IgE Epitope Mapping Research Articles

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.

Visualization of clustered IgE epitopes on α-lactalbumin

alpha-Lactalbumin (alpha-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. We performed molecular, structural, and immunologic characterization of alpha-La. Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.

Linear IgE-epitope mapping and comparative structural homology modeling of hazelnut and English walnut 11S globulins

Allergic reactions to walnuts and hazelnuts can be serious. The 11S globulins (legumins) have been identified as important allergens in these and other nuts and seeds. Here we identify the linear IgE-binding epitopes of walnut and hazelnut 11S globulins, and generate 3D 11S globulin models to map the locations of the epitopes for comparison to other allergenic homologues. Linear IgE-epitope mapping was performed by solid-phase overlapping 15-amino acid peptides probed with IgE from pooled allergic human sera. Several walnut (Jug r 4) and hazelnut (Cor a 9) 11S globulin peptides with reactivity to patient IgE were identified. Comparative alignment with cashew (Ana o 2), peanut (Ara h 3), and soybean G1 (Gly m 6.0101) and G2 (Gly m 6.0201) allergenic homologues revealed several shared allergenic ‘hot spots’. Homology modeling was performed based on the atomic structure of the soybean glycinin. Surface map comparisons between the tree nut and peanut homologues revealed structural motifs that could be important for IgE elicitation and binding and show that, contrary to predictions, the reactive epitopes are widely distributed throughout the monomeric subunits, both internally and externally, including regions occluded by quaternary subunit association. These findings reveal structural features that may be important to allergenicity and cross-reactivity of this protein class.

Development of a novel peptide microarray for large-scale epitope mapping of food allergens

The peptide microarray is a novel assay that facilitates high-throughput screening of peptides with a small quantity of sample. We sought to use overlapping peptides of milk allergenic proteins as a model system to establish a reliable and sensitive peptide microarray-based immunoassay for large-scale epitope mapping of food allergens. A milk peptide microarray was developed by using commercially synthesized peptides (20-mers, 3 offset) covering the primary sequences of alpha(s1)-casein, alpha(s2)-casein, beta-casein, kappa-casein, and beta-lactoglobulin. Conditions for printing and immunolabeling were optimized using a serum pool of 5 patients with milk allergy. Reproducibility of the milk peptide microarray was evaluated using replicate arrays immunolabeled with the serum pool, whereas specificity and sensitivity were assessed by using serial dilution of the serum pool and a peptide inhibition assay. Our results show that epitopes identified by the peptide microarray were mostly consistent with those identified previously by SPOT membrane technology, but with specific binding to a few newly identified epitopes of milk allergens. Data from replicate arrays were reproducible (r > or = 0.92) regardless of printing lots, immunolabeling, and serum pool batches. Using the serially diluted serum pool, we confirmed that IgE antibody binding detected in the array was specific. Peptide inhibition of IgE binding to the same peptide and overlapping peptides further confirmed the specificity of the array. A reliable peptide microarray was established for large-scale IgE epitope mapping of milk allergens, and this robust technology could be applied for epitope mapping of other food allergens.

The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum

Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides.Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine protease allergen-family, which stresses the importance of these proteins with respect to fungal IgE-cross-reactivity.

The IgE-Reactive Autoantigen Hom s 2 Induces Damage of Respiratory Epithelial Cells and Keratinocytes via Induction of IFN-γ

Hom s 2, the alpha-chain of the nascent polypeptide-associated complex, is an intracellular autoantigen that has been identified with IgE autoantibodies from atopic dermatitis patients. We investigated the humoral and cellular immune response to purified recombinant Hom s 2 (rHom s 2). rHom s 2 exhibited IgE reactivity comparable to exogenous allergens, but did not induce relevant basophil cell degranulation. The latter may be attributed to the fact that patients recognized single epitopes on Hom s 2 as revealed by IgE epitope mapping with rHom s 2 fragments. In contrast to exogenous allergens, rHom s 2 had the intrinsic ability to induce the release of IFN-gamma in cultured peripheral blood mononuclear cells from atopic as well as non-atopic individuals. IFN-gamma-containing culture supernatants from Hom s 2-stimulated peripheral blood mononuclear cells caused disintegration of respiratory epithelial cell layers and apoptosis of skin keratinocytes, which could be inhibited with a neutralizing anti-IFN-gamma antibody. Our data demonstrate that the Hom s 2 autoantigen can cause IFN-gamma-mediated cell damage.

Prediction of allergenic proteins and mapping of IgE epitopes in allergens

Prediction of allergenic proteins and mapping of IgE epitopes in allergens

A novel approach to IgE epitope mapping - virtual epitope mapping

A novel approach to IgE epitope mapping - virtual epitope mapping

Genetic Engineering of the Major Timothy Grass Pollen Allergen, Phl p 6, to Reduce Allergenic Activity and Preserve Immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.

AlgPred: prediction of allergenic proteins and mapping of IgE epitopes

In this study a systematic attempt has been made to integrate various approaches in order to predict allergenic proteins with high accuracy. The dataset used for testing and training consists of 578 allergens and 700 non-allergens obtained from A. K. Bjorklund, D. Soeria-Atmadja, A. Zorzet, U. Hammerling and M. G. Gustafsson (2005) Bioinformatics, 21, 39–50. First, we developed methods based on support vector machine using amino acid and dipeptide composition and achieved an accuracy of 85.02 and 84.00%, respectively. Second, a motif-based method has been developed using MEME/MAST software that achieved sensitivity of 93.94 with 33.34% specificity. Third, a database of known IgE epitopes was searched and this predicted allergenic proteins with 17.47% sensitivity at specificity of 98.14%. Fourth, we predicted allergenic proteins by performing BLAST search against allergen representative peptides. Finally hybrid approaches have been developed, which combine two or more than two approaches. The performance of all these algorithms has been evaluated on an independent dataset of 323 allergens and on 101 725 non-allergens obtained from Swiss-Prot. A web server AlgPred has been developed for the predicting allergenic proteins and for mapping IgE epitopes on allergenic proteins (). AlgPred is available at .

Information Management for the Study of Allergies

Microarrays and other large-scale screening technologies produce quantities of increasingly complex allergy data. These data link molecular and clinical measurements and observations and provide fertile ground for improving our understanding of the processes involved in allergic reactions. Information technology is employed in gathering, storage, retrieval and analysis of these data. The increasing proportion of allergy data are generated from genomics and proteomics approaches. The major activity focuses on characterization of allergens including IgE reactivity, structural properties, and mapping of IgE and T-cell epitopes. Because of the complexity of allergy data, their utilization requires bioinformatics approaches. Allergen data are stored in the general and specialist databases. At least a dozen of important allergen databases and data repositories have been developed to date. These data are analysed using general and specialist bioinformatics tools. The major applications of bioinformatics include support for allergen characterization, assessment of allergenicity, and identification of allergic cross-reactivity. These applications in turn support the development of vaccines and therapies for allergic disease. In this article we review allergen databases and tools for the analysis of allergens, and discuss the new directions in the field supported by large scale screening involving genomics, proteomics, and bioinformatics support.

IgE and IgG 4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2

Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping. We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2. Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies. By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG(4) signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping. Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.

Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library

Phl p 5 represents a major allergen of timothy grass pollen (Phleum pratense). Detailed knowledge about the structures responsible for IgE binding would allow the design of a novel generation of allergy vaccines. We aimed to characterize the IgE epitopes of Phl p 5a using phage display combined with a molecular modeling approach. Phl p 5a-specific IgE from sera of patients with grass pollen allergy was used for screening of a random peptide phage library displaying constrained decamers. Fifteen phage clones that shared sequence motifs and could be grouped into families were selected by using Phl p 5a-specific IgE. Peptide alignment with the solvent-accessible amino acids of Phl p 5a revealed 3 sequence sections with frequent hits of identical or similar amino acids. On the surface of Phl p 5a, these sections assembled in compact patches, most likely representing conformational IgE epitopes, whereas no matching clusters were found on the back sides of the 2 Phl p 5a halves. In surface plasmon resonance experiments, the high-affinity interaction between IgE and Phl p 5 could be competed by phage-displayed peptides up to 24%, indicating that they represent true epitope mimics (ie, mimotopes). Allergen-specific immunogenicity of the mimotopes was proved in Biozzi mice. The selected mimotopes facilitated the localization of conformational IgE epitopes of Phl p 5. We suggest them to be suitable candidates for the development of an epitope-specific immunotherapy.

Microarray immunoassay (MIA) for the parallel IgE and IgG4 epitope mapping of milk allergens

Rationale The natural history of young patients with milk allergy is variable. Characteristics such as IgE/IgG 4 ratio and specific epitope recognition patterns are being investigated for prognostic utility. Prospective, comprehensive analysis of patient epitope-specific antibody response is impractical with commonly used assays. We sought to develop a microarray-based immunoassay for parallel detection of whole allergen and peptide specific IgE and IgG 4. Methods Sets of 15-residue peptides, each with biotin-SGSG linkers, were designed corresponding to milk allergen sequences. These were printed together with biotinylated beta- and kappa-caseins to avidin-derivatized glass slides. Diluted human sera from milk-allergic patients and non-allergic controls were incubated over the arrayed peptides in a humidified chamber. Specific IgE and IgG 4 were simultaneously detected by incubation with a cocktail of monoclonal secondary antibodies labeled with distinct fluorochromes. Slides were read with a microarray fluorescence scanner. Results Compared to normal sera, IgE signal [binding] with patient sera was 1.5–2 log higher to beta- and kappa-caseins respectively (p<0.01). IgG 4 signal was also greater with patient sera (beta-casein, 1.5 log; kappa-casein 1 log; p<0.05), although IgG 4 signal was clearly detectable in normals. Inter-assay CVs were less than 10%. For 80% of patients, kappa-casein was the dominant allergen with higher absolute IgE and IgG 4 signals and higher IgE/IgG 4 ratios. Individual and pooled patient sera bound specific peptides. Conclusion MIA is a reproducible and specific assay for the investigation of allergen-specific antibody.

Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus.

Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens. This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated. Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10. The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal. Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.

Molecular and immunological characterization and IgE epitope mapping of Pen n 18, a major allergen of Penicillium notatum.

The mould genus, Penicillium, is a significant source of environmental aero-allergens. A major allergen from Penicillium notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of Penicillium citrinum, followed by matrix-assisted laser-desorption ionization-time-of-flight MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA encoded a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N- and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni(2+)-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mould-allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. When mapping of the allergenic epitopes was performed, at least nine different linear IgE-binding epitopes, located throughout the Pen n 18 protein, were identified. Of these, peptide C12, located in the N-terminal region of the molecule, was recognized by serum from 75% of the patients tested and therefore appears to be an immunodominant IgE-binding epitope.

Identification and Fine Mapping of IgG and IgE Epitopes in Ovomucoid

Ovomucoid is a major allergen in hen egg white which causes a serious IgE-mediated food allergy reaction. This study determined eight IgG epitopes, 5–11 amino acids in length, and nine IgE epitopes, 5–16 amino acids in length, within the primary sequence in ovomucoid using arrays of overlapping peptides synthesized on cellulose membranes. Pooled sera from eight egg-allergic patients were used to probe the membrane. We also analyzed the amino acids that are critical for antibody binding by substituting a single amino acid within each epitope. Mutational analysis of the epitopes indicated that charged amino acids (aspartic acid, glutamic acid, and lysine) and some hydrophobic (leucine, phenylalanine, and glycine) and polar (serine, threonine, tyrosine, and cystein) amino acids were important for antibody binding. These results provide useful information for the molecular design necessary to reduce the allergenicity of ovomucoid, and a better understanding of structure–function relationships of allergic epitopes in food proteins.

Soybean Glycinin G1 Acidic Chain Shares IgE Epitopes with Peanut Allergen Ara h 3

Background: The identification of IgE epitopes for proteins is the first step in understanding the interaction of allergens with the immune system. Proteins from the legume family have shown in vitro cross-reactivity in IgE-binding assays, but this cross-reactivity is rarely clinically significant. Resolution of this discrepancy requires IgE epitope mapping of legume family protein allergens. Methods: We constructed six fusion proteins representing overlapping regions of soybean glycinin G1 acidic chain. These fusion proteins were used in immunoblotting and a novel sandwich ELISA with pooled sera from soy-allergic individuals to reveal a common IgE-binding region. This region was the focus for IgE epitope mapping using overlapping synthetic peptides. Results: Data from the fusion protein experiments revealed an IgE-binding region consisting of residues F192–I265. Analysis of the overlapping synthetic peptides to this region indicated that IgE epitopes to glycinin G1 acidic chain consist of residues G217–V235 and G253–I265. The epitopes identified for glycinin G1 acidic chain are homologous to IgE epitopes previously identified for the peanut allergen Ara h 3 [<citeref rid="ref001">1</citeref>]. However, residues identified by alanine scanning in the peanut epitopes as being important for IgE binding are different in the natural soybean epitopes. Conclusions: The IgE epitopes identified for glycinin G1 acidic chain apparently represent an allergenic region of several legume family seed storage proteins. Our findings indicate that the identification of IgE epitopes and structural analysis of legume family proteins will provide valuable information to the study of food allergies.

The molecular basis for allergen cross-reactivity: crystal structure and IgE-epitope mapping of birch pollen profilin.

The profilins are a group of ubiquitous actin monomer binding proteins that are responsible for regulating the normal distribution of filamentous actin networks in eukaryotic cells. Profilins also bind polyphosphoinositides, which can disrupt the profilin-action complex, and proline-rich ligands which localize profilin to sites requiring extensive actin filament accumulation. Profilins represent cross-reactive allergens for almost 20 % of all pollen allergic patients. We report the X-ray crystal structure of birch pollen profilin (BPP) at 2.4 resolution. The major IgE-reactive epitopes have been mapped and were found to cluster on the N- and C-terminal alpha helices and a segment of the protein containing two strands of the beta sheet. The overall fold of this protein is similar to that of the mammalian and amoeba profilins, however, there is a significant change in the orientation of the N-terminal alpha helix in BPP. This change in orientation alters the topography of a hydrophobic patch on the surface of the molecule, which is thought to be involved in the binding of proline-rich ligands. Profilin has been identified as an important cross-reactive allergen for patients suffering from multivalent type I allergy. The prevalent epitopic areas are located in regions with conserved sequence and secondary structure and overlap the binding sites for natural profilin ligands, indicating that the native ligand-free profilin acts as the original cross-sensitizing agent. Structural homology indicates that the basic features of the G actin-profilin interaction are conserved in all eukaryotic organisms, but suggests that mechanistic differences in the binding of proline-rich ligands may exist. The structure of BPP provides a molecular basis for understanding allergen cross-reactivity.

Mapping of murine IgE epitopes involved in IgE‐Fcε receptor interactions

The generation of anti-IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (Fc epsilon R) interaction site has been determined using cross-inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the Fc epsilon R interaction site and the binding sites of various anti-IgE mAb, we employed site-directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant epsilon heavy chain (C epsilon) gene bearing a (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH region. Several site-specific mutants in the C epsilon 3 and C epsilon 4 domains of this recombinant C epsilon gene were prepared and expressed by transfection into the light chain-producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti-IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the C epsilon 3 domain, a mutant with a truncated C epsilon 4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C epsilon 3, and a chimeric human C epsilon in which the human C epsilon 3 was replaced by the homologous mouse C epsilon 3 domain. These mutants have permitted the localization, to the C epsilon 3 domain, of the epitopes recognized by the 84.1C and 95.3 anti-IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the Fc epsilon R binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc epsilon R binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE-Fc epsilon R interaction, has been mapped to the C epsilon 4 domain. When tested for binding to the Fc epsilon R on RBL-2H3 cells, the point mutant bound to the Fc epsilon R with twofold reduced affinity, while the C epsilon 3 deletion mutant and the mutant truncated in C epsilon 4 lost all receptor binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)