed consent and, for minors, assent.
Relative binding of serum IgE to Ara h 2 and Ara h 6 was measured by ELISA. Ninety-six well plates were coated with 10 ng of purified native Ara h 2 (a mixture, approx. 1:1, of Ara h 2.01 and 2.02) or Ara h 6 in 0.1 m bicarbonate buffer, pH 9.6, at 4°C overnight. The plates were washed and blocked. Subject sera, diluted from 1:10 to 1:1,280 with 1% BSA in PBS, was added to the wells and incubated at room temperature for 2 h. Plates were washed and incubated with mouse anti-human IgE-HRP (SouthernBiotech, Birmingham, Ala., USA) at room temperature for 1 h and developed. The optical density of IgE binding to Ara h 2 and Ara h 6 were compared at linear portions of binding curves to assess relative binding.
Quantification of Ara h 2 and Ara h 6 in our crude peanut extract was performed by modification of an inhibitory ELISA previously described independently by De Ceuninck et al.  and Schmitt et al. . Briefly, plates were coated with 10 ng of purified Ara h 2 (an approx. 1:1 mixture of Ara h 2.01 and 2.02) or Ara h 6 at a concentration of 0.2 µg/ml in 0.1 m bicarbonate buffer, pH 9.6, at 4°C overnight. The plates were washed and blocked. Varying concentrations of purified Ara h 2 and Ara h 6 (0–20 µg/ml) and CPE (0–500 µg/ml) were incubated with anti-Ara h 2 and Ara h 6 antibodies, respectively, for 1 h at room temperature before the samples were added to the wells. The plates were incubated for 3 h at room temperature and, after extensive washing, the plates were incubated with HPR goat anti-rabbit IgG antibody and developed. The content of allergens in CPE was calculated by reference to the purified allergen curve and expressed as the percentage of the total protein in the CPE.
CPEs were prepared from Georgia Green peanuts as previously described . Protein concentrations were determined with the Pierce BCA kit (Pierce, Rockford, Ill., USA).
All steps were performed at 4°C. Chromatography was carried out using an AKTA FPLC system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Gel filtration was performed on a HiLoad 26/60 Superdex 75 Prep-Grade Column as previously described . The fraction (13–25 kDa) consisting (approx. 97%) of Ara h 2 and Ara h 6 (referred to as the 20-kDa fraction or 20 kDa) was saved for further purification and the remaining fractions were recombined to yield a CPE without either Ara h 2 or Ara h 6 (CPE w/o 20 kDa). In previous studies we demonstrated that recombining all fractions from the gel filtration column including 20 kDa (CPE recombined) gave activity that was indistinguishable from the original CPE itself . For this reason, the original CPE was used as the control for these experiments.
All steps were performed at 4°C. The fractions from gel filtration (above) containing Ara h 2 and Ara h 6 were collected and dialyzed against 0.1 m phosphate, 1.5 m ammonium sulfate, pH 7.0 buffer and further purified by hydrophobic interaction chromatography (HIC) using HiLoad 16/13 Phenyl Sepharose column (Amersham Biosciences, Piscataway, N.J., USA). Elution was performed using linear and step-wise gradients of ammonium sulfate in 0.1 m phosphate, pH 7.0 at 2 ml/min. The resultant fractions were dialyzed against 1× PBS (Gibco) and stored in aliquots at approximately 1 mg/ml at –70°C.
Allergens were separated by SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed with anti-Ara h 2 and Ara h 6 antibodies, and followed by a goat anti-rabbit IgG conjugated to HRP (Bio-Rad, Hercules, Calif., USA) as previously described . Coomassie blue staining was performed using Bio-Safe Coomassie (Bio-Rad) as per the manufacturer’s instructions. For assessment of IgE binding, 2 µg of CPE, CPE recombined, or CPE w/o 20 kDa, and 0.5 µg of the 20-kDa fraction were run on the gel, probed with the human serum pool (1:10) and developed with biotinylated murine anti-human IgE (Invitrogen, Carlsbad, Calif., USA) and HRP-avidin (Sigma, St. Louis, Mo., USA) as previously described .
RBL SX-38 cells were grown, sensitized, triggered and the data analyzed as previously described except that they were first cultured in MEM medium containing 3% of human AB serum (Gemini Bio-products, West Sacramento, Calif., USA) and 7% of FBS for at least 1 month before being sensitized with the pooled serum [10,26,27]. Briefly, 2 × 105 cells/ml were labeled with 1 µCi/ml of tritium-5-hydroxytryptamine (5-HT, serotonin; PerkinElmer Life Sciences, Covina, Calif., USA) and passively sensitized overnight with MEM containing either 7.5% pooled serum or 5–10% of individual sera. The sensitized cells were then washed and challenged with CPE (200 ng/ml) or varying doses of Ara h 2 or Ara h 6. The data from replicate experiments were normalized to the amount of release with 200 ng/ml of CPE within each experiment and are presented as the percent of maximal (100%) release with CPE. Potencies of these proteins were compared by determining the concentration of each that gave 50% of the maximal degranulation seen with CPE (EC50) . For reconstitution experiments we studied the effect of adding Ara h 2 and/or Ara h 6 proportionately to the CPE w/o 20 kDa at a suboptimal CPE dose of 10 ng/ml. The amounts of purified Ara h 2 and Ara h 6 to be added to the CPE w/o 20 kDa were determined based on data from a competitive ELISA assay. For these experiments, the suboptimal signal at 10 ng/ml was set to 100% to allow comparison of replicate experiments.
One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to compare the EC50 values for Ara h 2.01, Ara h 2.02 and Ara h 6. All statistical comparisons were two tailed. Data were considered to be significantly different when the p value was <0.05. All statistical comparisons, the best-fit lines and EC50 values were generated with GraphPad Prism 5.0c for the MacIntosh (GraphPad Software Inc., San Diego, Calif., USA).
An IgE immunoblot of CPE, CPE recombined, CPE w/o 20 kDa and purified 20-kDa fraction containing Ara h 2 and Ara h 6 is shown in figure 1. This was probed with a pool of 10 peanut-allergic sera and developed with anti-IgE as described in Materials and Methods. The CPE (lane 1), the CPE recombined (lane 2) and the 20-kDa fraction (lane 4), all contain bands at 15, 17 and 19 kDa compatible with Ara h 6, Ara h 2.01 and Ara h 2.02, respectively. The presence of Ara h 2 and Ara h 6 in the 20-kDa fraction was confirmed by immunoblot with specific rabbit antibodies (data not shown). Similarly, the CPE w/o 20 kDa (lane 3) shows absence of Ara h 2 and Ara h 6 and the presence of Ara h 1.
Ara h 2.01, 2.02 and 6 were purified in 2 simple, nondenaturing steps (gel filtration and HIC chromatography) as described in Materials and Methods. Figure 2a shows the elution pattern from the HIC column. This method yielded highly purified (>99%) Ara h 2.01, Ara h 2.02 and Ara h 6 as detected by Coomassie blue staining (fig. 2b) and immunoblot analysis with rabbit anti-Ara h 2 antibody (fig. 2c) and anti-Ara h 6 antibody (fig. 2d).
To examine the allergenic activity of each purified allergen, RBL SX-38 cells were sensitized with IgE from a pool of 10 peanut-allergic sera and triggered with either purified Ara h 2.01, Ara h 2.02 or Ara h 6 at different concentrations (0.01–10 ng/ml) as described in Materials and Methods. The EC50 values of CPE, Ara h 2.01, Ara h 2.02 and Ara h 6 were 16.95 ± 2.55 (n = 3), 0.83 ± 0.1 (n = 3), 1.53 ± 0.26 (n = 4) and 2.57 ± 0.48 (n = 6) ng/ml, respectively (p < 0.0001 for all comparisons; fig. 3). These purified proteins are clearly more potent than is the CPE and their rank order of potency is Ara h 2.01 > Ara h 2.02 > Ara h 6, with Ara h 2.01, Ara h 2.02 and Ara h 6 having approximately 20-, 11- and 7-fold more potency than the original CPE, respectively. Although the differences in molecular size among the 2S albumins are small, we converted the data to moles of protein yielding EC50 values of 4.7 ± 0.12 × 10–11m, 8 ± 0.17 × 10–11m and 1.7 ± 0.19 × 10–10m for Ara h 2.01, Ara h 2.02 and Ara h 6, respectively (p < 0.0001 for all comparisons). However, it is important to point out that, given the several logs encompassed by the dose response curves, these differences, albeit statistically different, are not large.
The binding of anti-Ara h 2 and anti-Ara h 6 antibodies to immobilized Ara h 2 and Ara h 6 was specifically inhibited with CPE or purified allergens (fig. 4). To determine the relative content of each allergen in crude peanut extracts, the concentration of inhibitory allergens at 50% of inhibition (I50) were determined and compared. Based on these calculations, we estimated that the content of Ara h 2 in our CPE is 4 ± 0.3%, which is consistent with previous reports . Based on a similar assay, we estimate that Ara h 6 accounts for 6 ± 1.5% of the total protein in CPE.
Based on the preceding experiments, we considered the possibilities that Ara h 2 and Ara h 6 could act in a synergistic, additive or redundant fashion. We first repeated earlier work showing that, compared to CPE, the allergenic activity of CPE w/o 20 kDa is approximately 10% of the total activity of the original CPE [a shift in potency of approx. 1 log(10); fig. 5a] . In the reconstitution experiments, we decided to study the effect of adding Ara h 2 and/or Ara h 6 proportionately to the CPE w/o 20 kDa at a CPE dose of 10 ng/ml, which represents a suboptimal concentration slightly lower than 50% of the maximum allergenic activity (EC50) of CPE. Based on the data from figure 4, the concentrations of purified Ara h 2 and Ara h 6 in 10 ng/ml of CPE were approximately 0.4 ng/ml (4%) and 0.6 ng/ml (6%), respectively. With pooled sera (fig. 5b), adding either Ara h 2 or Ara h 6 alone at these concentrations restored the activity to approximately 80% of the activity in the original CPE (fig. 5b, bars 3 and 4) and adding both Ara h 2 and Ara h 6 together at these doses to CPE w/o 20 kDa fully restored the total allergenic activity of CPE (p < 0.01 compared with CPE w/o 20 kDa; fig. 5b, bar 5). When cells were sensitized with individual sera (fig. 5c–e), similar patterns were seen regardless of whether the IgE in these sera bound better to Ara h 2 (fig. 5c), equally to Ara h 2 and Ara h 6 (fig. 5d), or better to Ara h 6 (fig. 5e). Of note, in all graphs shown in figure 5 the maximal signal seen with repletion of both Ara h 2 and Ara h 6 was always numerically (but not statistically) greater than the original CPE. This likely reflects a minor overestimation of the content of Ara h 2 and Ara h 6 from the inhibitory ELISA in figure 4.
We have previously shown that removal of both Ara h 2 and Ara h 6, but neither alone, is sufficient to significantly reduce the allergenic activity of a CPE by 80–90% [9,10]. To study the relative contributions of Ara h 2 and Ara h 6 to the allergenic activity of CPE, we chose to study the activity of adding either Ara h 2 or Ara h 6 or both to a CPE preparation that was depleted of both Ara h 2 and Ara h 6 by gel filtration chromatography (CPE w/o 20 kDa). This approach was chosen to remove Ara h 2 and Ara h 6 rather than removal by immunodepletion since the removal of Ara h 2 and Ara h 6 by gel filtration is technically easier. In this report, we first described a simple, nondenaturing, two-step method for purification of Ara h 2.01, Ara h 2.02 and Ara h 6 (fig. 2). It is possible that our purified Ara h 2 and Ara h 6 contained minor impurities. However, the gels and immunoblots shown in figure 2 demonstrate that impurities are <1% of the recovered protein.
We then demonstrated that these allergens have strong allergenic activity (fig. 3). Because Ara h 2.02 isoform contains an extra copy of a reported immunodominant IgE-binding epitope, DPYSPS, it has been speculated that Ara h 2.02 could be more potent than Ara h 2.01 [16,29]. In fact, Hales et al.  reported an increased frequency of binding of IgE to Ara h 2.02 compared with Ara h 2.01 and that Ara h 2.02 was more efficient in an IgE competition assay. However, our finding that Ara h 2.01 has slightly greater potency than Ara h 2.02 is contrary to these earlier reports and underscores the concept that the capacity of an allergen to bind IgE in an immunochemical assay may not be indicative of its ability to cross-link IgE/FcεRI complexes on mast cells/basophils leading to mast cell activation. Furthermore, this finding suggests that the repeating linear sequence, DPYSPS, does not contribute substantially to the allergenic activity of Ara h 2. It is important to point out that the data in figure 3 were obtained with RBL SX-38 cells sensitized with IgE from a pool of 10 peanut-allergic sera. It is possible that some individual sera have a different rank order of sensitivity to Ara h 2.01, 2.02 and Ara h 6. However, given the small differences in the EC50 values, to determine this with accuracy would entail multiple assays with each serum and not accomplish very much. Finally, our repletion assays (fig. 5) did not reveal any significant serum-to-serum variability in the activity of Ara h 2 and Ara h 6 (see below).
We next found that, based on their concentrations in the original CPE, either Ara h 2 or Ara h 6 alone could greatly restore the allergenic activity (about 80–100%) of a CPE that had been depleted of both Ara h 2 and Ara h 6, and that the proportionate addition of Ara h 2 and 6 together to CPE depleted of Ara h 2 and Ara h 6 fully restored the allergenic activity (fig. 5; p < 0.01). It is interesting that the repletion of Ara h 2 and Ara h 6 consistently resulted in final activities that were slightly in excess of 100% (fig. 5). This could be due to underestimation of the concentrations in our purified samples or overestimation of the amount of allergen in the CPE, or possibly due to the unveiling of new epitopes in the purified materials that may have been inhibited by other proteins in the extract. These findings were true if the cells were sensitized with IgE from a pool of 10 sera or from individual sera containing IgE that bound more Ara h 2 than Ara h 6 and from sera containing IgE that bound more Ara h 6 than Ara h 2. Thus, these allergens have substantial redundant functional activity.
To our knowledge, this is the first study to examine the allergenic activity of purified native Ara h 2.01, Ara h 2.02 and Ara h 6 in parallel and to determine the relative contributions of Ara h 2 and Ara h 6 to the activity of a CPE. We did this quantitatively by removal of both allergens followed by reconstitution with purified allergens. These studies are also distinctive in that we used native allergens purified under gentle conditions. Of note, we did not determine the relative content of Ara h 2.01 and 2.02 and did not reconstitute with purified preparations of these isoforms. However, the Ara h 2 used in these studies did contain the same relative amounts of Ara h 2.01 and Ara h 2.02 as found in the original CPE.
To better understand the possible molecular basis of this redundancy, we aligned the primary amino acid sequences of Ara h 2.01 and Ara h 6 and found that three prominent IgE-binding linear epitopes of Ara h 2.01 previously reported in the literature are conserved in Ara h 6 (CEALQQIMENQ, KRELRNLP, QRCDLDVE) and may explain in great part the redundancy between Ara h 2 and Ara h 6 described in this study .
In conclusion, our studies demonstrate in a quantitative fashion that Ara h 2 and Ara h 6 account for the great majority of the allergenic activity in a CPE and have redundant biologic activity. Given their overall 60% sequence homology, we predict that the similarities between these proteins are more important than their differences. An important implication of the findings in this study and our previous work [9,10,22] is that depletion or genetic alteration of either Ara h 2 or Ara h 6 alone from peanuts should not have a significant impact on either reducing the clinical severity to peanut or on decreasing the threshold of reactivity. For the benefit of the general peanut-allergic population, it will be necessary to eliminate or dramatically alter both of these major peanut allergens.
Support in the form of divisional funds and RO1-AI052164 and a supplemental ARRA grant from the National Institute of Allergy and Infectious Diseases to Dr. Dreskin are acknowledged.
The authors also thank our study coordinator, Darcy G. Schlichting, and Drs. Allen Adinoff, Karen Andrews, Sandy Avner, Alan Bock, Leon Greos, John James, Grant Olson, Jerry Koepke, Jeffery Rumbyrt, and Catherine Van Kerkhove for referring patients, and our study subjects for their participation.
Correspondence to: Dr. Stephen C. Dreskin
Division of Allergy and Clinical Immunology
Department of Medicine, University of Colorado Denver
Aurora, CO 80045 (USA)
Tel. +1 303 724 7190, E-Mail firstname.lastname@example.org
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