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.     

Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen.

IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals. We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest. Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P. interpunctella larvae. cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated. Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography. Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions. It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients' IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel. Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens. Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals.     

Identification of a wheat allergen,Tri a Bd 36K,as a peroxidase

A 36-kDa allergen, Tri a Bd 36K, was purified from wheat albumin and characterized. The protein was similar to barley peroxidase BP-1 both in its amino acid sequence and peroxidase activity. The enzyme seemed to contain L-fucose and D-mannose and the glycan moiety reacted with IgE antibodies in a patient's serum.     

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.     

Trichophyton tonsurans allergen. I. Characterization of a protein that causes immediate but not delayed hypersensitivity.

Fungal infections of skin or nails are extremely common and often caused by dermatophyte fungi of the genus Trichophyton. These fungi are unusual in that they can give rise to delayed hypersensitivity (DH) or immediate hypersensitivity (IH) responses. Recently, IH to Trichophyton tonsurans has been demonstrated in patients by skin tests, serum IgE antibody test (RAST), and positive nasal and bronchial challenges. To further investigate the immunology of Trichophyton, a 30-kDa T. tonsurans allergen was isolated by gel filtration and hydrophobic interaction chromatography. This protein, Tri t I, gave a single band on SDS-PAGE, and the 30 amino-terminal amino acids have been determined. Among patients with positive IH skin tests, 34 of 48 (71%) had IgG antibody and 26 of 48 (54%) had IgE antibody to Tri t I. Among those who had positive responses to both skin tests and RAST, 22 of 30 (73%) had IgE antibodies to Tri t I; thus, this protein represents a major allergen. Twelve clones of murine IgG mAb antibodies were produced. Two clones, 2F2-F7 and 6B11-C2, were found to define separate epitopes on Tri t I and were used to develop an immunometric assay for the quantitation of Tri t I. Twenty-three of 38 volunteers with a history of athlete's foot were found to have either IH and/or DH to Trichophyton mix and underwent further testing with purified Tri t I. Of the nine found to have IH to the mix, eight were sensitive to Tri t I. Seven of these eight had IgG and IgE antibodies to Tri t I, by Ag-binding RIA, and all were RAST positive to the unpurified extract. An additional 14 had either DH alone (n = 7) or a wheal and flare response followed by DH at 48 h (n = 7). Of these 14 who had DH responses to Trichophyton mix, only one showed DH to an equivalent quantity of purified Tri t I; among this group, none showed IH or serum IgE antibodies and only one had detectable IgG antibody to Tri t I. The results suggest that the majority of subjects with DH to Trichophyton are responding to a protein other than Tri t I and that the wheal that precedes DH reactions is some patients is not associated with IgE antibodies.     

Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants

Detailed IgE‐binding epitope analysis is a key requirement for the understanding and development of diagnostic and therapeutic agents to address food allergies. An IgE‐specific linear peptide microarray with random phage peptide display for the high‐resolution mapping of IgE‐binding epitopes of the major soybean allergen Gly m 4, which is a homologue to the birch pollen allergen Bet v 1 is combined. Three epitopes are identified and mapped to a resolution of four key amino acids, allowing the rational design and the production of three Gly m 4 mutants with the aim to abolish or reduce the binding of epitope‐specific IgE. In ELISA, the binding of the mutant allergens to polyclonal rabbit‐anti Gly m 4 serum as well as IgE purified from Gly m 4‐reactive soybean allergy patient sera is reduced by up to 63% compared to the wild‐type allergen. Basophil stimulation experiments using RBL‐SX38 cells loaded with patient IgE are showed a decreased stimulation from 25% for the wild‐type Gly m 4 to 13% for one mutant. The presented approach demonstrates the feasibility of precise mapping of allergy‐related IgE‐binding epitopes, allowing the rational design of less allergenic mutants as potential therapeutic agents.     

The Transaldolase,a Novel Allergen of Fusarium proliferatum,Demonstrates IgE Cross-Reactivity with Its Human Analogue

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.     

A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.     

Expression, purification, and immunological characterization of Cr PI

An efficient preparation of Periplaneta americana nymphae allergen, Cr PI (54 kDa) is described. It was expressed as a GST-tag fusion protein in Escherichia coli, strain BL21 (DE3). Expression of recombinant Cr PI (rCr PI), denaturation/renaturation of the inclusion bodies and the effects of protein and L-arginine concentration on inclusion body aggregation were optimized. The fusion protein was purified by affinity chromatography and size exclusion chromatography, and Cr PI fusion protein was purified to >95%. rCr PI bound strongly to IgE in the sera of individuals with cockroach allergies as shown by western blot and ELISA. Highly refolded and purified recombinant protein was obtained, providing a basis for the large-scale preparation of Cr PI allergen.     

The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives

BACKGROUND: Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied. METHODS: IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana extracts was measured using intradermal skin testing. RESULTS: Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana. CONCLUSIONS: Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana.     

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.     

Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.

Type I allergies are immunological disorders that afflict a quarter of the world's population. Improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. We describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. We expressed our model allergen Bet v 1, the major birch pollen allergen, in the tobacco-related species Nicotiana benthamiana using a tobacco mosaic virus vector. Two weeks postinoculation, plants infected with recombinant viral RNA containing the Bet v 1 coding sequence accumulated the allergen to levels of 200 microg/g leaf material. Total nonpurified protein extracts from plants were used for immunological characterizations. IgE immunoblots and ELISA (enzyme-linked immunoassay) inhibition assays showed comparable IgE binding properties for tobacco recombinant (r) Bet v 1 and natural (n) Bet v 1, suggesting that the B cell epitopes were preserved when the allergen was expressed in N. benthamiana plants. Using a murine model of type I allergy, mice immunized with crude leaf extracts containing Bet v 1 with purified rBet v 1 produced in E. coli or with birch pollen extract generated comparable allergen-specific IgE and IgG1 antibody responses and positive type I skin test reactions. These results demonstrate that nonpurified Bet v 1 overexpressed in N. benthamina has the same immunogenicity as purified Bet v 1 produced in E. coli or nBet v 1. We therefore conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts. In addition, there may be a broad utility of this system for the development of new and low-cost vaccination strategies against allergy.     

Molecular and immunological characterization of tri a 36, a low molecular weight glutenin, as a novel major wheat food allergen

Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ～80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.     

Identification of a 55-kDa high-affinity calmodulin-binding protein from Electrophorus electricus

A high-affinity calcium-dependent calmodulin-binding protein (CaMBP) has been isolated from Electrophorus electricus main electric organ. This 55-kDa CaMBP has been purified to homogeneity by ion exchange and calmodulin-Sepharose affinity chromatography and electrophoretic elution from preparative sodium dodecyl sulfate-polyacrylamide gels. Antibodies against the 55-kDa CaMBP were raised in sheep and were affinity purified. A 47-kDa high-affinity CaMBP species was demonstrated by limited protease digestion and immunoblot analysis to be derived from the 55-kDa CaMBP. The 55-kDa CaMBP has also been isolated from skeletal muscle. It is not detectable by immunoblot analysis in nonexcitable tissues. Characterization of the 55-kDa high-affinity calmodulin-acceptor protein may further elucidate the role of calcium-calmodulin in the regulation of bioelectricity.     

Pen c 1, a novel enzymic allergen protein from Penicillium citrinum. Purification, characterization, cloning and expression.

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.     

Identification of the Soybean Allergenic Protein,Gly m Bd 30K,with the Soybean Seed 34-kDa Oil-body-associated Protein

The soybean allergenic protein, Gly m Bd 30K [Ogawa et al., J. Nutr. Sci. Vitaminol., 37, 555–565 (1991)] which is most strongly and frequently recognized by the IgE antibodies in sera of soybean-sensitive patients with atopic dermatitis, has been characterized. The allergen was isolated from the crude 7S-globulin fraction as an oligomeric form with a molecular weight of more than 3000,000 by gel-filtration chromatography. On two-dimensional gel electrophoresis, the native oligomeric allergen had an isoelectric point of about pH 4.5 and was dissociated into a monomeric form with a molecular weight of about 32,000 by the treatment with sodium dodecyl sulfate and 2-mercaptoethanol. The monomeric allergen had an N-terminal amino acid sequence and amino acid composition identical with those of the soybean seed 34-kDa oil-body-associated protein or the soybean vacuolar protein P34 with close homology to papain-like thiol proteinases [Kalinski et al., J. Biol. Chem., 267, 12068 (1992)]. The identity was further confirmed by the immunological cross-reactivity to the antibodies produced against each of the purified allargen and the 34-kDa oil-body-associated protein. By this observation, Gly m Bd 30K was shown to have about 30% sequence homology with Der pI, a house dust mite allergen that is a thiol proteinase from Dermatophagoides pteronyssius.     

Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease.

Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.     

Skin prick testing does not reflect the presence of IgE against food allergens in adult eosinophilic esophagitis patients: a case study

Skin prick testing is widely used to predict the presence of allergen-specific IgE. In eosinophilic esophagitis patients, who frequently exhibit polysensitization and broad reactivity upon skin prick testing, this is commonly used to aid avoidance recommendations in the clinical management of their disease. We present here the predictive value of skin prick testing for the presence of allergen-specific IgE, in 12 patients, determined by immunoblot against the allergen extracts using individual-matched serum. Our results demonstrate a high degree of predictive value for aeroallergens but a poor predictive value for food allergens. This suggests that skin prick testing likely identifies IgE reactivity towards aeroallergens in adult eosinophilic esophagitis but this is not true for foods. Consequently, IgE immunoblotting might be required for determining food avoidance in these patients.