IgE-binding Epitopes Research Articles

Analysis of potential immunoglobulin E epitopes of Gly m 4, a bet V 1-related allergen in soy beans

Binding and crosslinking of FcaRI-bound IgE to conformational epitopes on allergens causes hypersensitivity reactions in allergic patients. The knowledge and modulation of epitopes on the molecular level allows the design of both hypoallergenic recombinant variants of the allergen and specific antibodies for therapeutic and diagnostic purposes, respectively. We sought to identify and analyze the IgE-binding epitopes on Gly m 4, a soy bean allergen that is also responsible for anaphylactic reactions in individuals with birch pollen pollinosis.1 Utilizing a phage-display peptide library Mittag et al. identified peptides that bound Gly m 4-specific IgE.2 In the present study we reassessed these data and mapped 21 mimotopes onto the molecular surface of Gly m 4 (PDB code 2K7H). Using an algorithm to predict conformational epitopes on the protein surface we identified 8 major patches that might represent IgE epitopes.3 To verify the in-silico predicted IgE-binding surface areas of Gly m 4 we chose a mutagenesis approach and substituted alanine for lysine residues within the putative epitopes. The resulting recombinant Gly m 4 variants were expressed in Escherichia coli and IgE-binding was tested in western blot analyses and ELISA with a panel of sera of soy-allergic individuals sensitized to Gly m 4. rGly m 4 variants with low or no IgE-reactivity were purified and their molecular integrity was analyzed by static and dynamic light scattering as well as circular dichroism spectroscopy. Studies on the capability of the rGly m 4 variants to release mediators in humanized rat basophil cells as well as a comprehensive epitope analysis of Gly m 4 screening different phage-display peptide libraries are in progress. The benefit of a thorough IgE epitope analysis of Gly m 4 for diagnosis and therapy of soy allergy is discussed.

Asp159 is a critical core amino acid of an IgE-binding and cross-reactive epitope of a dust mite allergen Der f 7

Der f 7 and Der p 7 are important house dust mite allergens with known structure and suggested biological function recently. However, their IgE-binding determinants remain unknown. The purpose of this study is to identify the IgE-reactive epitopes of Der f 7 and the determinants of IgE-mediated cross-reactivity between Der f 7 and Der p 7. IgE-reactive determinants were identified by immunodot blot inhibition using synthetic overlapping peptides, allergen mutants, and a Der f 7 structural model. Our results showed that synthetic peptides with sequence 156SILDP160 on Der f 7 bind IgE in two of the 30 asthmatic serum samples tested. Recombinant Der f 7 I157A, L158A, or D159A mutants have reduced IgE-binding activity. Inhibition experiments confirmed Asp159 as a critical core residue for IgE-binding. Among Der p 7, Der f 7 and Der f 7 mutants with single substitution between residues 156 and 160, only the D159A mutant cannot inhibit significantly IgE-binding against Der p 7. Therefore, Asp159 contributes to IgE-mediated cross-reactivity between Der f 7 and Der p 7. The structural model constructed for Der f 7 suggests that the IgE-binding epitope forms a loop-like structure on the surface of the molecule. In conclusion, Asp 159 is a critical core residue of an IgE-binding and IgE-mediated cross-reactive epitope 156SILDP160 of Der f 7. Results obtained from this study provide more information on molecular and structural features related to allergenicity, underlying basis of IgE cross-reactivity between allergens, and in designing safer immunotherapy.

Cloning and Characterization of an 11S Legumin, Car i 4, a Major Allergen in Pecan

Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.

Mapping IgE binding epitopes of major shrimp (Penaeus monodon) allergen with immunoinformatics tools

Tropomyosin, a myofibrillar protein, was described as the major allergen in crustaceans. The objective of this study was to screen and identify the linear IgE epitopes of tropomyosin in Penaeus monodon. Three immunoinformatics tools were used to predict the potential epitopes and the resultant epitopes were confirmed by dot-blot inhibition with sera from shrimp allergic subjects. As the result, 10 peptides were predicted and assembled by solid-phase synthesis. Eight epitopes were identified by dot-blot inhibition test, among which peptide 4 and 8 had not been reported by previous researches. Moreover, peptide 6 and 10 had IgE binding capability with all sera, while peptide 5, 8 and 9 could bind with 80% of the sera. Amino acid analysis demonstrated that Y, E, R, F and S presented more frequently in epitopes. Although further research should be done, it seems that immunoinformatics-driven epitope prediction can benefit epitope identification with high accuracy and efficiency.

Immunoglobulin‐E‐binding epitopes of wheat allergens in patients with food allergy to wheat and in mice experimentally sensitized to wheat proteins

At present, B cell epitopes involved in food allergy to wheat are known only for a few allergens and a few categories of patients. To characterize the epitopes of different wheat kernel allergens: α-, γ, ω2, and ω5-gliadin, a low-molecular-weight (LMW) glutenin subunit, and a lipid transfer protein (LTP1) recognized by allergic patients and by sensitized mice and provide further understanding of the role of structure in determining allergic response. Sera were obtained from 39 patients suffering from food allergy to wheat. BALB/c mice were sensitized to gliadins or LTP1 by intraperitoneal immunizations. Continuous epitopes bound by IgE were delineated by the Pepscan technique. The response to reduced, alkylated LTP1 was compared with that of the native form to evaluate the importance of protein folding on IgE reactivity. Few continuous epitopes of LTP1 reacted with IgE from allergic patients and mice, but one of them was common to several patients and sensitized mice. The unfolded protein was not recognized by either patient or mouse IgE, emphasizing the major role of LTP1 folding and discontinuous epitopes in IgE-binding. In contrast, many continuous epitopes were detected by patient and mouse IgE especially for an ω5-gliadin, which is an unstructured protein, and to a lesser extent, for the other gliadins and a LMW-glutenin subunit. The conformation of LTP1 appeared to have a strong impact on the type of IgE-binding epitopes elicited by this protein in both man and mouse. The responses in mice sensitized to gliadins or LTP1 were sufficiently comparable with the human response in terms of IgE-binding epitopes to provide support for the use of the mouse model in further investigations.

Cloning, Expression and Patient IgE Reactivity of Recombinant Pru du 6, an 11S Globulin from Almond

Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.

Identification of Critical Amino Acids in the IgE Epitopes of Ric c 1 and Ric c 3 and the Application of Glutamic Acid as an IgE Blocker

BackgroundThe allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae) is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis.Methodology/Principal FindingsThe IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction.Conclusions/SignificanceThe identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.

Molecular diagnosis of egg allergy

Allergy to hen's egg is common in infancy and childhood. Oral food challenges are often required to diagnose egg allergy, because of the limitation in the diagnostic accuracy of skin test and specific IgE to egg white. New molecular diagnostic technologies have been recently introduced into allergological research. In this article, we will review the recent literature regarding the potential value of these tests for the clinical management of egg-allergic patients. Component-resolved diagnosis that can be combined with the microarray technology is promising as measurement of specific IgE antibodies to individual egg white components has been shown to predict different clinical patterns of egg allergy. Specific IgE to ovomucoid has been identified as a risk factor for persistent allergy and could indicate reactivity to heated egg. Ovomucoid and ovalbumin IgE and IgG4-binding epitope profiling could also help distinguish different clinical phenotypes of egg allergy. Particularly, egg-allergic patients with IgE antibodies reacting against sequential epitopes tend to have more persistent allergy. Using recombinant allergens, IgE-binding epitopes, and microarrays, molecular-based technologies show promising results. However, none of these tests is ready to be used in clinical practice and oral food challenge remains the standard for the diagnosis of egg allergy.

Epitope mapping and identification on a 3D model built for the tartary buckwheat allergic protein TBb

Allergic protein TBb, a major allergen in tartary buckwheat, was divided into four epitope-containing fragments and was named F1, F2, F3, and F4, respectively. Results of immunological assays revealed that F2 had the strongest IgE-binding activity to patient's sera, which indicated that it might contain the linear IgE-binding epitope of TBb. According to the results of sequence analysis and molecular modeling of tartary buckwheat allergen, three mutants of F2 gene (R139A, R141A, and D144A) were reconstructed using site-directed mutagenesis, and each mutant was expressed in Escherichia coli BL21 (DE3). Following purification by Ni(2+) affinity chromatography, enzyme-linked immunosorbent assay and dot blot were performed for wild-type F2 and its mutants using sera from buckwheat-allergic patients and a negative control (non-allergic patient). Results showed that mutants R139A and D144A had weaker IgE-binding activity to patient's sera than wild-type F2, implying that Arg(139) and Asp(144) might be involved in the allergic activity of TBb. However, R141A had the weakest IgE-binding activity, suggesting that Arg(141) may be the critical amino acid of TBb. This is the first report on the epitope mapping and identification of TBb. Our findings will contribute to the production of TBb hypoallergens and to allergen-specific immunotherapy for tartary buckwheat allergy.

IgE-binding epitopes: a reappraisal

Here, we discuss various questions related to IgE epitopes: What are the technical possibilities and pitfalls, what is currently known, how can we put this information into hypothetical frameworks and the unavoidable question: how useful is this information for patient care or allergenicity prediction? We discuss the information obtained by (i) 3D structures of allergen-antibody complexes; (ii) analysis of allergen analogues; (iii) mimics without obvious structural similarity; (iv) mAbs competing with IgE; (v) repertoire analysis of cloned IgEs, and other developments. Based on limited data, four suggestions are presented in the literature: (i) IgE might be more cross-reactive than IgG; (ii) IgE might be more often directed to immunologically 'uninviting' surfaces; (iii) IgE epitopes may tend to cluster and (iv) IgE paratopes might have a higher intrinsic flexibility. While these are not proven facts, they still can generate hypotheses for future research. The hypothesis is put forward that the IgE repertoire of switched B-cells is less influenced by positive selection, because positive selection might not be able to rescue IgE-switched B cells. While this might be of interest for the discussion about mechanisms leading to allergen-sensitization, we need to be modest in answering the 'clinical relevance' question. Current evidence indicates the IgE-epitope repertoire is too big to make specific IgE epitopes a realistic target for diagnosis, treatment or allergenicity prediction. In-depth analysis of a few selected IgE epitope-peptides or mimitopes derived from allergen-sequences and from random peptide libraries, respectively, might well prove rewarding in relation to diagnosis and prognosis of allergy, particularly food allergy.

Hev b 2 et Hev b 13, deux allergènes majeurs de l’allergie au latex et du syndrome latex-fruits

Among the major latex allergens (Hev b 1, Hev b 2, Hev b 4, Hev b 5, Hev b 6.01, Hev b 6.02, and Hev b 13), Hev b 2 and Hev b 13 have been poorly investigated and, accordingly, their involvement in both latex allergy and latex-fruit syndrome has to be addressed. Both allergens consist of ubiquitous glycoproteins widely distributed in plants, especially in fruits (banana, cherry, kiwi fruit). Hev b 2 consists of an endo-β1,3-glucanase involved in the glucan metabolism during the fruit ripening process. Hev b 13 consists of an esterase also with a metabolic function. Most of the IgE-binding epitopes identified on both allergens occur outside the putative N-glycosylation sites and do not apparently consist of glycotopes. The high prevalence of Hev b 2 and Hev b 13 in latex allergic patients make both allergens useful for improving the diagnosis of latex allergy and latex-fruit syndrome.

High Level Expression of the Acidic and Basic Subunits of Recombinant Ara h 3

RATIONALE: Ara h 3 is a multi-subunit, major peanut allergen. The acidic subunit is believed to contain the immunodominant, linear IgE binding epitopes of this allergen. Also, reports have shown that IgE binding epitopes are found on the basic subunit. Here, the acidic and basic subunit of Ara h 3 have been expressed at high levels for independent structural, functional and immunological analysis.

IgE Epitope Mapping of Ara h 2 and Ara h 6 by Microarray Immunoassay

RATIONALE: We have reported that Ara h 2 and Ara h 6 are important peanut allergens and together account for 60-90% of the effector activity of a crude peanut extracts (CPE). These molecules are 60% homologous at the amino acid level and have similar three-dimensional structures. We hypothesized that the IgE-binding linear epitopes of Ara h 2 will align with those of Ara h 6. METHODS: Twenty-mer peptides were commercially synthesized from full length Ara h 6 and Ara h 2 sequences with 3 overlapping amino acids. Each peptide was site-specifically arrayed in triplicate onto epoxy derivatized glass slides. Binding of IgE from a serum pool of four highly peanut allergic subjects were compared with IgE from a serum of a peanut-tolerant patient with the hyper-IgE syndrome. Specific IgE was detected with biotinylated (Bt) goat anti-Human IgE followed by gold labeled anti-biotin and finally by a silver-based chromogenic detection system (Gentel Biosciences, Madison, WI). RESULTS: Two of the four most intense IgE-binding peptides for Ara h 2 and Ara h 6 were highly homologous (85%) and two were very different (0 and 15% homology, respectively). CONCLUSIONS: Ara h 2 and Ara h 6 are highly homologous allergens that have shared and potentially complementary IgE binding epitopes. These findings may explain their relative contributions to the effector activity of CPE.

Localization On Caprine β-casein Of The Epitopes Specifically Recognized By Ige From Patients Allergic To Goat's Milk And Tolerant To Cow's Milk

RATIONALE: Patients allergic to goat's milk (GM) but tolerant to cow's milk (CM) display a much higher IgE specificity to caprine caseins than to bovine caseins, especially to the β-casein despite a sequence homology of 90%. We aimed to identify the IgE-binding epitopes of the caprine β-casein involved in this restricted specificity.

Silencing β1,2-xylosyltransferase in Transgenic Tomato Fruits Reveals xylose as Constitutive Component of Ige-Binding Epitopes.

Complex plant N-glycans containing β1,2-xylose and core α1,3-fucose are regarded as the major class of the so-called “carbohydrate cross-reactive determinants” reactive with IgE antibodies in sera of many allergic patients, but their clinical relevance is still under debate. Plant glycosyltransferases, β1,2-xylosyltransferase (XylT), and core α1,3-fucosyltransferase (FucT) are responsible for the transfer of β1,2-linked xylose and core α1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of β1,2-xylose-containing epitopes, expression of the tomato β1,2-xylosyltransferase was down-regulated by RNA interference (RNAi) in transgenic plants. Fruits harvested from these transgenic plants were analyzed for accumulation of XylT mRNA, abundance of β1,2-xylose epitopes and their allergenic potential. Based on quantitative real-time PCR analysis XylT mRNA levels were reduced up to 10-fold in independent transgenic lines as compared to untransformed control, whereas no xylosylated N-glycans could be revealed by MS analysis. Immunoblotting using anti-xylose-specific IgG antibodies revealed a strong reduction of β1,2-xylose-containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specific reduction in IgE-binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of β1,2-xylose-containing complex N-glycans skin prick tests were performed demonstrating a reduced responsiveness of tomato allergic patients, in vivo. This study provides strong evidence for the clinical relevance of β1,2-xylose-containing epitopes in vivo.

The specific IgE reactivity pattern of weed pollen-induced allergic rhinitis patients

Conclusion: Specific immunoglobulin E (IgE) reactivity towards the major mugwort allergen Art v 1 is a good indicator for Art v sensitization. Allergens from the ragweed species Amb t and Amb a possibly share common IgE-binding epitopes. Objective: The aim of this study was to investigate the reactivity pattern of IgE in Chinese patients with weed pollen-induced allergic rhinitis. Methods: Sera from 50 weed pollen-induced allergic rhinitis patients were tested for specific serum IgE reactivity against allergenic extracts of mugwort (Artemisia vulgaris, Art v), short ragweed (Ambrosia artemisiifolia, Amb a), giant ragweed (Ambrosia trifida, Amb t), and single allergens of Art v 1, Art v 3, Amb a 1, and profilin. Results: Sera from 88% of the patients demonstrated positive specific IgE reactivity to Art v, and of these 82% were positive to Art v 1. Sera from 38% of the patients showed positive specific IgE reactivity to both ragweed species Amb t and Amb a. A strong correlation was found between the specific IgE levels of Amb t and Amb a. Of the Amb a IgE-positive patients, 38% were positive for Amb a 1. Of all patient sera tested, 12% were specific IgE-positive to profilin.

Signification clinique des allergènes croisants de la noix de Cajou ( Anacardium occidentale)

The consumption of cashew as roasted nuts or as a hidden allergen in various foodstuffs, e.g. Asian meals, ice creams or cakes, is responsible for severe anaphylactic reactions in previously sensitized individuals. Like peanut allergy, allergy to Cashew nut has now become a worrying problem of public health due to the extreme severity of its clinical manifestations. The major cashew allergens consist of seed storage proteins embedded in the seed protein bodies, namely Ana o 1 (vicilin) and Ana o 2 (legumin) of the cupin family, and Ana o 3 (2S albumin). All of these cashew allergens strongly resist to heat denaturation (roasting) and hydrolysis by digestive proteases (pepsin, trypsin), and thus display a high allergenic potency. The IgE-binding epitopes have been identified on the molecular surface of both allergens. A high degree of IgE-binding cross-reactivity occurs between the cashew allergens and the corresponding allergens Pis v 1, Pis v 2 and Pis v 3, of the closely-related pistachio. Obviously, this cross-reactivity depends on the highly conserved character of both the sequence and three-dimensional fold among these allergens. Less frequent cross-reactions also occur with other tree nuts, like almond and hazelnut. Conversely, almost no cross-reaction was reported to occur between cashew and peanut. In most cases, however, this cross-reactivity is apparently devoid of any clinical significance and would simply reflect some phylogenetical relatedness among tree nuts. In this respect, it is noteworthy that cashew and pistachio belong to the same family of Anacardiaceae.

Identification of IgE sequential epitopes of lentil (Len c 1) by means of peptide microarray immunoassay

Lentils are often responsible for allergic reactions to legumes in Mediterranean children. Although the primary sequence of the major allergen Len c 1 is known, the location of the IgE-binding epitopes remains undefined. We sought to identify IgE-binding epitopes of Len c 1 and relate epitope binding to clinical characteristics. One hundred thirty-five peptides corresponding to the primary sequence of Len c 1 were probed with sera from 33 patients with lentil allergy and 15 nonatopic control subjects by means of microarray immunoassay. Lentil-specific IgE levels, skin prick test responses, and clinical reactions to lentil were determined. Epitopes were defined as overlapping signal above interslide and intraslide cutoffs and confirmed by using inhibition assays with a peptide from the respective region. Hierarchic clustering of microarray data was used to correlate binding patterns with clinical findings. The patients with lentil allergy specifically recognized IgE-binding epitopes located in the C-terminal region between peptides 107 and 135. Inhibition experiments confirmed the specificity of IgE binding in this region, identifying different epitopes. Linkage of cluster results with clinical data and lentil-specific IgE levels displayed a positive correlation between lentil-specific IgE levels, epitope recognition, and respiratory symptoms. Modeling based on the 3-dimensional structure of a homologous soy vicilin suggests that the Len c 1 epitopes identified are exposed on the surface of the molecule. Several IgE-binding sequential epitopes of Len c 1 have been identified. Epitopes are located in the C-terminal region and are predicted to be exposed on the surface of the protein. Epitope diversity is positively correlated with IgE levels, pointing to a more polyclonal IgE response.

Effects of enzymatic hydrolysis on lentil allergenicity

Enzymatic hydrolysis and further processing are commonly used to produce hypoallergenic dietary products derived from different protein sources, such as cow's milk. Lentils and chickpeas seem to be an important cause of IgE-mediated hypersensitivity in the Mediterranean area and India. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as soybean, chickpea, lentil, and lupine. Nevertheless, there are only a few studies carried out to evaluate the effect on IgE reactivity of these food-hydrolysis products with sera from patients with well-documented allergy to these foods. In this study, lentil protein extract was hydrolyzed by sequential action of an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to raw and hydrolyzed lentil extract was evaluated by means of IgE immunoblotting and ELISA using sera from five patients with clinical allergy to lentil. The results indicated that sequential hydrolysis of lentil results in an important proteolytic destruction of IgE-binding epitopes shown by in vitro experiments. However, some allergenic proteins were still detected by sera from four out of five patients in the last step of sequential hydrolyzation.

Elucidation of IgE-binding epitopes of Ani s 1: The major Anisakis simplex allergen

Ani s 1, the major allergen of Anisakis simplex, is expected to be a useful antigen in allergen-specific immunotherapy of Anisakis allergy. To avoid side-effects such as anaphylactic shock in immunotherapy, it is desirable to use not native allergens but hypoallergenic mutants devoid of IgE-binding epitopes. This study was hence aimed to elucidate IgE-binding epitopes of Ani s 1 toward the development of hypoallergenic Ani s 1 mutants. Mapping experiments using 32 peptides (P1–32) revealed that major IgE-binding epitopes are included in P24 (region 116–130) and P28 (region 136–150). Furthermore, Ala-scanning and truncation experiments established that eight (underlined in the sequence 116-ELFAREYEGVCKSGK-130) and nine amino acid residues (underlined in the sequence 136-RGSGWMMTILGKSCD-150) are crucial for the IgE-binding of P24 and P28, respectively. The determined crucial residues of P24 and P28 were assumed to be involved in electrostatic and hydrophobic interactions with IgE, respectively.