The expression of a mountain cedar allergen comparing plant-viral apoplastic and yeast expression systems

Jun a 3, a major allergenic protein in mountain cedar pollen, causes seasonal allergic rhinitis in hypersensitive individuals. Recombinant Jun a 3 was expressed in Nicotiana benthamiana interstitial fluid (300 microg/g leaf material) and Pichia pastoris (100 microg/ml media). Polyclonal anti-Jun a 3 and IgE antibodies from the sera of allergic patients both reacted with the recombinant protein. Of the two systems, recombinant protein from the plant apoplast contained fewer contaminating proteins. This method allows for a more convenient and inexpensive expression of the recombinant allergen, which will allow for further structural studies and may prove useful in diagnostic and/or immunotherapeutic strategies for cedar allergy.     

Variable expression of pathogenesis-related protein allergen in mountain cedar (Juniperus ashei) pollen

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.     

Purification, identification, and cDNA cloning of Jun a 2, the second major allergen of mountain cedar pollen

The second major allergen of Juniperus ashei (mountain cedar) pollen, Jun a 2, has been purified and its cDNA cloned. The purified protein has a molecular mass of 43 kDa and its N-terminal 9-residue amino acid sequence is highly homologous to those of Cry j 2 and Cha o 2, the second major allergen of Cryptomeria japonica and Chamaecyparis obtusa pollen, respectively. cDNA clones encoding Jun a 2 were isolated after PCR based amplification, and their nucleotide sequences were determined. The cDNA contains an open reading frame of 507 amino acid residues, and encodes a putative 54-residue signal sequence and a 453-residue intermediate, which releases a C-terminal fragment upon maturation. Three possible N-linked glycosylation sites and 20 cystein-residues are found in the deduced amino acid sequence. The amino acid sequence of Jun a 2 shows 70.7 and 82.0% identity with those of Cry j 2 and Cha o 2, respectively. Immunological observations that IgE antibodies in sera of Japanese pollinosis patients bind not only to Cry j 2 and Cha o 2 but also to Jun a 2 strongly suggest that Jun a 2 is an allergen of mountain cedar pollen, and that allergenic epitopes of these three allergens are similar.     

Assessment of 3D models for allergen research

Allergenic proteins must crosslink specific IgE molecules, bound to the surface of mast cells and basophils, to stimulate an immune response. A structural understanding of the allergen–IgE interface is needed to predict cross‐reactivities between allergens and to design hypoallergenic proteins. However, there are less than 90 experimentally determined structures available for the approximately 1500 sequences of allergens and isoallergens cataloged in the Structural Database of Allergenic Proteins. To provide reliable structural data for the remaining proteins, we previously produced more than 500 3D models using an automated procedure, with strict controls on template choice and model quality evaluation. Here, we assessed how well the fold and residue surface exposure of 10 of these models correlated with recently published experimental 3D structures determined by X‐ray crystallography or NMR. We also discuss the impact of intrinsically disordered regions on the structural comparison and epitope prediction. Overall, for seven allergens with sequence identities to the original templates higher than 27%, the backbone root‐mean square deviations were less than 2 Å between the models and the subsequently determined experimental structures for the ordered regions. Further, the surface exposure of the known IgE epitopes on the models of three major allergens, from peanut (Ara h 1), latex (Hev b 2), and soy (Gly m 4), was very similar to the experimentally determined structures. For the three remaining allergens with lower sequence identities to the modeling templates, the 3D folds were correctly identified. However, the accuracy of those models is not sufficient for a reliable epitope mapping. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Development of transgenic rice seed accumulating a major Japanese cedar pollen allergen (Cry j 1) structurally disrupted for oral immunotherapy

Rice seed-based edible vaccines expressing T-cell epitope peptides derived from Japanese cedar major pollen allergens have been used to successfully suppress allergen-specific Th2-mediated immunoglobulin E (IgE) responses in mouse experiments. In order to further expand the application of seed-based allergen-specific immunotherapy for controlling Japanese cedar pollinosis, we generated transgenic rice plants that specifically express recombinant Cry j 1 allergens in seeds. Cry j 1 allergens give low specific IgE-binding activity but contain all of the T-cell epitopes. The allergens were expressed directly or as a protein fusion with the major rice storage protein glutelin. Fusion proteins expressed under the control of the strong rice endosperm-specific GluB-1 promoter accumulated in rice endosperm tissue up to 15% of total seed protein. The fusion proteins aggregated with cysteine-rich prolamin and were deposited in endoplasmic reticulum-derived protein body I. The production of transgenic rice expressing structurally disrupted Cry j 1 peptides with low IgE binding activity but spanning the entire Cry j1 region can be used as a universal, safe and effective tolerogen for rice seed-based oral immunotherapy for cedar pollen allergy in humans and other mammals.     

Longitudinal study of effects of oral dosage of Bifidobacterium bifidum G9‐1 on Japanese cedar pollen‐induced allergic nasal symptoms in guinea pigs

Previous studies using experimental animal models have reported the beneficial effects of probiotics on allergic responses; however, their long‐term effects on allergic nasal symptoms in clinical settings have not yet been elucidated in detail. In the present study, a guinea pig allergic rhinitis model involving repeated inhalation challenges with a natural allergen, Japanese cedar pollen, was used to examine the longitudinal effects of Bifidobacterium bifidum G9‐1 (BBG9‐1) on allergic nasal symptoms. BBG9‐1 was administered orally once a day. Amelioration of nasal blockage was consistently observed throughout the experimental period in the BBG9‐1‐treated group. Although challenge‐induced sneezing was not significantly inhibited in the BBG9‐1‐treated group, prolonged treatment with BBG9‐1 slightly reduced the frequency of sneezing. Antigen‐specific IgE antibody production was also not inhibited in the BBG9‐1‐treated group. Increases in the numbers of eosinophils and neutrophils in nasal cavity lavage fluid collected after pollen challenge were almost completely suppressed by BBG9‐1 treatment, whereas those in mast cell mediators, histamine and cysteinyl leukotrienes were not. In contrast, increases in the levels of nitric oxide metabolites were potently suppressed. Furthermore, prolonged BBG9‐1 treatment markedly suppressed exogenous leukotriene D₄‐induced nasal blockage. Thus, prolonged oral administration of BBG9‐1 suppresses Japanese cedar pollen‐induced allergic nasal symptoms. The inhibitory mechanisms responsible may involve reductions in the responsiveness of target organs, such as endothelial cells in nasal mucosal blood vessels, to chemical mediators.     

Multiple Amb a I allergens demonstrate specific reactivity with IgE and T cells from ragweed-allergic patients.

The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels.     

Biochemical,Biophysical and IgE-Epitope Characterization of the Wheat Food Allergen,Tri a 37

Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptid were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients'' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential IgE epitopes indicates that sensitization to alpha-purothionin occurs via the gut. Both allergens can be used for in-vitro diagnosis of wheat food allergy. The induction of blocking IgG antibodies suggests the usefulness for immunotherapy.     

Identification of a villin-related tobacco protein as a novel cross-reactive plant allergen

In a paradigmatic approach we identified cross-reactive plant allergens for allergy diagnosis and treatment by screening of a tobacco leaf complementary DNA (cDNA) library with serum IgE from a polysensitized allergic patient. Two IgE-reactive cDNA clones were isolated which code for proteins with significant sequence similarity to the actin-binding protein, villin. Northern- and Western-blotting demonstrate expression of the villin-related allergens in pollen and somatic plant tissues. In addition, villin-related proteins were detected in several plant allergen sources (tree-, grass-, weed pollen, fruits, vegetables, nuts). A recombinant C-terminal fragment of the villin-related protein was expressed in Escherichia coli, purified and shown to react specifically with allergic patients IgE. After profilin, villin-related proteins represent another family of cytoskeletal proteins, which has been identified as cross-reactive plant allergens. They may be used for the diagnosis and treatment of patients suffering from multivalent plant allergies.     

Crystal structure of Jun a 1, the major cedar pollen allergen from Juniperus ashei, reveals a parallel beta-helical core

Pollen from cedar and cypress trees is a major cause of seasonal hypersensitivity in humans in several regions of the Northern Hemisphere. We report the first crystal structure of a cedar allergen, Jun a 1, from the pollen of the mountain cedar Juniperus ashei (Cupressaceae). The core of the structure consists primarily of a parallel beta-helix, which is nearly identical to that found in the pectin/pectate lyases from several plant pathogenic microorganisms. Four IgE epitopes mapped to the surface of the protein are accessible to the solvent. The conserved vWiDH sequence is covered by the first 30 residues of the N terminus. The potential reactive arginine, analogous to the pectin/pectate lyase reaction site, is accessible to the solvent, but the substrate binding groove is blocked by a histidine-aspartate salt bridge, a glutamine, and an alpha-helix, all of which are unique to Jun a 1. These observations suggest that steric hindrance in Jun a 1 precludes enzyme activity. The overall results suggest that it is the structure of Jun a 1 that makes it a potent allergen.     

IgE in allergy and asthma today

The spreading epidemic of allergies and asthma has heightened interest in IgE, the central player in the allergic response. The activity of IgE is associated with a network of proteins; prominent among these are its two principal receptors, FcepsilonRI (high-affinity Fc receptor for IgE) and CD23, as well as galectin-3 and several co-receptors for CD23, notably CD21 and various integrins. Here, we review recent progress in uncovering the structures of these proteins and their complexes, and in our understanding of how IgE exerts its effects and how its expression is regulated. The information that has emerged suggests new therapeutic directions for combating allergic disease.     

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.     

Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.     

Mountain cedar pollen induces IgE-independent mast cell degranulation, IL-4 production, and intracellular reactive oxygen species generation

Cedar pollens cause severe allergic disease throughout the world. We have previously characterized allergenic pollen glycoproteins from mountain cedar (Juniperus ashei) that bind to allergen-specific immunoglobulin E (IgE). In the present report, we investigated an alternative pathway of mast cell activation by mountain cedar pollen extract through IgE-independent mechanisms. We show that mountain cedar pollen directly induces mast cell serotonin and IL-4 release and enhances release induced by IgE cross-linking. Concomitant with mediator release, high levels of intracellular reactive oxygen species (ROS) were generated, and both ROS and serotonin release were inhibited by anti-oxidants. These findings suggest that alternative mechanisms exist whereby pollen exposure enhances allergic inflammatory mediator release through mechanisms that involve ROS. These mechanisms have the potential for enhancing the allergenic potency of pollens.     

Purification of allergenic proteins from peanut for preparation of the reactive solid phase of a specific IgE radioimmunoassay

Peanut is one of the most allergenic foods. Detection of specific IgE in the serum of allergic patients requires the purification of allergenic proteins. In the present work, proteins were recovered from peanut kernel after successive treatment in acetone and diethy ether. The proteins were dissolved in 0.05% TFA and analysed by RP-HPLC with a 0–100% gradient of methanol containing 0.05% TFA. The protein peaks were recovered and tested in SDS-PAGE. Eleven proteins were identified with a M_r ranging from 13 to 81. Western blotting was performed with sera from allergic patients. Allergenic proteins had a M_r of 15, 18, 19, 33, 41 and 67. By comparison, a protein fraction from peanut shell contained seven proteins with M_r ranging from 15 to 81. Only two proteins with M_r of 18 and 41 were detected in a Western blot. The protein fractions were coupled to epoxy-Sepharose and the gels were used as a solid reactive phase for detection by IgE-RIA of specific IgE from the serum of allergic patients.     

Use of secondary structural information and C alpha-C alpha distance restraints to model protein structures with MODELLER

Protein secondary structure predictions and amino acid long range contact map predictions from primary sequence of proteins have been explored to aid in modelling protein tertiary structures. In order to evaluate the usefulness of secondary structure and 3D-residue contact prediction methods to model protein structures we have used the known Q3 (alpha-helix,beta-strands and irregular turns/loops) secondary structure information, along with residue-residue contact information as restraints for MODELLER. We present here results of our modelling studies on 30 best resolved single domain protein structures of varied lengths. The results shows that it is very difficult to obtain useful models even with 100% accurate secondary structure predictions and accurate residue contact predictions for up to 30% of residues in a sequence. The best models that we obtained for proteins of lengths 37, 70, 118, 136 and 193 amino acid residues are of RMSDs 4.17, 5.27, 9.12, 7.89 and 9.69,respectively. The results show that one can obtain better models for the proteins which have high percent of alpha-helix content. This analysis further shows that MODELLER restrain optimization program can be useful only if we have truly homologous structure(s) as a template where it derives numerous restraints, almost identical to the templates used. This analysis also clearly indicates that even if we satisfy several true residue-residue contact distances, up to 30%of their sequence length with fully known secondary structural information, we end up predicting model structures much distant from their corresponding native structures.     

Allergenic lipid transfer proteins from plant-derived foods do not immunologically and clinically behave homogeneously: the kiwifruit LTP as a model

Background

Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.

Objective

Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.

Methods

Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.

Results

Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients'' diet.

Conclusion

The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention.     

Occurrence of Lewis a epitope in N-glycans of a glycoallergen, Jun a 1, from mountain cedar (Juniperus ashei) pollen

We have determined the structures of N-glycans linked to major allergens in the mountain cedar (Juniperus ashei) pollen, Jun a 1. First, two kinds of the pollen glycoallergen (Jun a 1-A and Jun a 1-B) were purified from partially purified Jun a 1 by cation exchange chromatography. The N-glycans were liberated by hydrazinolysis from the two glycoallergens and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC from Jun a 1-A and Jun a 1-B respectively. The structures were determined by a combination of exo- and endo-glycosidase digestions, two dimensional sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS) analysis. Structural analysis indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-) occurs in the N-glycans of the pollen allergens.     

Use of secondary structural information and C<Emphasis Type="Italic">α</Emphasis>-C<Emphasis Type="Italic">α</Emphasis> distance restraints to model protein structures with MODELLER

Protein secondary structure predictions and amino acid long range contact map predictions from primary sequence of proteins have been explored to aid in modelling protein tertiary structures. In order to evaluate the usefulness of secondary structure and 3D-residue contact prediction methods to model protein structures we have used the known Q3 (alpha-helix, beta-strands and irregular turns/loops) secondary structure information, along with residue-residue contact information as restraints for MODELLER. We present here results of our modelling studies on 30 best resolved single domain protein structures of varied lengths. The results shows that it is very difficult to obtain useful models even with 100% accurate secondary structure predictions and accurate residue contact predictions for up to 30% of residues in a sequence. The best models that we obtained for proteins of lengths 37, 70, 118, 136 and 193 amino acid residues are of RMSDs 4.17, 5.27, 9.12, 7.89 and 9.69, respectively. The results show that one can obtain better models for the proteins which have high percent of alpha-helix content. This analysis further shows that MODELLER restrain optimization program can be useful only if we have truly homologous structure(s) as a template where it derives numerous restraints, almost identical to the templates used. This analysis also clearly indicates that even if we satisfy several true residue-residue contact distances, up to 30% of their sequence length with fully known secondary structural information, we end up predicting model structures much distant from their corresponding native structures.     

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7.

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.