The cross-reactive calcium-binding pollen allergen,Phl p 7, reveals a novel dimer assembly

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.     

Divalent ion binding properties of the timothy grass allergen, Phl p 7

The timothy grass allergen, Phl p 7, was studied by calorimetry, spectroscopy, and analytical ultracentrifugation. As judged by isothermal titration calorimetry (ITC), the protein binds Ca (2+) cooperatively with stepwise macroscopic association constants of 1.73 x 10 (6) and 8.06 x 10 (6) M (-1). By contrast, Mg (2+) binding is sequential with apparent macroscopic association constants of 2.78 x 10 (4) and 170 M (-1). Circular dichroism and ANS fluorescence data suggest that Ca (2+) binding provokes a major conformational change that does not occur upon Mg (2+) binding. Conformational stability was assessed by differential scanning calorimetry (DSC). In phosphate-buffered saline (PBS) containing EDTA, the apoprotein undergoes two-state denaturation with a T m of 78.4 degrees C. In the presence of 0.02 mM Ca (2+), the T m exceeds 120 degrees C. Phl p 7 is known to crystallize as a domain-swapped dimer at low pH. However, analytical ultracentrifugation data indicate that the protein is monomeric in neutral solution at concentrations exceeding 1.0 mM, in both the apo and Ca (2+)-bound states.     

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.     

Solution structure of Phl p 3, a major allergen from timothy grass pollen

The major 97-aa timothy grass (Phleum pratense) allergen Phl p 3 was recently isolated from an extract of timothy grass pollen. Sequence comparison classifies this protein as a group 3 allergen. The solution structure of Phl p 3 as determined by nuclear magnetic resonance spectroscopy reveals that the protein consists of a core of hydrophobic amino-acid side chains from two beta-sheets of five and four anti-parallel beta-strands, respectively. This conformation is very similar to the crystal structure published for Phl p 2 and strongly resembles the known conformation of the carboxy-terminal domain of Phl p 1, the major difference being the loop orientations. Phl p 2 and Phl p 3 show virtually identical immunoreactivity, and comparison of the charged surface amino acids of the two proteins gives initial clues as to the IgE recognition epitopes of these proteins.     

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.     

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.     

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.     

Identification of an allergen related to Phl p 4, a major timothy grass pollen allergen,in pollens,vegetables, and fruits by immunogold electron microscopy

Group 4 grass pollen allergens represent 60 kDa glycoproteins recognized by 70% of patients sensitive to these pollens. An antiserum against purified Phl p 4 from timothy grass pollen was used to investigate various pollens, fruits, and vegetables for Phl p 4-related allergens by immunogold electron microscopy. In timothy grass, mugwort, and birch pollens, allergens were located in the wall, and in timothy grass and birch pollens additionally in the cytoplasm. In peanut, apple, celery root, and carrot root, only cytoplasmic areas were labeled. Group 4-related allergens thus occur in pollens of unrelated plants and in plant food and may therefore contribute to crossreactivities in patients allergic to various pollens and plant food.     

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.     

Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense)

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.     

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.     

An immunoglobulin-like fold in a major plant allergen: the solution structure of Phl p 2 from timothy grass pollen.

BACKGROUND: Grass pollen allergens are the most important and widespread elicitors of pollen allergy. One of the major plant allergens which millions of people worldwide are sensitized to is Phl p 2, a small protein from timothy grass pollen. Phl p 2 is representative of the large family of cross-reacting plant allergens classified as group 2/3. Recombinant Phl p 2 has been demonstrated by immunological cross-reactivity studies to be immunologically equivalent to the natural protein. RESULTS: We have solved the solution structure of recombinant Phl p 2 by means of nuclear magnetic resonance techniques. The three-dimensional structure of Phl p 2 consists of an all-beta fold with nine antiparallel beta strands that form a beta sandwich. The topology is that of an immunoglobulin-like fold with the addition of a C-terminal strand, as found in the C2 domain superfamily. Lack of functional and sequence similarity with these two families, however, suggests an independent evolution of Phl p 2 and other homologous plant allergens. CONCLUSIONS: Because of the high homology with other plant allergens of groups 1 and 2/3, the structure of Phl p 2 can be used to rationalize some of the immunological properties of the whole family. On the basis of the structure, we suggest possible sites of interaction with IgE antibodies. Knowledge of the Phl p 2 structure may assist the rational structure-based design of synthetic vaccines against grass pollen allergy.     

Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy

IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage lambda gt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities. Both isoforms represented calcium-binding proteins that belonged to the beta-lineage of parvalbumins. The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity. Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly alpha-helical secondary structure and a molecular mass of 11,416 Da, respectively. rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon. Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy. The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01. The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.     

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.     

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.     

A hypoallergenic hybrid molecule with increased immunogenicity consisting of derivatives of the major grass pollen allergens, Phl p 2 and Phl p 6

Allergen-specific immunotherapy is currently based on the administration of allergen extracts containing natural allergens. However, its broad application is limited by the poor quality of these extracts. Based on recombinant allergens, well-defined allergy vaccines for allergen-specific immunotherapy can be produced. Furthermore, they can be modified to reduce their allergenic activity and to avoid IgE-mediated side effects. Here, we demonstrate that the immunogenicity of two grass pollen-derived hypoallergenic allergen derivatives could be increased by engineering them as a single hybrid molecule. We used a hypoallergenic Phl p 2 mosaic, generated by fragmentation of the Phl p 2 sequence and reassembly of the resulting peptides in an altered order, and a truncated Phl p 6 allergen, to produce a hybrid protein. The hybrid retained the reduction of IgE reactivity and allergenic activity of its components as shown by ELISA and basophil activation assays. Immunization with the hybrid molecule demonstrated the increased immunogenicity of this molecule, leading to higher levels of allergen-specific IgG antibodies compared to the single components. These antibodies could inhibit patients' IgE binding to the wild-type allergens. Thus, the described strategy allows the development of safer and more efficacious vaccines for the treatment of grass pollen allergy.     

A contaminant trypsin-like activity from the timothy grass pollen is responsible for the conflicting enzymatic behavior of the major allergen Phl p 1

We intend to solve whether or not Phl p 1 can be regarded as a protease. A group reported that Phl p 1 has papain-like properties and later on, that this allergen resembles cathepsin B, while another one demonstrated that Phl p 1 lacks proteinase activity and suggested that the measured activity may rise either from a recombinant Phl p 1 contaminant or as a result of an incompletely purified natural allergen. A third group reported Phl p 1 to act by a non-proteolytic activity mechanism. We report the purification of the natural Phl p 1 by means of hydrophobic interaction, gel filtration and STI-Sepharose affinity chromatographies. The Phl p 1 purity was assessed by silver-stained SDS-PAGE and by ‘in-gel’ and ‘gel-free’ approaches associated to mass spectrometry analyses. The proteolytic activity was measured using Boc–Gln–Ala–Arg–AMC and Z-Phe–Arg–AMC as substrates. While amidolytic activity could be measured with Phl p 1 after rechromatography on gel filtration, it however completely disappeared after chromatography on STI-Sepharose. The contaminant activity co-eluting with Phl p 1 was not affected by cysteine proteases inhibitors and other thiol-blocking agents, by metalloproteases inhibitors and by aspartic proteases inhibitors. However, it was completely inhibited by low molecular weight and proteinaceous serine proteases inhibitors. TLCK, but not TPCK, inhibited the contaminant activity, showing a trypsin-like behavior. The pH and temperature optimum were 8.0 and 37 °C, respectively. These data indicated that Phl p 1 is not a protease. The contaminant trypsin-like activity should be considered when Phl p 1 allergenicity is emphasized.     

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.     

Solution structure and backbone dynamics of Calsensin, an invertebrate neuronal calcium-binding protein

Calsensin is an EF-hand calcium-binding protein expressed by a subset of peripheral sensory neurons that fasciculate into a single tract in the leech central nervous system. Calsensin is a 9-kD protein with two EF-hand calcium-binding motifs. Using multidimensional NMR spectroscopy we have determined the solution structure and backbone dynamics of calcium-bound Calsensin. Calsensin consists of four helices forming a unicornate-type four-helix bundle. The residues in the third helix undergo slow conformational exchange indicating that the motion of this helix is associated with calciumbinding. The backbone dynamics of the protein as measured by (15)N relaxation rates and heteronuclear NOEs correlate well with the three-dimensional structure. Furthermore, comparison of the structure of Calsensin with other members of the EF-hand calcium-binding protein family provides insight into plausible mechanisms of calcium and target protein binding.