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.     

Quantification of the major allergen from cypress (Cupressus arizonica) pollen, Cup a 1, by monoclonal antibody-based ELISA

BACKGROUND: Cypress pollen allergy is an important cause of rhinoconjunctivitis and asthma in Mediterranean countries. Cypress allergenic extracts are difficult to produce since they have low protein and high carbohydrate content, thus accurate standardization of them is essential to guarantee their quality. The aim of this study is to develop a sandwich ELISA for the quantification of Cup a 1, the major allergen of cypress (Cupressus arizonica) pollen extract. METHODS: Monoclonal antibodies directed to purified Cup a 1 were produced. Two of them (9C7 as capture antibody and 3D2 as the tracer) were selected to develop a quantitative sandwich ELISA. This ELISA was subsequently evaluated and compared with other techniques. RESULTS: The described ELISA is very sensitive with a detection limit of 8.7 ng/ml and a practical working range of 62.5-1,000 ng/ml. The assay is also highly reproducible with intra-assay and interassay coefficients of variation of less than 10%. The purified Cup a 1, used as standard, presents pectate lyase enzymatic activity. The assay also detected Cup a 1-like proteins in pollen from other Cupressaceae. A good correlation was obtained between Cup a 1 content of 12 C. arizonica pollen extracts and their IgE-binding activity. CONCLUSIONS: The described Cup a 1 ELISA is sensitive, specific and reproducible and can be used for the quantification of Cup a 1 in C. arizonica and other related pollen extracts. It also provides a reliable indication of the allergenic activity of the whole cypress pollen extract.     

Allergenic relevance of Cupressus arizonica pollen extract and biological characterization of the allergoid

BACKGROUND: Cupressaceae (cypress) pollens can cause pollinosis in winter. However, the lack of specific commercial extracts combined with the early pollination period of cypress trees make a precise diagnosis difficult. The need for a reliable and effective cypress extract for diagnostic and therapeutic purposes is increasingly felt. METHODS: Mixed or single Cupressus arizonica, lusitanica and sempervirens pollen extracts precipitated with ammonium sulfate (PPT) were compared by direct RAST, RAST inhibition and SDS-PAGE techniques. The major allergen of C. arizonica (Cup a 1), purified by anion exchange chromatography, was checked by immunoblotting experiments before chemical modification, in parallel with a C. arizonica extract, with potassium cyanate (KCNO) to obtain a monomeric allergoid. The allergoid extract was characterized for its biological, chemico-physical and immunological features by RAST inhibition, SDS-PAGE and ELISA assays. RESULTS: Direct RAST, RAST inhibition, and SDS-PAGE data indicated that the PPT C. arizonica pollen extract showed the most allergenic potential, and it can be considered representative of the Cupressus spp. Immunoblotting data confirmed Cup a 1 as a major allergen. RAST inhibition and ELISA showed that modified PPT C. arizonica extract had less IgE reactivity than the native, non-modified extract, while preserving the immunogenic capacity typical for an allergoid. Finally, the SDS-PAGE profile of Cup a 1 allergoid was similar to native Cup a 1 allergen, suggesting the modified C. arizonica extract shows the characteristics of a monomeric allergoid. CONCLUSIONS: The PPT C. arizonica pollen extract shows good in vitro diagnostic potential and its chemically modified form offers the features of a monomeric allergoid. It might therefore lend itself to the development of a product to be administered by the sublingual or oromucosal route for immunotherapy of individuals with cypress pollinosis.     

Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen

BACKGROUND: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. METHODS: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. RESULTS: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. CONCLUSION: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.     

Role of carbohydrate moieties in IgE binding to allergenic components of Cupressus arizonica pollen extract

BACKGROUND: A reduction of IgE immunoreactivity after periodate-treatment has been previously reported for various glycoprotein allergens. OBJECTIVE: The aim of this study was to investigate the role of glycan moiety of a C. arizonica extract in the binding of patients' IgE and to identify the carbohydrates possibly involved. METHODS: The reactivity of IgE with C. arizonica extract, before and after periodate-treatment, was evaluated by immunoblotting and ELISA inhibition. The specificity of carbohydrate-reactive IgE was evaluated by ELISA using unrelated glycoproteins with known sugar composition and structure, such as pineapple bromelain, honeybee venom phospholipase A2, and ovalbumin, before and after periodate treatment. RESULTS: When periodate-treated C. arizonica extract was probed after SDS-PAGE and immunoblotting with patients' IgE, no reactivity could be detected. Furthermore, a very poor inhibitory activity of the periodate-treated C. arizonica extract as compared with the untreated sample could be observed in the ELISA inhibition experiments performed using C. arizonica extract as antigen. When phospholipase A2 and bromelain were used as antigens in ELISA, they were recognized by patients' IgE, whereas ovalbumin was negative. Treatment of phospholipase A2 and bromelain with periodate completely abolishes the IgE reactivity. CONCLUSION: A large portion of the IgE reactivity of Cupressaceae-allergic subjects appears to be associated with sugar moieties of C. arizonica extract which appear to be shared by bromelain and phospholipase A2, thus suggesting that the IgE of patients reacting with such epitopes probably react with beta 1 --> 2 xylose, alpha 1 --> 3 fucose and/or alpha 1 --> 6 fucose.     

Identification of italian cypress (Cupressus sempervirens) pollen allergen Cup s 3 using homology and cross-reactivity.

BACKGROUND: The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe. OBJECTIVE: To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens. METHODS: Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3. RESULTS: Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen. CONCLUSION: Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.     

Effects of air pollution and other environmental factors on birch pollen allergens

To determine the effects of anthropogenic pollution on water-soluble proteins and specifically allergens in birch ( Betula pendula and B. pubescens ) pollen, we analyzed extracts of pollen from the pollution gradient around a factory complex (emitting sulfur oxides and heavy metals) by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE) and IgE immunoblotting. In addition, tree density-associated shading of the tree habitat, and quantity and quality of proteins and allergens in pollen of the two birch species were studied. The two studied birch species gave identical allergen profiles even though their protein profiles differed. Distance from the factory did not affect the amount of birch pollen major allergen. Bet v 1 (17 kDa), or of two other strong allergens (23 and 36 kDa). Trees growing in shaded places had significantly stronger responses to Bet v 1 and to the 23-kDa allergen than trees growing in open or half-open environments. Thus, we propose that combined heavy metal and sulfur dioxide pollution does not have an important effect on birch pollen allergens. Instead, other factors, e.g., shading and soil properties of the tree habitat, as well as the genetic background of the tree, may have a stronger influence on the quantity and relative composition of allergens.     

Identification of a polygalacturonase (Cup s 2) as the major CCD‐bearing allergen in Cupressus sempervirens pollen

As IgE glyco‐epitopes, also referred to as cross‐reactive carbohydrate determinants (CCDs), can share significant structural homologies between different plants, they are prone to extensive cross‐reactivity among allergen pollen extracts. Here, cypress pollen allergens, especially a polygalacturonase (PG), were further characterized using double one‐dimensional electrophoresis (D1‐DE). The presence of specific IgE directed against CCDs was investigated by bromelain IgE inhibition and concanavalin A binding assays using sera of cypress pollen‐sensitized patients. Our results showed that IgE reactivity to CCDs in Cupressus sempervirens pollen extracts is mainly related to bromelain‐type epitopes of a newly identified cypress PG. This glycoprotein has been further characterized through an immunoproteomic approach and officially indexed as Cup s 2 by the WHO/IUIS allergen nomenclature. Cup s 2 could thus be associated with the increased prevalence of IgE reactivity to cypress pollen extracts because of CCD interference.     

Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution

Background Grass pollen allergens are known to be present in the atmosphere in a range of particle sizes from whole pollen grains (approx. 20 to 55 μim in diameter) to smaller size fractions < 2.5 μ (fine particles, PM2.5). These latter particles are within the respirable range and include allergen-containing starch granules released from within the grains into the atmosphere when grass pollen ruptures in rainfall and are associated with epidemics of thunderstorm asthma during the grass pollen season. The question arises whether grass pollen allergens can interact with other sources of fine particles, particularly those present during episodes of air pollution. Objective We propose the hypothesis that free grass pollen allergen molecules, derived from dead or burst grains and dispersed in microdroplets of water in aerosols, can bind to fine particles in polluted air. Methods We used diesel exhaust carbon particles (DECP) derived from the exhaust of a stationary diesel engine, natural highly purified Lol p 1, immunogold labelling with specific monoclonal antibodies and a high voltage transmission electron -microscopic imaging technique Results DECP are visualized as small carbon spheres, each 30–60 nm in diameter, forming fractal aggregates about 1–2μ in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p I. binds to one defined class of fine particles, DECP. Conclusion DECP are in the respirable size range, can bind to the major grass pollen allergen Lol p I under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.     

Profilin is a cross-reactive allergen in pollen and vegetable foods.

Sera with IgE antibodies against grass pollen often contain IgE against vegetable foods. We investigated the role of the ubiquitous protein profilin in this cross-reactivity. Profilin was purified from Lolium perenne grass pollen by means of affinity purification with Sepharose-coupled poly(L-proline). This solid phase was also used as capturing agent for profilin from pollen and food extracts for application in a radioallergosorbent test. It was shown that profilin is an allergen in grass pollen and in a wide range of vegetable foods, like potato and celery. Within a grass-pollen-sensitive population, patients with IgE to vegetable foods have a high incidence of antibodies against profilin. IgE antibodies against grass pollen profilin were shown to be cross-reactive with respect to vegetable foods.     

Secretion of proinflammatory eicosanoid-like substances precedes allergen release from pollen grains in the initiation of allergic sensitization

It is commonly believed that allergic sensitization starts when an allergen contacts the surface of an antigen-presenting cell in mucosal or skin epithelia. Most studies dealing with this aspect use allergen extracts as stimulus. Under natural exposure conditions, however, the bioavailability of allergen depends on allergen liberation from internal binding sites within the allergen carrier, e.g. pollen grains. In comparing total protein and major allergen release from timothy grass (Phleum pratense L.) pollen freshly collected on rural meadows or near high-traffic roads, there was a striking difference between the pollen, with higher allergen release rates from rural meadow pollen grains. Thus, allergen release does not explain the higher prevalence rates of atopic sensitization and disease observed in many epidemiological studies in children exposed to automobile exhaust. Therefore, other possible effectors from pollen grains were investigated. Pollen grains incubated in protein- free buffer were found to secrete significant amounts of eicosanoid-like substances, namely leukotriene (LT) B(4)-like and prostaglandin E(2)-like substances, in a pH-, time- and temperature-dependent fashion. The highest values of eicosanoid secretion were found in birch, grass and mugwort pollen, while pine (Pinus sylvestris L.) pollen showed only marginal eicosanoid-like secretion. Additionally, the release of these substances was significantly higher from pollen which had been collected near roads with heavy traffic, indicating a stronger proinflammatory activity of these pollen grains. In order to investigate the effects of air pollutants, native pollen grains were exposed in a dose- and time-dependent fashion in a fluidized bed reactor to traffic-related pollutants, e.g. volatile organic compounds (toluene, m-xylene), leading again to a significant increase in the secretion of LTB(4)-like immunoreactivity, in contrast to exposure with sulfur dioxide. This finding opens a new dimension of understanding of the early events in allergic sensitization, indicating that proinflammatory effects of the allergen carrier, e.g. the pollen grain itself, can lead to activation of the mucosal membrane. These findings might help to also explain the higher prevalence rates of pollen allergy in areas with high automobile exhaust emissions. Furthermore, the allergenic 'potency' of various allergens has to be redefined at the allergen carrier level with regard to different stages of allergen and mediator release prior to the contact with the host's immune system.     

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.     

Residential outdoor air pollution and allergen sensitization in schoolchildren in Oslo, Norway

BACKGROUND: Epidemiological studies that have investigated the association between air pollution and atopy have found inconsistent results. Furthermore, often exposure to outdoor air pollution has had limited quality, and more individual exposure is needed. OBJECTIVE: To investigate the relations between early and lifetime exposure to residential outdoor air pollution and allergen sensitization in 9-10-year-old children in Oslo, Norway. METHODS: Sensitization to common allergens was measured by skin prick tests (SPTs), which were performed in 2244 children who had lived in Oslo since birth. Several definitions of positive SPT were used. Information on potential confounding variables was collected by a parental questionnaire. Exposure to outdoor air pollution was assessed by the EPISODE dispersion model, which calculates hourly concentrations of nitrogen dioxide (NO2), particulate matter (PM) with aerodynamic diameter <10 microm (PM10) and <2.5 microm (PM2.5), respectively. RESULTS: We found no associations between long-term air pollution exposure and sensitization to any allergen, any indoor or any pollen allergen. However, lifetime air pollution exposure was associated with sensitization to the house dust mite Dermatophagoides farinae. One interquartile increase of lifetime exposure to NO2, PM10 and PM2.5 was associated with 1.88 (adjusted odds ratio) (1.02, 3.47) [95% confidence interval (CI)], 1.61 (0.96, 2.72) and 1.46 (0.96, 2.22), respectively, for D. farinae. Lifetime exposure was also associated with sensitization to cat in a subpopulation. Both associations diminished after adjusting for a contextual socio-economic factor. CONCLUSION: Long-term exposure to traffic-related pollutants was generally not associated with allergen sensitization in 9-10-year-old Oslo children. However, lifetime exposure was associated with sensitization to D. farinae, and with sensitization to cat in a subpopulation, which may be explained by socio-economic confounding or multiple comparisons. The air pollution levels in Oslo may be too low to reveal associations with sensitization.