Reduction in allergenicity of grass pollen by genetic engineering

BACKGROUND: Hay fever and allergic asthma triggered by grass pollen allergens affect approximately 20% of the population in cool temperate climates. Ryegrass is the dominant source of allergens due to its prodigious airborne pollen production. Lol p 5 or group 5 is among the most important and widespread grass pollen allergen because it reacts with IgE antibodies of more than 90% of grass pollen-allergic patients, contains most of the grass pollen-specific IgE epitopes and elicits strong biological responses. Significant efforts have been made in developing diagnostic and therapeutic reagents for designing new and more effective immunotherapeutic strategies for treatment of allergic diseases. An alternative approach to this problem could be to reduce the amount of allergen content in the source plant. METHODS: High velocity microprojectile bombardment was used to genetically engineer ryegrass. Antisense construct targeted to one of major allergen, Lol p 5, was introduced. The expression of antisense RNA was regulated by a pollen-specific promoter. Pollen was analysed for IgE reactivity. RESULTS: Analysis of proteins with allergen-specific monoclonal and polyclonal antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE binding capacity of pollen extract as compared to control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development. CONCLUSIONS: Our studies showed that it is possible to selectively 'switch off' allergen production in pollen of ryegrass demonstrating feasibility of genetic engineering of plants for reduced allergenicity.     

Pollen allergens are restricted to few protein families and show distinct patterns of species distribution

BACKGROUND: Inhalative allergies are elicited predominantly by pollen of various plant species. However, a classification of the large number of identified pollen allergens is still missing. OBJECTIVE: To analyze pollen allergen sequences with respect to protein family membership, taxonomic distribution of protein families, and interspecies variability. METHODS: Protein family memberships of all plant allergen sequences from the Allergome database were determined by using the Protein Families Database of Alignments and Hidden Markov Models. The taxonomic distribution of pollen allergens was established from the Integrated Taxonomic Information System. Members of abundant pollen allergen families were compared with allergenic and nonallergenic homologues by database similarity searches and multiple sequence alignments. RESULTS: Pollen allergens were classified into 29 of 7868 protein families. Expansins, profilins, and calcium-binding proteins constitute the major pollen allergen families, whereas most plant food allergens belong to the prolamin, cupin, or profilin families. Pollen allergens were revealed to be ubiquitous (eg, profilins), present in certain plant families (eg, pectate lyases), or limited to a single taxon (eg, thaumatin-like proteins). Allergenic plant profilins constitute a highly conserved family with sequence identities of 70% to 85% among each other but low identities of 30% to 40% with nonallergenic profilins from other eukaryotes, including human beings. Similarly, allergenic polcalcins possess sequence identities of 64% to 92% but show low identities of 39% to 42% to related nonallergenic calmodulins and calmodulin-like proteins from vegetative plant tissues and man. CONCLUSION: This classification of pollen allergens into protein families will aid in predicting cross-reactivity, designing comprehensive diagnostic devices, and assessing the allergenic potential of novel proteins.     

Cross-reactivity of pollen allergens: impact on allergen immunotherapy.

OBJECTIVE: To provide guidelines for the rational formulation of allergen immunotherapy extracts based on knowledge of pollen allergen and epitope cross-reactivity. DATA SOURCES: A PubMed search was performed for articles published from 1966 to 2007 using the keywords pollen, allergen, and cross-reactivity. Older literature was found through cross-referencing of older articles and older reviews on pollen cross-reactivity. Study Selection: Articles that dealt with crude pollen extracts and characterized allergens that addressed cross-reactivity were selected for inclusion in this review. RESULTS: In addition to unique allergens, several families of botanic proteins have similarities that allow them to act as pan-allergens. Although frequently these are minor allergens, in some circumstances they may also be major allergens. Recent studies have investigated nonspecific lipid transfer proteins, calcium-binding proteins, pathogenesis-related protein families, and profilins. Calcium-binding proteins and nonspecific lipid transfer proteins are responsible for pollen-fruit interactions and pollen cross-reactivity. Clarification of pollen allergen enzymatic activity helps explain the ubiquitous nature of these proteins. CONCLUSION: Characterization of specific pollen allergens and their protein families has provided insight into cross-reactivity. Clarification of these relationships allows for consolidation or substitution in formulation of inhalant extracts.     

Digestibility of allergens extracted from natural rubber latex and vegetable foods

BACKGROUND: Several cross-reactive allergens are now known to be involved in the defense responses of higher plants. Such proteins are drawing the attention of plant breeders because of their antimicrobial or stress-alleviating activities. Plants genetically modified to express defense-related proteins are being developed. The current concern is focused on the allergenicity of these intentionally expressed proteins. OBJECTIVE: It is believed that food allergens are proteins resistant to digestion. Digestibility tests have been accepted as an appropriate method for evaluating the allergenicity of newly introduced proteins. In this study we investigated the usefulness of this method for detecting allergens from natural rubber latex and vegetable foods. METHODS: Proteins were extracted from rubber latex, potato, and 5 kinds of fruits. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were used for the digestibility test. An aliquot of each digest was periodically withdrawn and analyzed. Allergens were detected with pooled sera from individuals with latex allergy or patients given a diagnosis of oral allergy syndrome. RESULTS: Most latex and vegetable food proteins were digested by the SGF within 4 minutes. Numerous allergens were also decomposed by the SGF within 8 minutes. Although vegetable food allergens were relatively stable in the SIF, kiwi allergens were substantially degraded by the SIF within 16 hours. CONCLUSION: The pronounced lability of the plant-derived allergens was thought to reflect the discrete sensitization and elicitation processes of patients with latex-fruit syndrome or oral allergy syndrome. These results indicate that the allergenicity of a newly expressed protein should be carefully evaluated according to not only its digestibility but also other important properties.     

Cross-reactivity of pollen allergens: recommendations for immunotherapy vaccines

PURPOSE OF REVIEW: This review will summarize recent research on pollen allergen and epitope cross-reactivity. Knowledge of these relationships aids in the rational formulation of allergen immunotherapy vaccines. RECENT FINDINGS: There has been further clarification of panallergens and their roles as both major and minor allergens. Recent studies have targeted non-specific lipid transfer proteins and calcium-binding proteins (polcalcins), as well as pathogenesis-related protein families and profilins. Polcalcins and non-specific lipid transfer proteins are responsible for pollen-fruit interactions as well as pollen cross-reactivity, in some cases, but not all, accounting for major allergenicity. Delineation of the enzymatic activity of certain allergens explains the ubiquitous nature of these pollen proteins. SUMMARY: Characterization of specific pollen allergens and their protein families has provided insight into the grounds for cross-reactivity. Continuing clarification of these relationships will allow the substitution and consolidation of inhalant extracts as described in the conclusion.     

Molecular cloning and characterization of hazel pollen protein (70 kD) as a luminal binding protein (BiP): a novel cross-reactive plant allergen

BACKGROUND: Tree pollen contains many allergens showing cross-reactivity to proteins from pollen, seeds, and fruits of different plant species. Amongst Fagales, responsible for several allergenic responses, hazel provides the best material to study pollen as well as food allergens in one species. The aim of this study was to identify and characterize the physiological function of an allergen from hazel pollen and to determine possible cross-reactivity to proteins from hazelnut. METHODS: Monoclonal antibodies (mAbs) against hazel pollen crude extract were produced. On the basis of IgE binding, demonstrated by sera from patients allergic to hazel pollen, one mAb indicating the best correlation has been selected, and the putative allergen was purified by preparative gel electrophoresis. Isoforms were investigated by two-dimensional PAGE, and for molecular identification a hazel pollen cDNA library was constructed. In situ localization of the allergen during pollen development was performed by immunofluorescence labelling. RESULTS: Immunological staining of crude hazel pollen extract with specific IgE and mAb revealed a 70-kD protein. Immunoblot studies with mAb showed cross-reactive proteins of 70-72 kD in different plant tissues and species. After protein purification, the IgE-binding reactivity of the allergen has been reconfirmed, and two isoforms were detected. Molecular cloning identified the allergen as a luminal binding protein (BiP) of the Hsp70 family with 88-92% sequence identity in various plants. Further immunocytological studies indicated involvement of BiP during pollen development. CONCLUSIONS: Chaperons like BiP play an important role in protein synthesis and in the protection of cellular structures during stress-related processes. Because of their highly conserved protein sequences, we propose that such allergens could be responsible for at least a part of the allergenic cross-reactivity between proteins from different pollens and plant foods.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family.

Pear is known as an allergenic food involved in the 'oral allergy syndrome' which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n = 16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.     

Reappraisal of inhalant allergens available for allergy practice in Japan. A need to increase allergen extracts available for immunotherapy

In an attempt to assess whether or not the availability of inhalant allergens for diagnosis and treatment of allergic diseases in Japan is adequate, we tested 21 Japanese patients with allergic rhinitis or bronchial asthma against 75 inhalant allergens commonly used in the United States of America. These 21 patients had never lived outside of Japan and came to the United States for the purpose of diagnosis and treatment of their allergic diseases. We determined the importance of each allergen by calculating an allergenicity index. Allergenicity index for a given allergen was defined as a sum of a multiple of each of 4 grades of prick test reactions to the allergen and a number of patients reacting to the allergen with each corresponding grade. Mite had the highest allergenicity index, followed by house dust. All 14 pollen allergens occupying the 3rd through 12th ranks including many grass and weed pollens are not available for immunotherapy in Japan. Japanese cedar pollen which is considered the most important pollen allergen in Japan ranked the 13th. Of a total 25 allergens occupying from the 1st through 13th ranks 22 allergens are not available for immunotherapy including the most important allergen, mite. These results suggest that we should expand the list of allergens available for diagnosis and treatment of allergic diseases in Japan.     

The analysis of specific allergenicity of food allergens families

Classification of food allergens based on protein's structure and function contributes to the study of the relationship between bioinformatics and potential allergenicity of allergens, as well as the evaluation of novel proteins' allergenicity. Some researches were focused on classification within plant or animal food allergens respectively, but there is not any classification of the wholefood allergens. In this article, we classified all the food allergens included in the SDAP into different food allergen families and analysed their specific allergenicity. According to allergen families in AllFam Database, food allergens taken from SDAP (Structural Database of Allergenic Proteins) are classified into different allergen families. Moreover, Protean of DNAStar was applied to analyzing the allergenicity of food allergens. 60% of food allergens are included in the five allergen families: Prolamin superfamily, EF hand domain, Cupin superfamily Profilin and Bet v 1-related protein. Besides, three other cross-food allergen families are found: EF hand domain, Serpin serine protease inhibitor and Triose phosphate isomerase, which include food allergens from both plants and animals. Common characters of food allergens from the same family were easy to find. For instance, they share the same specific peptides and potential T lymphocyte antigen epitope. This classification and characterization of allergen allergenicity in different food allergen families provide foundation for studying the structure and function of novel food allergens as well as the data for the assessment of potential allergenicity.     

Phl p 3: Structural and immunological characterization of a major allergen of timothy grass pollen

BACKGROUND: The relevant importance of individual allergens for allergic sensitization is only partially understood. More detailed information on allergen structure and how it influences immunological responses can lead to better diagnosis of disease and improved preparations for allergen-specific immunotherapy. Grass pollen contains several different allergens, and although the group 3 allergens have been classified long ago, their structure and allergenicity have been poorly investigated. OBJECTIVE: To characterize Phl p 3 from timothy grass pollen and compare it with Phl p 2 with respect to biochemical structure and allergenicity. METHODS: Natural Phl p 2 and Phl p 3 were separated from a pollen extract by chromatography and characterized by 2D electrophoresis and protein sequencing. The complete sequences were determined by DNA cloning and detected in natural pollen extracts by mass spectrometry. Further comparisons of the allergens were made for IgE-binding and cross-reactivity, allergenicity was determined by basophil CD203c activation and skin prick test and 3D structures were compared by molecular modelling. RESULTS: Phl p 3 reveals molecular masses of 10.958 and 10.973 kDa and pIs of 8.9 and 9.3, respectively, Phl p 2 a molecular mass of 10.816 kDa and a pI of 4.6. The sequence identity is 58%. In spite of these differences in the primary structures, both allergens reveal similar conformational structures, resulting in similar immunological and allergological moieties. CONCLUSIONS: The group 3 and group 2 allergens are major allergens with similar 3D structures. Although they differ considerably in their protein sequences and their pIs, they show only a slightly higher immunological reactivity for Phl p 3 on the B-cell level (conformational epitopes). But distinct differences between the sequences may influence reactivity at the T cell level.     

Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins.

BACKGROUND: Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1. CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.     

Cloning and expression of a major allergen from Cupressus arizonica pollen, Cup a 3, a PR-5 protein expressed under polluted environment

BACKGROUND: This paper describes the cloning and expression of the Cupressus arizonica pollen protein Cup a 3. In addition, we present its modulation under polluted environmental conditions. Species of the Cupressaceae family are important because of their high sensitization prevalence. METHODS: Cup a 3 cloning is based on the sequence of the homologous protein Jun a 3. Cup a 3 was expressed with good yield in the methylotropic yeast Pichia pastoris. RESULTS: Recombinant Cup a 3 (rCup a 3) contains 199 amino acids, 10 potential phosphorylation sites and no glycosylation sites. By immunoblot 63% of cypress allergic patients had specific immunoglobulin E antibodies against rCup a 3 (n = 104). This major allergen is homologous to members of the pathogenesis-related proteins (PR-5 group) and contributes to the overall allergenicity of C. arizonica pollen. Our results show that the increased expression of Cup a 3 is dependent on the pollution in the area where the pollen has been collected, being higher under polluted conditions. CONCLUSIONS: Cup a 3 is a PR-5 protein derived from C. arizonica pollen. The expression of the protein under polluted conditions has a direct incidence on the pollen allergenicity, as has been demonstrated by skin tests and Radioallergosorbent test inhibition.     

Nondiffusional release of allergens from pollen grains of Artemisia vulgaris and Lilium longiflorum depends mainly on the type of the allergen

BACKGROUND: Upon contact with a wet surface, mature pollen grains hydrate and release proteins including allergens. Knowledge of the release mechanism of allergens that are mainly localized intracellularly may allow the design of strategies for inhibition of allergen release and the consequent sensitization process. METHODS: An improved pollen chromatography was performed with Artemisia vulgaris and Lilium longiflorum pollen. Using three elution media of different pH, osmolality and salt concentration mimicking various types of wet surfaces, the time-dependent elution profiles of total protein, a cell wall-bound acid phosphatase activity (acPase), allergenic (profilin, Art v 1) and nonallergenic molecules (14-3-3 protein, actin) were monitored. RESULTS: The release kinetics of total protein and cell wall-bound acPase followed an exponential decrease in both pollen species indicating a diffusion-based protein release, whereas the elution profiles of profilin, Art v 1 and 14-3-3 protein showed nondiffusion characteristics. No general dependence on pH, osmolality or salt concentration of the elution media was observable in the elution profiles. Under the applied conditions, actin was not released indicating that the pollen grains remained intact during the elution. CONCLUSION: The elution profiles of pollen allergens indicated that substantial amounts of these proteins do not diffuse from the cell wall or are released from intracellular compartments during imbibitional leakage. Instead, a mechanism seems to operate that involves translocation from the pollen cytoplasm to the extracellular environment by crossing an intact plasma membrane. Such a mechanism would probably allow the use of pharmaceuticals for inhibition of allergen release.     

Strong allergenicity of Pru av 3, the lipid transfer protein from cherry, is related to high stability against thermal processing and digestion

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) have been identified as major fruit allergens in patients from the Mediterranean area. Sensitization to nsLTPs is accompanied by severe reactions, possibly because of specific biophysical and biochemical properties of this allergen family. OBJECTIVE: To assess the protein stability and allergenic potency of nsLTP from fruits in comparison with birch pollen-related allergens from the same allergenic source. METHODS: Stability of natural and recombinant cherry allergens Pru av 3 (nsLTP), Pru av 1 (Bet v 1 homologue), and Pru av 4 (profilin) to pepsin digestion and to thermal processing and stability of allergens in skin prick test reagents was investigated by immunoblotting and/or circular dichroism spectroscopy. Moreover, allergenicity of processed and fresh fruits in regard to Pru av 1 and Pru av 3 was analyzed by histamine release assays. RESULTS: Lipid transfer proteins showed the highest resistance to digestion by pepsin (rPru av 3 > rPru av 1 > rPru av 4). Immunologically active Pru av 3 was detectable after 2 hours of digestion by pepsin, whereas IgE reactivity of Pru av 1 and Pru av 4 was abolished within less than 60 minutes. In contrast with Pru av 1, IgE reactivity to nsLTPs was not diminished in thermally processed fruits, and secondary structures of purified Pru av 3 were more resistant to heating. Moreover, nsLTPs were stable components in skin prick test reagents. Histamine release assays confirmed the strong allergenicity of nsLTPs, which was not affected by protease treatment or thermal processing of fruits. CONCLUSION: In contrast with birch pollen-related allergens, nsLTPs are highly stable to pepsin treatment and thermal processing and show higher allergenic potency. Therefore, nsLTPs have the potential to act as true food allergens, probably eliciting severe systemic reactions by reaching the intestinal mucosa in an intact and fully active form.     

Abortive pollen germination: a mechanism of allergen release in birch,alder, and hazel revealed by immunogold electron microscopy

BACKGROUND: Pollen from early-flowering trees (eg, birch, alder, hazel) represent major seasonal allergen sources. The effects of rain on the release of allergens from tree pollen has thus far not been studied at the ultrastructural level. OBJECTIVE: This study was designed to investigate the effects of rain on the morphology of pollens from early-flowering trees and of potential rain-induced mechanisms of allergen release. METHODS: Freshly collected pollen grains (birch, alder, and hazel) were exposed under controlled conditions to rainwater. Changes of pollen morphology and the release of allergens were analyzed by scanning electron microscopy. The release of allergen-bearing submicronic particles was studied by field emission scanning electron microscopy and transmission electron microscopy in conjunction with immunogold staining by using antibodies with specificity for the major allergens. RESULTS: Scanning electron microscopy showed that freshly isolated pollen grains from birch, alder, and hazel have abortive germination in rainwater. Abortive pollen germination is characterized by the formation of short pollen tubes, which rupture at their tips and release micronic and submicronic particles containing major allergens. Immunogold transmission electron microscopy provided evidence that the allergens are transported through the pollen tubes during germination. CONCLUSIONS: Rainwater-induced release of allergen-bearing submicronic particles from abortively germinated tree pollens may represent a mechanism of allergen release, with important implications on the induction of asthma as well as on current methods for measuring environmental allergen exposure.     

Profilins: mimickers of allergy or relevant allergens?

Profilins are ubiquitous proteins, present in all eukaryotic cells and identified as allergens in pollen, latex and plant foods. The highly conserved structure justifies the cross-reactive nature of IgE antibodies against plant profilins and their designation as pan-allergens. Primary sensitization to profilin seems to arise from pollen sensitization with later development of cross-reactive IgE antibodies against plant food (and possibly latex) profilins. The role of profilin in inducing allergic symptoms needs to be evaluated and raises important issues in allergy diagnosis due to cross-reactivity. IgE cross-reactivity among profilins is associated with multiple pollen sensitization and with various pollen-food syndromes. In respiratory allergy, sensitization to pollen to which the patient has virtually no environmental exposure has been identified as a manifestation of profilin sensitization. As a food allergen, profilin usually elicits mild reactions, such as oral allergy syndrome, is not modified by processing and is especially important in allergy to some fruits, such as melon, watermelon, banana, tomato, citrus fruit and persimmon. Purified natural and recombinant profilins for in vitro and in vivo allergy tests are helpful in the diagnostic work-up. Herein we review the current state of knowledge about the allergen profilin and its implications in the diagnosis and treatment of allergic diseases. We conclude that, although its role in triggering allergic symptoms is still controversial, profilin is undoubtedly a relevant allergen. As a pan-allergen, profilin is associated with multiple pollen sensitization and pollen-food-latex syndromes that the allergist has to be aware of in order to accomplish an accurate diagnosis and successful treatment of allergic diseases.     

1,3-β-glucanases as candidates in latex–pollen–vegetable food cross-reactivity

BACKGROUND: 1,3-beta-glucanases (group 2 of pathogenesis-related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens. OBJECTIVE: The aim of this work was to study the potential implication of 1,3-beta-glucanases in cross-reactivities among latex, pollen and vegetable foods. METHODS: The cDNA encoding the N-terminal domain (NtD) of Ole e 9, a major allergenic 1,3-beta-glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N-terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD-specific polyclonal antiserum were used. RESULTS: The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3-beta-glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD-specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3-beta-glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell-pepper, banana and latex. CONCLUSION: rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.     

Plant food allergens homologous to pathogenesis-related proteins

In general, pathogenesis-related (PR) proteins are expressed by plants in response to stress conditions like infection, exposure to certain chemicals, wounding and environmental conditions. In some plant tissues, however, PR proteins are constitutively expressed, e.g. in pollens or fruits, tissues that are more likely to be attacked (by insects or fungi) or exposed to atmospheric conditions (e.g. UV irradiation). PR proteins display multiple effects within the plant and possess antimicrobial activity, and can thus be regarded as a part of the plant's defense system. Analyzing known amino acid sequences and functions of characterized (cloned) food allergens, it is remarkable that many of these molecules can be classified as PR proteins. Many PR proteins are stable at low pH, and display considerable resistance to proteases, requirements to act as food allergens. According to sequence characteristics and their enzymatic or biologic activity, PR proteins can be divided into 14 groups. Seven of these 14 groups contain proteins with allergenic properties, six groups contain food allergens.     

Cross-reactivity and epitope analysis of Pru a 1, the major cherry allergen.

A high percentage of birch pollen allergic patients experiences food hypersensivity after ingestion of fresh fruits and vegetables. The cross-reactivity of the major allergens of sweet cherry (Pru a 1), apple (Mal d 1), pear (Pyr c 1), celery tuber (Api g 1) and carrot (Dau c 1) is due to structural similarities which are reflected by high amino acid sequence identities with Bet v 1a, the major birch pollen allergen. Apart from a strong cross-reactivity to Bet v 1a, IgE inhibition experiments with Mal d 1, Pru a 1 and Api g 1 demonstrated the presence of common and different epitopes among the tested food allergens. Secondary structure prediction of all investigated allergens indicated the presence of almost identical structural elements. In particular, the 'P-loop' region is a common domain of the pollen related food allergens and of pathogenesis related proteins. To identify the IgE binding epitopes, five overlapping recombinant Pru a 1 fragments representing the entire amino acid sequence with lengths of approximately 60-120 residues were investigated. Weak IgE binding capacity was measured exclusively with Pru a IF4 (1-120) by immunoblotting, whereas none of the fragments showed allergenicity in the rat basophil leukaemia cell mediator release assay. Site-directed mutagenesis experiments with Pru a 1 revealed that amino acid S112 is critical for IgE binding of almost all patients sera tested. This reduced IgE binding was also observed with a single point mutant of Bet v 1a (S112P) and thus indicated serine 112 as an essential residue for preserving the structure of a cross-reactive IgE epitope. Moreover, two Pru a 1 mutants with an altered 'P-loop' region, showed a lowered IgE binding capacity for IgE from a subgroup of allergic patients. The investigation of essential features for preserving cross-reactive IgE-epitopes provides the structural basis for understanding the clinically observed cross-allergenicity between pollen and fruits. Moreover, non-anaphylactic allergen fragments or variants derived from the IgE-inducing pollen allergens may serve as useful tools for a new strategy of specific immunotherapy.     

Nonspecific lipid-transfer proteins in plant foods and pollens: an important allergen class

PURPOSE OF REVIEW: Here we focus our attention on the structural stability and physicochemical properties of plant nonspecific lipid-transfer proteins (nsLTPs) as keys to their allergenicity. We further present the current opinions on the route of sensitization and include the latest additions to the nsLTP allergen family. RECENT FINDINGS: Plant nsLTPs are small cysteine-rich lipid-binding proteins that play a key role in plant resistance to biotic and abiotic stress. Besides their relevance for plant-pathogen interactions, nsLTPs have attracted interest as true food allergens which are of high importance to atopics in Mediterranean countries. It is now becoming clear that their molecular properties such as the remarkable stability to proteolysis and thermal denaturation are intrinsically linked to their allergenicity. These properties also facilitate sensitization via the gastrointestinal tract which allows these molecules to act as allergens independently of prior exposure to pollen. In addition, a group of allergenic pollen nsLTPs exists which seem to be only partially linked to the food nsLTPs by cross-reactivity. SUMMARY: Research into the family of nsLTPs will continue to provide insights about the particular molecular properties that make an nsLTP an allergen and how primary sensitization occurs.