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Background Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease. Objective To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-β-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis. Methods Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays. Results Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively. Conclusions Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.

Background Among allergenic spices, mustard represents 1 of the most important in terms of allergenic potency and widespread use. An 11S globulin (Sin a 2) has been isolated from yellow mustard seeds and identified as a new major allergen. Objective Cloning and sequencing a cDNA encoding a Sin a 2 subunit and producing the allergen as a recombinant protein. Methods Sin a 2 subunit–encoding cDNA was amplified by polymerase chain reaction, cloned, and sequenced. The allergen was produced as a recombinant protein in Escherichia coli and used for enzyme-linked immunosorbent assay, immunoblotting, and inhibition experiments. Sera from patients with mustard allergy and an anti-polyhistidine monoclonal antibody were used. Results Sin a 2–specific cDNA comprises an open reading frame that encodes a protein of 510 amino acids, in which the first 23 residues correspond to the signal peptide. Sequence alignment with other allergenic 11S globulins showed levels of sequence identity ranging between 27% and 38%. Three peptides described as epitopes in Ara h 3 were moderately conserved in Sin a 2. Approximately 87% of the IgE binding to natural Sin a 2 was inhibited by the recombinant allergen using sera from patients with mustard allergy. Conclusion The recombinant 11S globulin from yellow mustard seeds produced in E coli retained the IgE-binding capability of the natural allergen. Clinical implications The availability of Sin a 2 sequence and its recombinant production could help to develop future therapeutic approaches and might well open new investigation lines to resolve whether 11S globulins are proteins implicated in cross-reactivity processes involving mustard seeds.

10 páginas, 6 figuras, 1 tabla -- PAGS nros. 4425-4434, Employing transgenic plants as alternative systems to the conventional Escherichia coli, Pichia pastoris or baculovirus hosts to produce recombinant allergens may offer the possibility of having available edible vaccines in the near future. In this study, two EF-hand-type Ca2+-binding allergens from olive pollen, Ole e 3 and Ole e 8, were produced in transgenic Arabidopsis thaliana plants. The corresponding cDNAs, under the control of the constitutive CaMV 35S promoter, were stably incorporated into the Arabidopsis genome and encoded recombinant proteins, AtOle e 3 and AtOle e 8, which exhibited the molecular properties (i.e. MS analyses and CD spectra) of their olive and/or E. coli counterparts. Calcium-binding assays, which were carried out to assess the biochemical activity of AtOle e 3 and AtOle e 8, gave positive results. In addition, their mobilities on SDS/PAGE were according to the conformational changes derived from their Ca2+-binding capability. The immunological behaviour of Arabidopsis-expressed proteins was equivalent to that of the natural- and/or E. coli-derived allergens, as shown by their ability to bind allergen-specific rabbit IgG antiserum and IgE from sensitized patients. These results indicate that transgenic plants constitute a valid alternative to obtain allergens with structural and immunological integrity not only for scaling up production, but also to develop new kind of vaccines for human utilization, This work was supported by grants SAF2002-02711 to RR and BIO2004–00628 to JS from the Ministerio de Ciencia y Tecnología (Spain) and CPE03-006-C6-1 to JS. from INIA. We thank Alejandro Baleriola for language revision

Background: Clustered severe adverse reactions to immunotherapy with olive pollen extracts have been occasionally reported in areas where olive trees are extensively grown. Allergic patients from these areas, in addition to the major olive pollen allergen Ole e 1, frequently recognize a recently described allergen, Ole e 9. Objective: We aimed to develop an immunoassay to measure Ole e 9 concentration and to study the variability of this allergen in olive pollen extracts. Methods: Monoclonal antibodies (mAb) to Ole e 9 were produced from mice immunized with the pure allergen. One of these mAbs was used to develop a sandwich ELISA with an anti-olive pollen extract rabbit serum as the tracer. Olive pollen batches from several suppliers were analyzed using this method. These batches were also analyzed for Ole e 1 content and biological activity. Results: A 10-fold variation between the extreme values was found for the biological activity of the batches analyzed. Ole e 1 concentration showed a 25-fold variation. Variability of Ole e 9 concentration was extremely high, up to 161 times. The ratio Ole e 1/Ole e 9 varied in a range from 0.6 to 390.4. Conclusion: The availability of a mAb-based ELISA for Ole e 9 made it possible for us to detect an important source of variability in olive pollen batches. This variability may be the cause of outbreaks of adverse reactions in the course of immunotherapy treatments, which have sometimes been observed among olive-allergic patients living in areas with very high levels of airborne olive pollen.

Background:Olive pollen is an important cause of allergy in Mediterranean countries. More than 50% of olive-pollen-allergic patients are sensitized against the 1,3-β-glucanase Ole e 9. To date, prophylactic and therapeutic treatments using purified recombinant allergens have not been studied in animal models of olive pollen allergy. Methods: BALB/c mice were immunized against Ole e 9 combining intraperitoneal injections of the allergen in Al(OH)3 with airway allergen challenges. A prophylactic treatment was performed by intranasal administration of a mixture of the recombinant fragments of the allergen prior to Ole e 9 sensitization. Serum levels of specific IgE, IgG1, IgG2a and IgG2b were measured by ELISA, and total IgE levels by sandwich ELISA. Bronchoalveolar lavage and lungs from mice were collected to study airway inflammation by light microscopy. Results: BALB/c mice immunized against Ole e 9 developed a predominantly Th2-like immune response with allergen-specific immunoglobulin induction and airway inflammation accompanied by the infiltration of eosinophils, lymphocytes, and neutrophils in the lung. Prophylactic treatment by intranasal application of the recombinant fragments of Ole e 9 avoids airway inflammation induced by sensitization with this allergen although the levels of Ole e 9-specific antibodies remain unchanged. Conclusions: Prophylactic intranasal treatment with recombinant fragments of Ole e 9 prevents airway inflammation triggered by immunization to this allergen in a murine model of type I allergy.

Background Although mustard seed allergy has been largely reported during the preceding 20 years, currently only 2 allergens, Sin a 1 and Bra j 1, have been identified. Objective To improve the characterization of the allergenic profile of yellow mustard seeds by reporting the identification and biochemical characterization of an 11S globulin as a new major allergen. Methods Mustard seed proteins were separated using size exclusion and ion-exchange chromatographic columns, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 2-dimensional polyacrylamide gel electrophoresis. Separation of different polypeptide chains was achieved by reverse-phase high-performance liquid chromatography. Mass spectrometry after tryptic digestion and Edman degradation were used to determine amino acid sequences of peptides. IgE binding assays were performed with 13 serum samples from mustard allergic patients in immunoblotting and enzyme-linked immunosorbent inhibition assays. Results A protein of 51 kDa was recognized as a major allergen by patients allergic to mustard and called Sin a 2 . The allergen was dissociated in 2 chains of 36 and 23 kDa, which also bound IgE. N-terminal end and internal amino acid sequences allowed identification of the new allergen as a seed storage 11S globulin belonging to the Cupin super family. Purified allergen was able to inhibit the IgE binding of sera from allergic patients to mustard seeds extract in up to 55% of the responses. Conclusions An 11S globulin storage protein has been isolated and identified as a novel major allergen of mustard seeds.

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.

Olive pollen allergy is a clinical disorder that affects around 20% of the population in Mediterranean areas. The major olive pollen allergen, Ole e 10, is involved in cross-reactivity phenomena and asthma induction in allergic patients, and, besides its clinical interest, Ole e 10 is the first member of a new family of plant proteins. Ole e 10-specific cDNA has been cloned in the plasmid pPICZαA and expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein has been purified in a two chromatographic-step procedure. N-Terminal sequencing, mass spectrometry, IgG, and IgE binding assays were employed to characterize the recombinant allergen. These analyses revealed that the product undergoes a proteolytic cleavage in the N-terminal end with the loss of the first six residues. Different strategies were used to solve this problem, such as changes in the fermentation conditions and the employment of protease-deficient yeast strains. Proteolytic cleavage was minimized and about 51% of rOle e 10 was obtained as a full-length protein. Moreover, a covalent modification was found in the N-terminal end of the full-length rOle e 10. Peptide mapping and mass spectrometry analyses pointed to the existence of a phosphorylation located in a serine residue of the N-terminal segment of rOle e 10 and it was confirmed after treatment of the sample with alkaline phosphatase. Finally, both full-length and truncated rOle e 10 retained most of the IgG- and IgE-binding capabilities of the natural protein isolated from the pollen.

Ole e 1 is a major allergen from olive pollen with an IgE-binding frequency around 80% among allergic population. Its diagnostic value has been demonstrated, and cross-reactive allergens have been found in ash, lilac and privet. We sought to determine IgE- and IgG-binding regions of Ole e 1. Ole e 1-specific polyclonal antiserum and sera from patients allergic to olive pollen were used to analyze IgG and IgE epitopes, respectively. Short overlapping synthetic peptides covering the complete sequence of Ole e 1 and point mutants of these peptides bound to membranes, as well as long recombinant peptides fused to GST were used in dot blot immunostaining and ELISA. Skin prick tests were performed on 14 allergic patients to assay the response in vivo to the recombinant fusion peptides. Residues at positions 8–11, 29, 32, 33, 55–59, 70, 107–110, 112, 120, 123, 141 of Ole e 1 sequence were found to be antigenically relevant in the IgG-binding. Although amino acids K137, L138, G139, Y141 and P142 were involved in the IgE-recognition of a pool of sera from allergic individuals, the response to the IgEs seemed to be preferentially conformational. IgE-binding capability of recombinant GST-fused peptide T114–M145 was demonstrated by in vivo (prick test) and in vitro (ELISA) experiments. Major IgG and IgE-binding regions of Ole e 1 have been identified being the C-terminal an immunodominant region. These data could help to design hypoallergenic forms of the allergen.

Background: The clinical characteristics in olive pollen allergy are dependent on the antigenic load, the allergens profile, and the genetic restrictions. Our objective was to determine specific response pattern in Ole e 2 and Ole e 10 sensitization at those levels. Methods: We studied 146 patients with seasonal rhinitis and/or asthma and positive prick test to Olea europaea pollen. IgE against Ole e 2 and Ole e 10 were detected by skin prick test and ELISA. HLA-DRB1 and HLA-DQB1 loci were typed by polymerase chain reaction sequence-specific primers method. Results: A total of 102 (69.9%) and 79 (54.0%) patients showed significant IgE antibody response against Ole e 2 and Ole e 10, respectively. There was a significant association between Ole e 2 (OR 2.2, P = 0.04) and Ole e 10 reactivities (OR 2.8, P = 0.007) with asthma. In addition, total and specific IgE antibody levels significantly correlated with asthma (P

Chenopod pollen is one of the major sources of allergens in some locations in the US, southern Europe and desert countries, and pollen profilin (Che a 2) is a major allergen. Recombinant Che a 2 (rChe a 2) has been produced in Escherichia coil cells with a final yield of 25 mg/l of cell culture. The expressed protein was isolated and structurally characterized by means of mass spectrometry, Edman degradation and circular dichroism. rChe a 2 displayed a molecular mass of 13 959 Da, which agrees with that of the amino acid sequence. The N-terminal amino acid sequence indicated the correct processing of the recombinant product. The immunological analysis of rChe a 2 showed IgG- and IgE-binding capabilities equivalent to those of its natural counterpart, Che a 2, isolated from the pollen. Inhibition experiments showed high cross-reactivity degrees with different allergenic sources. Inhibition degrees of >95% and >80% were obtained for chenopod profilin and, respectively, latex and pollen extracts, whereas 10-95% of inhibition was observed for different plant-derived foods. Due to its close relation to other allergenic profilins from pollens, plant-derived foods and latex, rChe a 2 could be a useful tool in clinical trials to detect profilin-allergic patients and perhaps, depending on its clinical relevance, in specific immunotherapy of these hypersensitive individuals.

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

Ole e 1 is the most prevalent allergen of olive (Olea europaea) pollen. The suitability of poly( d , l -lactide-co-glycolide) (PLG) microparticles as a vaccine delivery system for Ole e 1 in therapy against olive allergy has been evaluated. BALB/c mice were immunized intraperitoneally with Ole e 1 in PLG microparticles. Encapsulated Ole e 1 induced specific and long-lasting antibodies that were predominantly of the IgG2a isotype. Splenic cells from immunized mice secreted in vitro IFN-γ, but not IL-4, after stimulation with Ole e 1. These results suggest that Ole e 1-containing PLG microparticles elicit a specific Th1-type immune response and may become a promising concept for allergy vaccine.

Several allergenic proteins, such as the 1,3-beta-glucanases, have been associated with plant defence responses. Ole e 9 (46 kDa) is a 1,3-beta-glucanase and major allergen from olive pollen, which is a principal cause of allergy in Mediterranean countries. Its C-terminal segment (101 amino acid residues) has been produced as a recombinant polypeptide in the yeast Pichia pastoris. The cDNA encoding the polypeptide was inserted into the plasmid vector pPICZalpha-A and overexpressed in KM71 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reversed-phase HPLC. Edman degradation, MS and CD were used to determine molecular properties of the recombinant polypeptide, which exhibited 16% alpha-helix and 30% beta-sheet as regular elements of secondary structure. Disulphide bridges of the molecule were determined at positions Cys-14-Cys-76, Cys-33-Cys-94 and Cys-39-Cys-48. The high IgE-binding capability of the recombinant C-terminal segment of Ole e 9 against sera from Ole e 9-sensitive individuals, which was determined by immunoblotting and ELISA inhibition, supported the proper folding of the polypeptide and the maintenance of antigenic properties that it exhibits as a part of the whole allergen. These data indicated that this portion of Ole e 9 constitutes an independent domain, which could be used to study its three-dimensional structure and function, as well as for clinical purposes such as diagnosis and specific immunotherapy. Since it shows sequence similarity with portions of 1,3-beta-glucanases from plant tissues and the Gas/Phr/Epd protein families involved in yeast morphogenesis, we suggest that this domain could play an equivalent functional role within these enzymes.

Ole e 1 is the main allergen of olive pollen, which is a major cause of pollinosis in countries of the Mediterranean area. Nine Ole e 1-specific murine monoclonal antibodies (mAbs), as well as two Ole e 1-isoforms and two Ole e 1-like allergens from lilac and privet, all of them obtained in Pichia pastoris by recombinant methods, have been used as tools to determine the role of the three-dimensional (3D)-folding, the glycan component and several point changes of the amino acid sequence in the binding of murine IgG mAbs and human IgE to the olive allergen. Seven mAb families (F1-F7) were established, two of which (F1 and F2) recognize continuous epitopes. The carbohydrate moiety of Ole e 1 was involved in the binding to F2 and F4, whereas F3 and F7 were able to bind to all Ole e 1 variants. The remaining families of IgG murine antibodies exhibited different affinities for the antigens assayed in a native or denatured conformation. Although the binding of human IgE to Ole e 1 was not affected by heat treatment, it was shown to be strongly dependent on the integrity of the disulfide bridges and was partially inhibited by F3-F7 IgG antibodies, their individual values ranging from 12 to 31% and reaching 53% with their mixture. The IgE from sera of olive-allergic patients showed a significant diversity of binding capacity to the members of the Ole e 1-like family due to the microheterogeneity of their polypeptide sequences, in spite of their highly conserved primary structures. Whereas one of the isoforms of Ole e 1 exhibits a highly similar behavior to the natural form, being a putative molecule for diagnostic purposes, other ones can be considered as hypoallergenic variants of this allergen and, thus, potential candidates to be used in immunotherapy.

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.

Background: Olive pollen is one of the main causes of allergy in Mediterranean countries, where it is widely distributed. One inconvenience in studying new immunotherapies for olive pollen allergy is the lack of suitable animal models. The aim of this study was to develop a murine model of IgE sensitization to Ole e 1, the major allergen of olive pollen, which mimics the immunological features of olive pollinosis in humans and to investigate the in vivo antigenicity of the recombinant form of the allergen. Methods: BALB/c mice were sensitized by intraperitoneal administration of natural Ole e 1 (nOle e 1) and recombinant Ole e 1 (rOle e 1) in Al(OH)3, respectively. Serum levels of specific IgE, IgG1 and IgG2a, cytokine production and the proliferative response of splenocytes after in vitro stimulation with nOle e 1 were analyzed by ELISA and flow cytometry. The binding capacity of rOle e 1-specific IgG1 was examined by ELISA and immunoblotting. Results: Sensitization with nOle e 1 or rOle e 1 induced high levels of specific IgE and IgG1 versus low IgG2a antibody levels. Splenocytes from sensitized mice exhibited a proliferative response to nOle e 1. In vitro stimulated splenic cells from nOle e 1-primed mice produced IL-4 and low or nondetectable levels of IFN-γ. Specific IgE and IgG1 antibodies of immunized mice bound to the same Ole e 1 isoforms and showed a similar degree of cross-reactivity as observed for human IgE. Mouse specific nOle e 1 IgG1 was strongly inhibited by IgE from allergic patients. The IgG1 antibodies elicited by rOle e 1 reacted with both the recombinant and natural forms of the allergen. Conclusions: A murine model of Ole e 1 sensitization has been established. rOle e 1 shows similar allergenicity and antigenicity to its natural form. This model should provide a useful tool for evaluating antigenic molecules and exploring new therapeutic approaches in order to treat IgE-mediated olive pollinosis.

Pollinosis to Chenopodium album has been reported, but no data are available on its allergenic proteins.An allergen from C. album pollen has been isolated by means of gel permeation and reverse-phase high-performance liquid chromatography. Molecular characterization was achieved by concanavalin A reaction, mass spectrometry, Edman degradation and cDNA sequence. Antigenic analyses were performed by immunoblotting, ELISA, and ELISA inhibition, using sera from allergic patients, two Ole e 1-specific monoclonal antibodies and an Ole e 1-specific polyclonal antiserum.The isolated allergen, Che a 1, is a glycoprotein of molecular mass 17.088 kD and 143 amino acid residues, whose sequence exhibits 27-45% identity with known members of the Ole e 1-like protein family. 77% of sera from patients allergic to chenopod pollen were reactive to Che a 1. No correlation was found between the IgE reactivities to Che a 1 and Ole e 1, the major allergens from olive pollen, and both allergens display low, although detectable, IgE and IgG cross-reactivities.Che a 1, a relevant allergen from chenopod pollen, is structurally related to the Ole e 1-like protein family, but exhibits significant differences on its polypeptide sequence that could explain its different antigenic behavior and limited cross-reactivity.

Ole e 1, the main allergen of the olive pollen, was entrapped in poly ( dl -lactide glycolide) microparticles by a solvent evaporation double emulsion technique. The physical properties of the microparticles, such as particle size, surface morphology, allergen entrapment rate and antigen release pattern were investigated. Microparticles with a spherical morphology displayed a size range of less than 2 μm in diameter and antigen loading up to 60–70% (w/w). SDS-PAGE and immunoblotting of the released Ole e 1 confirmed that the molecular integrity and the antigenic properties of Ole e 1 remained unaltered by the encapsulation process or polymer degradation. This finding suggests that microparticles displaying small particle sizes, rapid antigen release and a high allergen/polymer ratio may be a suitable delivery system for antigen in hyposensitization therapy against allergy.

Background: Sera of patients allergic to olive (Olea europaea) pollen were used to analyze the IgE cross-reactivity between olive-pollen extract and other pollens obtained from phylogenetically unrelated species. Methods: We used IgE immunostaining of pollen extracts blotted to nitrocellulose membranes after SDS–PAGE and inhibition analysis of this binding. Results: A high inhibition of the IgE binding on olive-pollen extract was exhibited by birch, mugwort, pine, and cypress pollens, suggesting that these extracts contain proteins which share common epitopes and thus can be recognized by olive-allergic sera. IgE binding to Gramineae pollen extracts was not inhibited by olive-pollen extract, indicating a primary sensitization of the patients to these species. From the inhibition assays, the presence of an allergen of 45 kDa in the olive pollen, which has no homologous counterparts in other allergenic species, has been inferred. Conclusions: Olive pollen contains allergens which cross-react with pollens from unrelated species, a fact that could simplify the diagnosis and treatment of pollinosis.

A novel allergenic member of the family of Ca 2+ -binding proteins has been cloned from olive tree pollen. The isolated DNA codes for a protein of 171 amino acid residues, which displays four EF-hand sequence motifs. The encoded protein was overproduced in Escherichia coli and purified. The protein (18 795 Da), which binds Ca 2+ and IgE antibodies from patients allergic to olive pollen, undergoes Ca 2+ -dependent conformational changes. It is retained on a phenyl-Sepharose column, which indicates the existence of regulatory EF-hand domains. This fact suggests its involvement in Ca 2+ -dependent signal transduction events of the pollen grain. This allergen could be considered as a member of a new subfamily of EF-hand Ca 2+ -binding proteins since it displays a low amino acid sequence similarity with the so far known proteins.

Background: Olive pollen is a major cause of inhalant allergy in countries around the Mediterranean sea. The major allergen of olive pollen is Ole e 1. Measurement of the major allergen content of allergen products for diagnosis and therapy is becoming an essential element of standardization protocols. This study aimed at the development of a monoclonal antibody (mAb) sandwich ELISA for Ole e 1. Methods: Balb/c mice immunized with Ole e 1 were used for the production of mAbs. Screening of mice and hybridomas was performed in a RIA with radiolabeled purified Ole e 1. Purified mAbs were used as catching and/or (biotinylated) detecting antibodies in sandwich ELISA. Results: Four mAbs (IgG1κ) directed to nonoverlapping epitopes on Ole e 1 were obtained: 1A12, 5C1, 10A12 and 3H8. Both 1A12 and 10A12 were successfully used for affinity purification of Ole e 1 from olive pollen extract. Two sandwich ELISAs were developed, with 1A12 and 10A12 as catching, and 5C1 and 3H8 as detecting antibodies, respectively. Both catching and detecting antibodies were used in similar concentrations, ranging from 60 to 100 ng/well. For both ELISAs, the sensitivity was approximately 1 ng/ml of Ole e 1. The measuring range was from 1 to 25 ng/ml. No significant differences were observed, when the performance of both ELISAs in standardization of olive pollen extracts was compared. Conclusions: Two sensitive sandwich ELISAs for the major olive pollen allergen Ole e 1 were developed. They will prove to be useful tools in allergen standardization protocols.

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.

9 páginas, 5 figuras., The distribution of Ole e I (the major olive pollen allergen) and its transcripts was investigated in the anther from premeiotic stages until the dehiscent pollen stage. Crude protein extracts were analyzed by immunoblotting and probed with a monoclonal antibody to Ole e I. The protein, with three variants, was found to accumulate from the early microspore stage onwards. In addition to the previously reported localization of the protein, Ole e I has been immunolocalized for the first time within the pollen wall and in the tapetum. Reverse transcription-polymerase chain reaction analysis using specific oligonucleotides and RNA extracted from whole anthers revealed that the Ole e I gene is expressed from the late tetrad stage onwards. No expression was found in control tissues such as petals, roots or leaves. Light microscopy in situ hybridization on developing flower buds and dehiscent pollen confirmed the transcripts to be present in both the microspores and the sporophytic tissue (tapetum). Labeling was found primarily in the tapetum, reaching the highest concentration in the cytoplasm of the developing and mature pollen, once tapetum started to degenerate. In situ hybridization at the transmission electron microscope level showed the transcripts to accumulate on ribosomes of the rough endoplasmic reticulum. These studies, together with others carried out previously by us, indicated that both synthesis and storage of Ole e I take place in the endoplasmic reticulum, coincidentally with the conspicuous changes suffered by this membrane system during pollen development. This process is most likely controlled at the transcriptional level. The localization of the protein in the pollen ectexine bring new insights into the function of the allergen, which are discussed., This work was supported by the European Commission, Contract FMBI-CT95-0470 and by DGES (Spanish MEC) Project PB95-0080.

Summary Background Seed proteins have been found to cause hypersensitivity by ingestion or inhalation. Rapeseed fiour was responsible for allergic symptoms in a patient, who develops into allergy to mustard spice. Objective To determine the presence of allergenic proteins in rapeseed fiour, and analyse the structure of the main component and its crossreactivity with the mustard allergeti. Methods SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequent immunoblotting with a serum from a rapeseed allergic patient were performed to detect IgE-binding proteins. Proteolytic digestiotis and high performance liquid chromatography were used to obtain the peptides from the allergenic BnIII napin from rapeseed flour. Automatic Edman degradations were carried out to determine their amino acid sequences, which were compared with other sequences in nucleotide and amino acid sequence databases. Crossreactivity assays were carried out by ELISA inhibition using sera from a rapeseed allergic patient and from patients allergic to mustard. Results The 2S albumins of rapeseed were recognized by the serum from a patient allergic to this seed. The most abundant isoform of the allergenic napins, Bnlll, was used for structural and immunological analysis. The protein consists of two different chains of 9.5 and 4,5 kDa. Their complete amino acid sequences were determined. The protein exhibited structural relationships with other napin-like storage proteins from seeds. IgE and IgG crossreactivity between rapeseed and mustard allergens was also demonstrated. Considering the structural and immunological data, certain polypeptide regions are suggested to be involved in the allergenicity of these proteins. Conclusions Rapeseed contains 2S storage proteins which may cause allergy in hypersensitive individuals. These proteins exhibit great sequence similarity with 2S albumins from different seeds. Crossreactivity between mustard and rapeseed flours can be explained by sequetice homology.

U E S D A Y 839 Evaluation of in Vivo and in Vitro Responses to Nole e1 in Systemic and Local Allergic Rhinitis Paloma Campo, MD, PhD, Carmen M. Rondon Segovia, MD, Mayte Villalba, PhD, Cristobalina Mayorga, PhD, M. J. Sanchez-Quintero, BS, Oscar Palomares, PhD, Luisa Galindo, RN, Lidia Melendez, Lab. Tech., Jose Luis Rodriguez-Bada, Lab. Tech., Miguel Blanca, MD, PhD; Allergy Service Carlos Haya Hospital, Malaga, Spain, Carlos Haya Hospital, Malaga, Spain, Department of Biochemistry and Molecular Biology, Faculty of Chemistry, Madrid, Spain, Research Laboratory, Carlos Haya Hospital, Malaga, Spain, Research Laboratory, Hospital Carlos Haya, Malaga, Spain. RATIONALE: The aim of this study is evaluating in vivo and in vitro responses to purified allergen nOle e1 in local allergic rhinitis (LAR) compared to systemic allergic rhinitis (SAR). METHODS: nOle e1 was purified from olive tree pollen by RP-HPLC chromatography. Subjects with positive skin prick test (SPT), specific IgE (sIgE) to olive >0.35 kU/L, and positive nasal challenge with commercial olive pollen extract were recruited (SAR subjects). Also, subjects with negative SPT, sIgE to olive _ 2 SI). Ethical committee approved the study. RESULTS: Six SAR subjects and 4 LAR subjects were selected for the study. SPTwith nOle e1 was positive in 100% SAR subjects and all LAR subjects had negative skin tests. All SAR and LAR subjects had positive responses to nOle e1 nasal challenge. Of SAR subjects, 100% of them had positive BATwith whole commercial olive extract and 75% with nOle e1. LAR subjects showed a 75% positive BAT responses to whole commercial olive extract but no positive responses to nOle e1. CONCLUSIONS: Subjects with LAR showed positive responses to nOle e1 in vivo. In vitro responses to whole olive pollen extract were detected in a significant percentage of subjects with LAR.

An allergen has been isolated from a saline extract of olive tree (Olea europaea) pollen. The protein consists of a single polypeptide chain of 9.2-kDa, as determined by mass spectrometry. It contains neither tryptophan nor tyrosine residues, and displays an acidic isoelectric point. The secondary structure of the protein, estimated from the analysis of the circular-dichroism spectrum in the peptide-bond region, is composed of 52% alpha-helix, 10% beta-strand, 29% beta-turn and 9% non-regular conformation. The N-terminal end of the protein is blocked. Amino-acid-sequence data have been obtained from peptides produced by CNBr treatment of the native allergen. A partial sequence of 36 amino acids has thus been elucidated. The protein exhibits sequence similarity with pollen allergens from Brassica species and contains a Ca(2+)-binding motif. The isolated protein displays IgE-binding activity against sera of patients allergic to olive-tree pollen. It has been named Ole e 3, according to the recommendations of the IUIS Nomenclature Committee. IgG ELISA inhibition assays with polyclonal antibodies specific for Ole e 3 reveal the presence of proteins similar to Ole e 3 in the pollen from non-related plant species, which may explain allergic cross-reactivity processes.

Ole e 1, the major allergen from olive pollen, is a glycoprotein containing a single Asn-linked glycan moiety. Rabbit antiserum against this protein has been obtained; and its immunologic cross-reactivities in Western blotting with ascorbate oxidase, horseradish peroxidase, bromelain, ovalbumin, and honeybee venom phospholipase A 2 have been studied. Ascorbate oxidase, peroxidase, and bromelain are recognized by the Ole e 1 antiserum. When these three proteins are deglycosylated by periodate treatment, such an immunologic reaction does not occur. The relative affinities of these proteins have been analyzed by direct and inhibition ELISA experiments. A commercially available antibody against horseradish peroxidase has also been considered in these studies. This antibody reacts with Ole e 1 but not with the periodate-deglycosylated allergen. Horseradish peroxidase, bromelain, and ascorbate oxidase are recognized by the IgE of sera from patients who are hypersensitive to olive tree pollen. This binding is also abolished by periodate treatment. The results are interpreted in terms of the presence of an epitope in the carbohydrate moiety of Ole e 1, which would contain a xylose involved in recognition by both IgE and IgG antibodies. (J ALLERGY CLIN IMMUNOL 1996;97:1264-71.)

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.

Two napins (nIa and nIb), isolated from Brassica napus (rapeseed) seeds, have been sequenced. The two proteins show the common structural pattern of the 2 S albumins, since they are composed of two disulfide-linked chains of different size, yet they exhibit an atypical low molecular weight (12.5 kDa vs. 14.5 kDa of the major napins). High sequence similarity has been found between these 2 proteins, but only 54% similarity can be estimated from their comparison with the 14.5 kDa major napins. Thus, nIa and nIb are considered representatives of a new distinct group of rapeseed napins since all the previously known napins exhibit 95% sequence similarity. Unexpectedly, the similarity increases when compared with the 2 S proteins from other species.

Allergen measurements are used extensively in the formulation of allergy diagnostics and vaccines, yet no purified international allergen standards are available for calibration purposes. The aims of the European Union CREATE project were to develop international standards with verifiable allergen content. Purified natural and recombinant allergens were analyzed by means of SDS-PAGE, mass spectrometry, circular dichroism spectra, and small-angle x-ray scattering. IgE reactivity was assessed by means of direct RAST, RAST inhibition, immunoblotting, and basophil histamine release with sera from 961 allergic patients. Three recombinant allergens, rBet v 1, rPhl p 5a, and rDer p 2, were structurally indistinguishable from their natural counterparts and showed excellent IgE reactivity suitable for use as certified reference materials. A second tier of allergens (rPhl p 5b, rOle e1, rDer p 1, rDer f 1, and rDer f 2) was identified that could provide suitable candidates for certified reference materials with minor improvements to the recombinant proteins. Only rPhl p 1 was considered unsuitable as a reference material. Quantitative ELISAs were identified that accurately measured each allergen, except for rPhl p 1. The CREATE project has provided a major step forward in allergen standardization and provides a model for the development of a comprehensive panel of international reference preparations that will harmonize allergen measurements worldwide.

A method to isolate the major allergen from olive pollen ( Ole e I) in high yeild is described. The allergenic fraction has been separated into 3 subfractions by reversephase HPLC. All these fractions were reactive to allergic sera from olive-sensitized patients, giving similar responses. No significant differences were observed between the amino acid compositions of these three proteins. The amino acid sequence of the first 27 amino acid residues from the N-terminal end is given. No homologies have been detected between Ole e I and other known allergens obtained from pollen.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus . It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 μM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 μM myo -inositol, glucosamine, galactose, myo -inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylaton of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 × 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 × 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.

6 pages, 5 figures., When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed., This work was supported by grants from CAICYT, FISS and Europharma.

We have obtained a rabbit antiserum that specifically immunoprecipitates the 50K and 25K proteins of rat liver phospholipid methyltransferase. Exposure of intact rat hepatocytes preincubated with [32P]phosphate to glucagon induces a time-dependent phosphorylation of the 50K protein of phospholipid methyltransferase. The incorporation of 32P into the 50K protein was only on phosphoserine. These data support the concept that the activation of rat liver phospholipid methyltransferase by glucagon is mediated by phosphorylation of the enzyme.

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. S-Adenosylmethionine/S-adenosylhomocysteine ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio S-adenosylmethionine/S-adenosylhomocysteine can modulate phosphorylation of a specific protein.

Background: Olive tree ( Olea europaea ) pollen is an important cause of pollinosis in countries of the Mediterranean area and California. Objective: The aim of this study was to identify and purify a new allergen of olive tree pollen. Methods: Detection of a pollen allergen was done with individual allergic sera by immunoblotting and ELISA tests. Two allergenic fractions were isolated from olive pollen extract by using gel filtration and reverse-phase HPLC. Molecular characterization was achieved by acid hydrolysis and amino acid analysis, as well as by mass spectrometry. Sequencing of the N-terminal end of the allergen was carried out by Edman degradation of the polypeptide chain. Allergenic characterization was performed with sera from subjects with olive allergy by means of ELISA and immunoblotting after SDS-PAGE. Results: The new allergen Ole e 7 exhibits a high degree of polymorphism. Its molecular mass is in the range of 9875 d to 10,297 d. Twenty-one amino acid residues from the N-terminal end of 2 isoforms of the allergen have been sequenced revealing no homology with proteins contained in database banks. Ole e 7 has an average frequency of about 47% in patients with olive allergy. The strategy of purification of Ole e 7 can be useful on the isolation of new allergens. Conclusions: A new olive pollen allergen of clinical significance has been purified and characterized, contributing to the study of the complete allergogram of the olive tree pollen. (J Allergy Clin Immunol 1999;104:797-802.)