Grantee Research Project Results
2010 Progress Report: Clinically Relevant IgE-Cross-Reactivity of Nut Allergens
EPA Grant Number: R834066Title: Clinically Relevant IgE-Cross-Reactivity of Nut Allergens
Investigators: Schein, Catherine H. , Teuber, Susanne , Maleki, Soheila
Institution: The University of Texas Medical Branch - Galveston , University of California - Davis , USDA
EPA Project Officer: Aja, Hayley
Project Period: December 1, 2008 through November 30, 2011
Project Period Covered by this Report: December 1, 2008 through June 30,2010
Project Amount: $409,927
RFA: Exploratory Investigations in Food Allergy (2007) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
Nuts provoke some of the strongest allergic reactions in sensitive individuals, and account for a major percentage of hospitalizations for anaphylaxis. Between 30-50% of patients allergic to peanuts also are sensitive to tree nuts, and vice versa. For this reason, it is important to understand what regions of nut proteins could account for cross-reactions. One way to predict such areas is to determine sequences (and surface exposure) of tree nut allergens that are significantly similar to known epitopes of the more extensively studied peanut allergens. The main goal of our project is to determine whether sequences of walnut allergens that were predicted using the property distance (PD) tool in the Structural Database of Allergenic Proteins (SDAP) could indeed bind patient IgE and be cross-reactive with areas of nut allergens. We had 3 specific aims for the grant:
(1) Test the ability of computational tools to identify cross-reactive epitopes of nut allergens using Western blotting and peptide SPOTS membranes developed with serum IgE from patients allergic to peanut, walnut, almond and/or cashew.
(2) Synthesize individual peptides representative of the strong cross-reactive IgE epitopes identified in Aim 1 and determine their comparative dissociation constants (Kd) for inhibition of binding of serum IgEs to purified peanut or walnut allergens with biophysical methods.
(3) Relate the observed Kd values to clinically relevant cross-reactivity, based on patient history and basophil activation testing and incorporate the data into SDAP.
Progress Summary:
Research progress under each of these aims is described below and summarized in the modified Gant Chart at the end of the report.
Table 1. Characteristics of the patient sera.
| | |||||||
| Subject Number | Age of onset (yrs) | Reaction to Peanut |
Reaction to Walnut | ImmunoCAP IgE to Peanut (KU/L) | ImmunoCAP IgE to Walnut (KU/L) | Peanut Proteins detected on WB | Walnut Proteins detected on WB |
|---|---|---|---|---|---|---|---|
| 1. | 1 | Yes, anphx | No | 21.5 | 0.24 | Ara h 1, Ara h 2, Ara h 3, Ara h 6 | Jug r 2, Jug r 4 |
| 2. | 2 | Yes | Yes, anphx | 1.8 | 2.21 | Arha h 1, Ara h 2 Ara h 3 | Jug r 1, Jug r 2, Jug r 4 |
| 3. | 1 | Yes, anphx | Yes, throat swelling, angioedema | >100 | 8.7 | Ara h 1, 2, 3 | Jug r 1, Jug r 2, Jug r 4 |
| 4. | <2 | Yes, anphx | No | 80 | 0.01 | Ara h 2 | none |
| 5. | < 4 | Yes, anphx | No | 23.1 | 0.12 | Ara h 1, Ara h 2, Ara h 6 | none |
| 6. | 2 | No | Yes, anphx | 0.94 | 10.2 | Ara h 1, Ara h 2, Ara h 3, Ara h 6 | Jug r 1 Jug r 2 |
Aim 1 - Summary: We have shown that, as anticipated, walnut sequences that are similar, according to their property distance (PD value, a tool incorporated into the SDAP database to detect sequences similar to known linear IgE epitopes), to known peanut epitopes in homologous allergens are indeed recognized by patient sera. In some cases the predicted epitope was more consistently recognized than the original peanut sequence. Mapping of the sequences on models of the isolated N-terminal domain of the Jug r 2 allergen, and on one prepared for Ara h 2, showed that the sequences were similar in predicted structure to the relevant area of the peanut allergen and were surface exposed.
Aim 2 - Summary: Four peptides were synthesized initially at 20 mg level for solution assays to measure the Kd of binding of the individual peptides (which cannot be accurately assessed in SPOT assays) and to determine whether they could compete for binding to IgE with Ara h 2 (in ELISAs, Aim 2 and basophil degranulation assays, Aim 3). Solubility problems were encountered with a peptide chosen as control. Another set of peptides were ordered and these now are being assayed for their behavior in the two assays. So far, we can see that both a previously known peanut epitope and its related walnut homologue (SPOTS 1 and 6 in Table 2 and Figure 1.3a), detected using the PD tool, can compete in ELISA assays with Ara h 2 for binding to IgE in patient sera. We will complete the competition assays, using appropriate controls.
Table 2. PD values of similar sequences in other peanut (Ara h 6 and 7) and walnut (Jug n 2 and Jug r 2) allergens to a weak (line 1) or strong (line 4) Ara h 2 epitope^(used for membrane). Red residues have significant surface expsure (re GETAREA) in the relevant models for the allergenic proteins.
| Spot # Membrane | Allergen name | PD value | Sequence and psition in allergen | ||
|---|---|---|---|---|---|
| 1 | Ara h 2 | 0.00 | 24 | QWELQGDR | 31 known IgE epitope |
| 2 | Jug n 2 | 8.03 | 351 | SFEDQGRR* | 358 |
| 3 | Jug r 2 | 8.21 | 463 | DYEGQGRR* | 470 |
| 4 | Ara h 2 | 0.00 | 27 | DRRCQSQLER | 36 Known IgE epitope |
| 5 | Ara h 2 | 5.23 | 42 | DDQCQRQLQR | 51 |
| 6 | Jug r 2 | 5.87 | 140 | QRQCQQRCER | 149 |
| 7 | Ara h 2 | 7.08 | 96 | EQRCCNELNR | 105 |
| 8 | Jug r 2 | 8.00 | 76 | YEQCQQQCER | 85 |
| 9 | Jug r 2 | 8.12 | 49 | DQRSQEERER | 58 |
| 10 | Jug r 2 | 8.22 | 101 | QRRQQEERER | 110 |
| 11 | Ara h 2 | 8.48 | 94 | DRQMVQHFKR | 103 |
| 12 | Jug r 2 | 8.65 | 532 | QNNIINQLER | 541 |
Aim 3 - Summary: The data were observed in terms of the degree of cross reactivity of the patients for peanuts and tree nuts. It was clear that the walnut sensitive individuals recognized sequences selected from walnut allergens better than the patients who were sensitive only to peanuts, or cross-reactive with a mild allergy to walnut. The peptides described in Aim 2 are undergoing evaluation of their ability to inhibit degranulation of a mast cell line and basophil cell line. Originally, the grant had proposed use of human basophils, however, the variability between donors was too high to visualize the slight differences in basophil activation that could be expected by inhibition of only one IgE epitope using the synthesized peptides. There also were difficulties with solubility of the peptides but work was done with the most soluble, peptide 1, as of this progress report. This peptide, a known IgE binding sequence from Ara h 2, could somewhat inhibit degranulation by Ara h 2 from the LAD2 mast cell line. The ability of sera to sensitize basophils or the mast cell line to Ara h 2 also was dependent on recognition of the whole protein in the Western Blots (Figure 1.1).
Figure 1.1. Immunoblots of IgE binding by peanut- and/or walnut allergic individuals. Patients
are depicted by nubers (1-6 as above in Table 1) and the clinical allergy to peanut (PNT)
or walnut (WAL). A serum from patient with pollen allergy, MW: protein standard markers.
We have been able to prove that the PD tool is indeed useful for predicting epitopes in homologous allergenic proteins, for the specific case of cross reactivity to walnut and peanut. Walnut sequences that had a low PD (high similarity) to known strong peanut epitopes did indeed bind IgE in patient sera (Figure 1.3 and associated).
Figure 1.3. The surface exposure of sequences from other nut allergens that were similar (low
PD value) to a known epitope of Ara h 2 (Peptid 4) are plotted on models of allergen structures. The
models, from SDAP or prepared for this study, are shown in ribbon format, with the surface exposed
areas of the potential epitopes shown space filling in red. THe surface exposed residues (>30%
solvent exposure according to GETAREA) in each sequence are highlighted by red type. Novel
epitopes 6,9, and 10 are in the N-terminal extension of Jug r 2, which we modeled for this worki.
Peptid 12, which has a higher PD value (lower similarity) to Peptid 4 than the others and did not
react with IgE in patient sera, is situated in the mature, vicilin region of Jug r 2 (far right figure,
drawn to scale), which has a cupin fold. Peptid PD values are shown in the second part of
Table 2; reactivity in SPOTs in section 1.3.
The closer the sequence similarity (the lower the PD value), the more likely it was that the peptide would bind IgE. We also could correlate patient sensitivity to the nut with the sequences recognized. We now are expanding the number of patient sera used for the studies, to confirm out initial observations, and including other tree nut proteins in our tests.
One unanticipated result of these studies is that our isolated peptides, which can compete with Ara h 2 for binding to IgE in patient sera (Figure 2.1), also reduce degranulation of basophils induced by a major peanut allergen, Ara h 2. (Figure 3.1).
Figure 2.1. Both a peptid of a known epitope of Ara h 2 (DRRCQSQLER,
blue line) and a walnut peptide with low PD to the peanut one
(QRQCQQRCER, orange) can compete with purified, recombinant
ARA h 2 for biding to IgE from patient sera. However, combining the
peptides (green line0 does not add to the inhibition, suggesting both
peptides bind to the same pool of antibodies in the sera.
It is thus essential that we continue with the basophil degranulation studies, as slow and cumbersome as this assay is, to determine which of our peptides could be most useful for preventing degranulation in the presence of Ara h 2, and eventually expand the group to see which would be additive in preventing degranulation induced by whole peanut extract. A solution of such inhibitory peptides, which in themselves could not trigger a response (Figure 3.2), but could bind IgE specific for a whole nut allergen, could be a way to defend against unanticipated environmental encounters with nuts.
Figure 3.1
Figure 3.2
Future Activities:
(Aim 1) The original characterization was done with 6 patients, and one control selected to have high serum IgE but primary allergy to pollen, rather than nuts. We are expanding the patient pool, and will characterize the new group of sera as below, using strips of selected sequences on spot assays, Western blotting, and, where appropriate, competition assays.
(Aim 2) Other characterized sera will be tested in these ELISA competitions.
(Aim 3) The finding that the Peptide 1, a known IgE binding sequence from Ara h 2, could inhibit basophil degranulation stimulated by Ara h 2, suggests that this might aid in a novel form of de-sensitization (or antibody blocking) therapy using peptides that can bind to the IgE but not trigger degranulation. Dr. Teuber’s group now is trying the other peptides, in combination with different sera, to see how specific the effect is for the chosen peptide.
After the initial SPOTs data were clear, we sorted through data on IgE recognition of specific nut proteins for a group of patients with sensitivity to multiple nut allergens. An additional group of sera from selected, severely allergic patients (history of anaphylaxis and multiple hospitalizations) have been selected for characterization and then to measure IgE binding to a group of selected epitopes (on small strips). These sera also will be classified according to their binding to the full screen of peptides in copies of our previous SPOTs membranes (Section 1.3, 1.4).
References:
Barre, A., Borges, J. P., Culerrier, R., and Rouge, P. (2005). Homology modelling of the major peanut allergen Ara h 2 and surface mapping of IgE-binding epitopes. Immunol Lett 100:153-158.
Ivanciuc, O., Midoro-Horiuti, T., Schein, C. H., Xie, L. P., Hillman, G. R., Goldblum, R. M., and Braun, W. (2009). The property distance index PD predicts peptides that cross-react with IgE antibodies. Molecular Immunology 46:873-883.
Oezguen, N., Zhou, B., Negi, S. S., Ivanciuc, O., Schein, C. H., Labesse, G., and Braun, W. (2008). Comprehensive 3D-modeling of allergenic proteins and amino acid composition of potential conformational IgE epitopes. Molecular Immunology 45:3740-3747.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 14 publications | 6 publications in selected types | All 6 journal articles |
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| Type | Citation | ||
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Maleki SJ, Teuber SS, Cheng H, Chen D, Comstock SS, Ruan S, Schein CH. Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts. Allergy 2011;66(12):1522-1529. |
R834066 (2010) R834066 (Final) |
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Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.