Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.

Concentrations of the major birch tree allergen Bet v 1 in pollen and respirable fine particles in the atmosphere.

BACKGROUND: Birch tree pollen allergens are an important cause of early spring hay fever and allergic asthma. Pollen counts provide a guide for individuals with birch pollen allergy. However, birch pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet birch pollen allergens are known to be associated with respirable particles present in the atmosphere. OBJECTIVE: We sought to determine the concentration of major allergen Bet v 1 in birch pollen and respirable particles in the atmosphere during the birch pollen season. METHODS: We used a two-site monoclonal antibody-based assay (ELISA) to quantitate Bet v 1 in pollen extracts and high-volume air sampler filters collecting particles larger and smaller than 7.2 microm. RESULTS: Bet v 1 (0.006 ng) is detectable per birch pollen grain, of which 0.004 ng is present in aqueous extracts (13.9% of soluble proteins). Atmospheric Bet v 1 concentrations are correlated with birch pollen counts. Heavy rainfall tended to wash out pollen and particles, indicated by a mean daily Bet v 1 concentration of 0.12 ng/m3 (20 pollen equivalents), but light rainfall produced a dramatic increase in allergen-loaded respirable particles with Bet v 1 concentrations of 1.2 ng/m3 (200 pollen equivalents). CONCLUSION: These results highlight the different environmental risk factors for hay fever and allergic asthma in patients sensitized to Bet v 1. Light rainfall causes an increase in respirable particles; hence, this is an important risk factor for asthma.

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 microns in diameter) to smaller size fractions < 2.5 microns (fine particles, PM25). 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 1-2 microns in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p 1, 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 1 under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.

Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen.

A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca2+-binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca2+-binding sites (EF hand domains). Depletion of Ca2+ with EGTA led to a loss of IgE-binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca2+-binding proteins.

Generative cells of Lilium longiflorum possess translatable mRNA and functional protein synthesis machinery.

Metabolic labelling with [35S]-methionine demonstrated that generative cells of Lilium longiflorum possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. The isolated generative cells synthesised ten proteins, of which six were unique to these specialised cells. Isolation of generative cells from pollen grains after [35S]-methionine labelling resulted in an identical protein profile, therefore the synthesis of these proteins was not due to isolation shock. Addition of cycloheximide, abolished TCA-precipitable counts, whilst actinomycin D had no qualitative effect on the observed protein profile, indicating active translation of pre-existing mRNAs by the generative cells.

Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen.

BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.

Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

Recombinant expression and epitope mapping of grass pollen allergens.

We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens. Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients. Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus. However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE. We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding. cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding. Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes. The allergenic epitopes were localised towards the C-terminal half of the molecule. Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7. At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens. These results aid in the design of diagnostics and in grass pollen immunotherapy.

Bcp1, a gene required for male fertility in Arabidopsis.

Male fertility in flowering plants is dependent on production of viable pollen grains within the anther. Genes expressed exclusively in the anther are likely to include those that control male fertility. On the basis of their tissue specificity, such genes have been isolated, yet in none of them has this function been demonstrated. Here we report that one such gene, Bcp1, is active in both diploid tapetum and haploid microspores and is required for pollen fertility. Perturbation of this gene in either tapetum or microspores prevents production of fertile pollen in transgenic Arabidopsis plants. When tapetum expression of this gene is perturbed, mature anthers contain dead shriveled pollen. On the other hand, when microspore expression is perturbed, anthers show 1:1 segregation of viable/aborted pollen. These findings identify a class of sporophytic/gametophytic genes controlling male fertility and, hence, reproduction in flowering plants.

Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

Isolation and characterization of group-I isoallergens from Bermuda grass pollen.

Two isoallergens of Cyn d I were isolated using preparative isoelectric focussing, and were designated Cyn d Ia and b. These isoallergens differ in their pI, molecular weight (Cyn d Ia, 32 kD and Cyn d Ib, 31 kD) and their NH2-terminal sequence. Four monoclonal antibodies (Mabs) were produced using Cyn d Ia as antigen. These Mabs recognized both Cyn d Ia and b. One of the Mabs recognized four different pI forms of Cyn d I on 2D gels. The Mabs also recognized cross-reactive epitopes on proteins from eight other grasses including rye grass, timothy grass, Kentucky bluegrass and Johnson grass.

Localization of the two major allergens in rye-grass pollen using specific monoclonal antibodies and quantitative analysis of immunogold labelling.

The intracellular localization of the two major allergens, Lol p I and Lol p IX, in rye-grass anthers was examined using monoclonal antibodies FMCA1 (specific for Lol p I) and FMCA7 (specific for Lol p IX) with immunocytochemical techniques and quantitative analysis. A newly developed anhydrous fixation technique in a mixture of glutaraldehyde, paraformaldehyde and 2,2-dimethoxypropane followed by embedding in LR Gold resin resulted in both improved infiltration of pollen grains compared with existing techniques and the localization of these water-soluble antigens in their original sites compared with diffusion artefacts following aqueous methods. After anhydrous fixation, Lol p I was predominantly located in the electron-opaque regions of the cytosol of the vegetative cell of the tricellular pollen grains (24 counts microns-2), whereas Lol p IX was detected mainly within starch granules (16 counts microns-2). For both Lol p I and Lol p IX, similar labelling was detected in the cells of the endothecium and middle layer (18 counts microns-2), but none was found in the tapetal cells or orbicules.

Immunological relationships among group I and group V allergens from grass pollen.

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from rye-grass pollen that demonstrates specific antigenic immunoreactivity.

We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572]. Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix. There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco.

The anther-specific cDNA clone Bcp1 from Brassica campestris is expressed in both the haploid pollen and diploid tapetum, as shown by in situ hybridization. We have isolated Bgp1, a genomic clone homologous to Bcp1. The coding region and extensive 5' flanking sequences of Bgp1 have been sequenced, and the coding region shows 88% identity with Bcp1. RNA gel blot analysis confirmed the expression of Bgp1-specific transcripts in B. campestris pollen. A 767 bp 5' DNA fragment was fused to the reporter gene beta-glucuronidase (gus) and introduced into both Arabidopsis thaliana and Nicotiana tabacum by transformation. This 5' fragment directed high-level expression in the pollen and tapetum of transgenic Arabidopsis. In transgenic tobacco however, the same construct was expressed only in pollen. A series of 5' deletion constructs has been created and used to transform A. thaliana to analyse the 5' region of Bgp1. The results indicate that Bgp1 expression in the tapetum and pollen of Arabidopsis requires the presence of different 5' DNA sequences.

Identification of canary grass (Phalaris aquatica) pollen allergens by immunoblotting: IgE and IgG antibody-binding studies.

The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen in the external environment of southern Australia. Seventeen allergenic fractions of canary grass pollen, ranging in mol. mass from 14 to 100 kDa, have been identified by immunoblotting, using IgE antibodies from sera of 24/30 grass-pollen-allergic subjects. The highest frequency of IgE binding (77%) was to a major 34-kDa fraction (tentatively designated Pha a I). This protein has been partially purified and identified as a group I allergen by immunodepletion experiments, with partially purified Lol p I (from rye-grass pollen), atopic serum, and Lol p I-specific MAb. In addition, microsequencing of the N-terminus of Pha a I showed an amino acid sequence identical to Lol p I. In a separate study, IgE binding to Western blots of Pha a I, Lol p I, and Cyn d I was investigated in 24 sera and found to occur in 19/24, 18/24, and 9/24, respectively. IgE binding to all three major allergens, and to both Pha a I and Lol p I, occurred in 8/24 sera. Our findings suggest that while the N-terminal sequence of Pha a I is identical to Lol p I, there may be specific allergenic epitopes exclusive to this allergen that are important for allergenicity in southern Australia.

Peptide mapping analysis of group I allergens of grass pollens.

Group I allergens, from eight different clinically important grass pollens of the Pooideae (rye-grass, canary, Kentucky bluegrass, orchard and timothy), Chloridoideae (Bermuda grass) and Panicoideae (Johnson grass, maize) were isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and subjected to limited partial proteolysis and cyanogen bromide cleavage. The generated digests were visualised on gels by silver staining. Replicate gels were blotted and screened with Lolp I-specific mAb FMC A1 and IgE binding to an allergic human serum pool, to determine the degree of antigenic and IgE-binding similarities, respectively. The highest antigenic and IgE-binding similarities were between orchard and rye-grass pollens, both in the same tribe Poeae, and among the closely related grass genera. No peptide mapping of major grass pollen allergens has previously been undertaken. This study proves peptide mapping is a powerful method for the preliminary identification and internal amino acid microsequencing of common antigenic and IgE-binding determinants of allergens, providing information that is useful in the development of reagents for the treatment of grass-pollen-associated allergies.

Mechanism of grass-pollen-induced asthma.

Many asthmatics are sensitive to rye-grass pollen, but pollen grains are too large to penetrate the lower airways. Our aim was to investigate the mechanism by which rye-grass pollen causes asthma. A major allergen of rye-grass pollen, Lol pIX, is located in intracellular starch granules within pollen grains. In-vitro tests showed that pollen grains are ruptured in rainwater by osmotic shock, each grain releasing about 700 starch granules into the environment. These granules are small enough to enter the airways (less than 3 microns in diameter). The starch granules were present in atmospheric samples taken during the pollen season, and showed a 50-fold increase in atmospheric concentration on days following rainfall. Isolated granules elicited IgE-mediated responses in asthmatic patients, and 4 patients with rainfall-associated asthma who underwent an inhalation challenge test had striking bronchial constriction after exposure to starch granules. Starch granules released from rye-grass pollen seem to be capable of causing asthma.

Isolation and developmental expression of Bcp1, an anther-specific cDNA clone in Brassica campestris.

Differential screening of a mature Brassica campestris pollen cDNA library has identified five cDNA clones that represent transcripts expressed exclusively, or at elevated levels, in pollen. We show here that the expression of one of these, clone Bcp1, is tissue specific and temporally regulated. The gene is activated during microspore development, as detected by in situ hybridization. Expression is enhanced at the time of pollen maturation and during pollen germination. In situ hybridization has also shown that Bcp 1 is activated in the tapetal cells in early anther development and continues to be expressed until tapetal dissolution. Homologous transcripts are present in pollen of other taxa of Brassicaceae including Arabidopsis, but not in pollen of any other families tested.