B cell epitopes of the major timothy grass pollen allergen, phl p 1, revealed by gene fragmentation as candidates for immunotherapy.

Group 1 grass pollen allergens are recognized by IgE antibodies of almost 40% of allergic individuals and therefore belong to the most important elicitors of Type I allergy worldwide. We have previously isolated the cDNA coding for the group 1 allergen from timothy grass, Phl p 1, and demonstrated that recombinant Phl p 1 contains most of the B cell as well as T cell epitopes of group 1 allergens from a variety of grass and corn species. Here we determine continuous B cell epitopes of Phl p 1 by gene fragmentation. IgE antibodies of grass pollen allergic patients identified five continuous epitope-containing areas that on an average bound 40% of Phl p 1-specific IgE antibodies and were stably recognized in the course of disease. In contrast to untreated patients, patients undergoing grass pollen immunotherapy started to mount IgG(4) antibodies to the recombinant IgE-defined fragments in the course of immunotherapy. The protective role of these IgG(4) antibodies is demonstrated by observations that 1) increases in rPhl p 1 fragment-specific IgG(4) were in parallel with decreases in Phl p 1-specific IgE, and 2) preincubation of rPhl p 1 with patients sera containing rPhl p 1 fragment-specific IgG(4) blocked histamine release from basophils of an untreated grass pollen allergic patient. We propose to use recombinant Phl p 1 fragments for active immunotherapy in order to induce protective IgG responses against IgE epitopes in grass pollen allergic patients. This concept may be applied for the development of allergy vaccines whenever the primary sequence or structure of an allergen is available.     

Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen.

Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.     

Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.     

Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.

Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.     

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.     

Three-dimensional structure of the cross-reactive pollen allergen Che a 3: visualizing cross-reactivity on the molecular surfaces of weed, grass, and tree pollen allergens

Two EF-hand calcium-binding allergens (polcalcins) occur in the pollen of a wide variety of unrelated plants as highly cross-reactive allergenic molecules. We report the expression, purification, immunological characterization, and the 1.75-A crystal structure of recombinant Che a 3 (rChe a 3), the polcalcin from the weed Chenopodium album. The three-dimensional structure of rChe a 3 resembles an alpha-helical fold that is essentially identical with that of the two EF-hand allergens from birch pollen, Bet v 4, and timothy grass pollen, Phl p 7. The extensive cross-reactivity between Che a 3 and Phl p 7 is demonstrated by competition experiments with IgE Abs from allergic patients as well as specific Ab probes. Amino acid residues that are conserved for the two EF-hand allergen family were identified in multiple sequence alignments of polcalcins from 15 different plants. Next, the three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 were used to identify conserved amino acids with high surface exposition to visualize surface patches as potential targets for the polyclonal IgE Ab response of allergic patients. The essentially identical three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 explain the extensive cross-reactivity of allergic patients IgE Abs with two EF-hand allergens from unrelated plants. In addition, analyzing the three-dimensional structures of cross-reactive Ags for conserved and surface exposed amino acids may be a first approach to mapping the conformational epitopes on disease-related Ags that are recognized by polyclonal patient Abs.     

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.     

A hypoallergenic vaccine obtained by tail-to-head restructuring of timothy grass pollen profilin, Phl p 12, for the treatment of cross-sensitization to profilin

Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive alpha-helical contents of the protein but retained its beta-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients' IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.     

Allergen Microarray Indicates Pooideae Sensitization in Brazilian Grass Pollen Allergic Patients

Background

Grass pollen, in particular from Lolium multiflorum is a major allergen source in temperate climate zones of Southern Brazil. The IgE sensitization profile of Brazilian grass pollen allergic patients to individual allergen molecules has not been analyzed yet.

Objective

To analyze the IgE sensitization profile of a Brazilian grass pollen allergic population using individual allergen molecules.

Methods

We analyzed sera from 78 grass pollen allergic patients for the presence of IgE antibodies specific for 103 purified micro-arrayed natural and recombinant allergens by chip technology. IgE-ELISA inhibition experiments with Lolium multiflorum, Phleum pratense extracts and a recombinant fusion protein consisting of Phl p 1, Phl p 2, Phl p 5 and Phl p 6 were performed to investigate cross-reactivities.

Results

Within the Brazilian grass pollen allergic patients, the most frequently recognized allergens were Phl p 1 (95%), Phl p 5 (82%), Phl p 2 (76%) followed by Phl p 4 (64%), Phl p 6 (45%), Phl p 11 (18%) and Phl p 12 (18%). Most patients were sensitized only to grass pollen allergens but not to allergens from other sources. A high degree of IgE cross-reactivity between Phleum pratense, Lolium multiflorum and the recombinant timothy grass fusion protein was found.

Conclusions

Component-resolved analysis of sera from Brazilian grass pollen allergic patients reveals an IgE recognition profile compatible with a typical Pooideae sensitization. The high degree of cross-reactivity between Phleum pratense and Lolium multiflorum allergens suggests that diagnosis and immunotherapy can be achieved with timothy grass pollen allergens in the studied population.     

Purification,structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen,Phl p 4, with cross-reactive potential

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.     

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.     

Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients' IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients' IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen's three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.     

Microheterogeneity of the major grass group 6 allergen Phl p 6: Analysis by mass spectrometry

The precise structural characterization of allergens is a basic requirement to improve diagnostics and to find therapeutic strategies against allergic disorders. Natural grass pollen allergens exhibit a wide variety of isoforms and it is still unknown whether this microheterogeneity is essential for the allergic reaction or has a functional effect on sensitization. Well-defined recombinant allergens are considered to replace natural allergens for clinical trials. For the major timothy grass pollen allergen Phl p 6 (approximately 12 kDa) and a recombinant rPhl p 6 we determined the structural microheterogeneity by two-dimensional electrophoresis (2-DE), high-resolution electrospray ionization-Fourier transform-mass spectrometry (ESI-FT-MS) of the intact molecules, and by tryptic peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Natural Phl p 6 is a mixture of mainly two isoforms that differ by two amino acids leading to a mass difference of 5 Da. For each of this two isoforms six variants were identified with modifications at the C- and/or N-terminus. The recombinant Phl p 6 comprises the same structure as one of the main isoforms indicating that it represents a major part of the natural Phl p 6.     

A major IgE epitope-containing grass pollen allergen domain from Phl p 5 folds as a four-helix bundle

Phl p 5, a 29 kDa major allergen from timothy grass pollen, is one of the most reactive members of group 5 allergens. Its sequence comprises two repeats of a novel alanine-rich motif (AR) whose structure and allergenic response are still mostly unknown. We report here a structural characterization of an immunodominant fragment of Phl p 5, Phl p 5(56-165) which comprises the first AR repeat. Recombinant (r)Phl p 5(56-165) was expressed in Escherichia coli, purified to homogeneity and shown to be sufficient to react with serum IgE from 90% of grass pollen allergic patients. Using NMR spectroscopy, we show conclusively that the fragment forms a compact globular domain which is, however, prone to degradation with time. The rPhl p 5(56-165) fold consists of a four-helix bundle held together by hydrophobic interactions between the aromatic rings and aliphatic side chains. This evidence gives clear indications about the structure of the full-length Phl p 5 and provides a rational basis for finding ways to stabilize the fold and designing therapeutic vaccines against grass pollen allergy.     

Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens

Specific IgE binding to carbohydrate moieties of glycosylated allergens has been known for years, but the importance of these structures for the elicitation of allergic reactions is still a matter of debate. Because of their conserved carbohydrate structures, especially N-glycans have always been prime candidates for IgE cross-reactivity between allergens from unrelated species. The aim of our study was to determine whether carbohydrate structures on glycoproteins can by themselves elucidate allergic reactions. We characterized in detail the carbohydrate moieties of the major allergens Phl p 1 and Phl p 13 of timothy grass pollen (Phleum pratense L.) by performing tryptic digests followed by HPLC, N-terminal sequencing, sugar analysis, MALDI-TOF- and ESI-ICRFT-MS. Phl p 1 contains one N-glycan with one of the two glycoforms MMXF3 and M0XF3 and a single furanosidic arabinose, which is bound to a hydroxyproline residue in direct vicinity to the N-glycan. This O-glycosylation is probably due to an arabinosylation consensus sequence found in the N-terminal part of Phl p 1 and other group 1 allergens, but displayed no IgE-reactivity. Thus, Phl p 1 is monovalent with respect to its IgE-binding carbohydrate epitopes and showed no mediator release. In contrast, the carbohydrate moiety of Phl p 13, which carries four of the same N-glycans (like Phl p 1), can cross-link IgE-receptors via carbohydrate chains and elicits IL-4 release from basophils.     

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.     

Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.     

Molecular and immunological characterization of a wheat serine proteinase inhibitor as a novel allergen in baker's asthma

IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly beta-sheets, random coils, and an alpha-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an approximately 50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma.     

Proteome analysis of maize pollen for allergy-relevant components

Over the last few decades, the cultivation of maize (Zea mays) has strongly increased in Central Europe. We therefore decided to study the allergen composition and the allergenic potency of its pollen in comparison with pollen from timothy grass (Phleum pratense), a typical representative of the native grasses. We found that 65% of the sera reactive to timothy pollen also bound to maize pollen proteins. By using 2-DE immunoblotting, followed by incubation with mAbs directed against known allergens or protein sequencing, those IgE-reactive components were further classified. Although novel, maize-specific pollen allergens could not be found, the presence of crossreacting allergens belonging to groups 1 and 13 (Zea m 1 and 13), both having high IgE prevalence, as well as the presence of the less important group 3 and 12 allergens was found. The structural variability of Zea m 1 and Zea m 13 was determined by sequencing clones isolated from a maize pollen cDNA library. This revealed sequence identities of 72 and 70%, respectively, to the corresponding Phl p 1 and Phl p 13 allergens of timothy grass pollen. IgE-crossreactivity was further studied using immunoblot inhibition tests. Here, timothy pollen extract completely blocked IgE binding to maize, whereas maize pollen extract blocked IgE reactivity to only some timothy pollen allergens.