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Background Ingestion of food allergens present in maternal milk during breastfeeding has been hypothesized as a gateway to sensitization to food; however, this process could develop during pregnancy, as the maternal–fetal interface develops a Th2- and Treg-mediated environment to protect the fetus. We hypothesized that in these surroundings, unborn children are exposed to food allergens contained in the mother's diet, possibly giving rise to first sensitization. Methods The presence of allergens in utero was studied by analyzing amniotic fluid (AF) samples in two different stages of pregnancy: at 15–20 weeks and after delivery at term. An antibody microarray was developed to test for the most common food allergens. The array detects the presence of ten allergens from milk, fruit, egg, fish, nuts, and wheat. Results AF from 20 pregnant women was collected: eight after delivery at term and 12 from women who underwent diagnostic amniocentesis between weeks 15 and 20 of gestation. The presence of allergens was detected in all samples. Samples from amniocentesis had a higher allergen concentration than samples after delivery at term. Conclusions We demonstrated the presence of intact major food allergens in AF samples. This early contact could explain subsequent sensitization to foods never eaten before.

Background: Peanut allergens are common responsible of food allergy. Analyses of sensitization patterns, relationships with other allergens, clinical symptoms and variation with age are needed. We studied sensitization to Ara h 2, Ara h 9 and Pru p 3 in a population of children/adolescents allergic to peanut, and the relation with peach and pollen. Methods: Patients allergic to peanut were classified into two groups: A) allergic to peanut, and B) allergic to peach and peanut. The IgE response was measured to Ara h 2, Ara h 9 and Pru p 3. Results: Of 456 subjects evaluated, 28% were allergic to peanut. From this group 69% were sensitized to pollen. Urticaria was the most frequent entity followed by anaphylaxis and OAS. Fifty-nine percent had Ara h 2 and/or Ara h 9 specific IgE. Over half reported symptoms with peanut alone (Group A); 46% to peanut and peach (Group B). We observed significant differences in sex, age, onset of symptoms and sensitization to Artemisia. IgE response to Ara h 2 was more frequent in Group A; Ara h 9 and Pru p 3 in Group B. We observed a decrease in sensitization to Ara h 2 and an increase to Ara h 9 and Pru p 3 with increasing age. Conclusion: Peanut allergy is frequent in subjects with allergy to plant-foods, with Ara h 2 and Ara h 9 being two important allergens. In younger patients Ara h 2 predominates over Ara h 9. The reverse was observed in older patients.

Summary Reasons for performing study: Lidocaine and ketamine are administered to horses as a constant rate infusion (CRI) during inhalation anaesthesia to reduce anaesthetic requirements. Morphine decreases the minimum alveolar concentration (MAC) in some domestic animals; when administered as a CRI in horses, morphine does not promote haemodynamic and ventilatory changes and exerts a positive effect on recovery. Isoflurane-sparing effect of lidocaine, ketamine and morphine coadministration has been evaluated in small animals but not in horses. Objectives: To determine the reduction in isoflurane MAC produced by a CRI of lidocaine and ketamine, with or without morphine. Hypothesis: Addition of morphine to a lidocaine-ketamine infusion reduces isoflurane requirement and morphine does not impair the anaesthetic recovery of horses. Methods: Six healthy adult horses were anaesthetised 3 times with xylazine (1.1 mg/kg bwt i.v.), ketamine (3 mg/kg bwt i.v.) and isoflurane and received a CRI of lidocaine-ketamine (LK), morphine-lidocaine-ketamine (MLK) or saline (CTL). The loading doses of morphine and lidocaine were 0.15 mg/kg bwt i.v and 2 mg/kg bwt i.v. followed by a CRI at 0.1 mg/kg bwt/h and 3 mg/kg bwt/h, respectively. Ketamine was given as a CRI at 3 mg/kg bwt/h. Changes in MAC characterised the anaesthetic-sparing effect of the drug infusions under study and quality of recovery was assessed using a scoring system. Results: Mean isoflurane MAC (mean ± s.d.) in the CTL, LK and MLK groups was 1.25 ± 0.14%, 0.64 ± 0.20% and 0.59 ± 0.14%, respectively, with MAC reduction in the LK and MLK groups being 49 and 53% (P

Summary Background Patients allergic to mustard are frequently sensitized to peach. Objective To identify and analyse new yellow mustard allergens that could be involved in IgE cross-reactivity. Methods Sera from mustard-allergic patients with symptoms to peach were studied. Mass spectrometry analyses provided sequences of IgE-reactive proteins. cDNAs encoding Sin a 3 and Sin a 4 were amplified by polymerase chain reaction, cloned and sequenced. The recombinant allergens were obtained in Pichia pastoris and Escherichia coli, respectively, and used for ELISA, immunoblotting and inhibition experiments. Sequence alignment was used to identify common IgE epitopes. Results Sin a 3- and Sin a 4-specific cDNAs encode for mature proteins of 92 and 131 amino acids that belong to nsLTP and profilin protein families, respectively. Sin a 3 and Sin a 4 showed 54% and 80% identity with allergenic nsLTP from peach and profilin from melon, respectively. Both recombinant allergens were IgE-reactive in ELISA and immunoblotting. Peach pulp and peel, and melon extracts nearly abolished the IgE binding to recombinant Sin a 3 or recombinant Sin a 4 in immunoblotting. Conclusion Sin a 3 (nsLTP) and Sin a 4 (profilin) were identified as new mustard allergens and showed IgE cross-reactivity with fruits such as peach or melon, respectively. The knowledge of these two allergens will contribute towards better understand with cross-reactivity between mustard and other plant food allergens, and their availability will provide physicians with useful tools for molecular diagnosis.

Summary Background Biodegradable microparticles, in particular poly(lactide-co-glycolide) (PLGA), have been shown as potential delivery vehicles for intranasal (i.n.) vaccines in animal models. Objectives To evaluate whether i.n. administration of PLGA microparticles containing a peptide with the major T cell epitope of Ole e 1, the main allergen of olive pollen, prevented mice from allergic sensitization to the whole protein. Methods Peptide-PLGA microparticles were prepared by a solvent evaporation double emulsion method. Microparticles in a size range of 0.8 μm were evaluated for peptide loading and in vitro antigen release. Stability and immunogenicity of the entrapped peptide were retained, as determined by dot blot and ELISA inhibition. BALB/c mice were intranasally treated with peptide-PLGA microparticles for 3 consecutive days, 1 week before sensitization/challenge to Ole e 1. Blood, lungs and spleen were collected and analysed for immune response. Biodistribution of microparticles was investigated using confocal microscopy. Results I.n. pretreatment of BALB/c mice with peptide-PLGA microparticles before sensitization to Ole e 1 led to a significant inhibition of serum allergen-specific IgE and IgG1 antibody levels, but a marked increase of specific IgG2a antibodies as compared with sham-pretreated mice. Moreover, IL-5 and IL-10 levels in spleen cell cultures were suppressed in peptide-PLGA pretreated mice. The airway histopathologic parameters associated with inflammation were significantly suppressed by the pretreatment. Conclusion: These results demonstrate that i.n. immunization with peptide T-PLGA microparticles is effective in preventing subsequent allergic sensitization to Ole e 1. Our data indicate that peptide-PLGA microparticles may be promising candidates for the design of nasal vaccines against allergic diseases in humans.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Summary Background The C-terminal region of Ole e 1, a major allergen from olive pollen, is a dominant IgE-reactive site and offers a target for site-directed mutagenesis to produce variants with reduced IgE-binding capability. Objective To evaluate in vitro and in vivo the immunogenic properties of three engineered derivatives of Ole e 1. Methods One point (Y141A) and two deletion (135Δ10 and 140Δ5) mutants were generated by site-directed mutagenesis of Ole e 1-specific cDNA and produced in Pichia pastoris. Ole e 1 mutants were analysed for IgE reactivity by ELISA using sera from olive pollen-allergic patients. Their allergenicity was also investigated in both a mouse model of allergic sensitization and in basophil activation assays. IgG1 response was assayed by immunoblotting and competitive ELISA. T cell reactivity was evaluated by proliferation assays and cytokine production in splenocyte cultures. Results The 135Δ10 mutant showed the strongest reduction in the IgE-binding capability of sera from olive pollen-allergic patients. Rat basophil leukaemia assays identified the deletion mutant 135Δ10 as the variant with the lowest β-hexosaminidase-releasing capacity. Furthermore, the same 135Δ10 mutant induced the lowest IgE levels in a BALB/c mouse model of sensitization. All Ole e 1 mutants retained their allergen-specific T cell reactivity. Immunization of mice with the mutants induced IgG1 antibodies, which cross-reacted with Ole e 1 and Ole e 1-like allergens from ash, lilac and privet pollens. The ability of the human IgE to block the binding of anti-Ole e 1 mutant-specific mouse IgG1 antibodies to natural Ole e 1 demonstrated that Ole e 1 mutants are able to induce in vivo antibodies reactive to the natural allergen. Conclusion The 135Δ10 mutant with reduced allergenicity, intact T cell reactivity and capacity to induce blocking antibodies could provide a suitable candidate vaccine for efficient and safer therapy of olive pollen allergy.

Rationale Food allergy is an increasing health problem with many proteins involved that belong mainly to a limited number of families. They show a high level of cross-reactivity. In the Mediterranean area, the most prevalent food allergens are those of vegetal origin. Although tomato (Solanum lycopersium L.) is one of the implicated foods, studies on the identification and relevance of their allergens have not been carried out in detail. This could be particularly relevant for tomato seeds as happen in other fruits like kiwi. The aimed was to analyse the sensitisation pattern to tomato seeds in patients from two hospitals integrated in the RIRAAF.

Background: Polcalcins are pollen-specific allergens with two EF-hand calcium-binding sites that exhibit strong cross-reactivity. Our objective was to isolate and express the cDNA coding of the EF-hand calcium-binding allergen from lilac pollen and to study cross-reactivity with other polcalcins from related and nonrelated pollen sources with different specific antibodies and sera from two different populations. Methods: Specific cDNA was amplified by PCR, cloned and expressed in Escherichia coli. Purification was achieved by gel permeation and ion exchange chromatographies. ELISA titration and inhibition assays were performed using the recombinant forms of Syr v 3, Ole e 3, Che a 3 and Phl p 7 with sera from two Spanish regions with different sensitization profiles, as well as Phl p 7- and Ole e 3-specific polyclonal rabbit antisera, and an Ole e 3-specific monoclonal antibody. Results: Syr v 3 displays two EF-hand consensus sites and 8863 Da of theoretical molecular mass. The allergen consists of 80 residues with identities ranging from 66 to 87% with polcalcins included in this study. Syr v 3, Ole e 3, Che a 3 and Phl p 7 showed a similar IgG- and IgE-binding capacity although differences at quantitative level were observed depending on the population of patients’ sera. Conclusion: Syr v 3 is a polcalcin with structural and antigenic similarities to the members of this family. Diagnosis of polcalcin-sensitized patients could be performed whatever polcalcin used, whereas for immunotherapy, primary sensitization to a particular allergenic source should be considered.

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 α-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 Aspf1Δ(7–22) have been produced as recombinant proteins; the deleted region corresponds to an exposed β-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. Aspf1Δ(7–22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted β-hairpin is involved in ribosome recognition and is a significant allergenic region.

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

Summary Background Several Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. Objective To study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. Methods Two olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunobloting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. Results Thirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. Conclusion EF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross-reactivity among members of the same family.

In a first approach, Ole e 8, a novel Ca2+-binding protein from olive pollen, was cloned and produced in Escherichia coli. We have obtained the natural form of Ole e 8 (nOle e 8) from the pollen and examined its immunologic equivalence with its recombinant form (rOle e 8). Size exclusion chromatography and a phenyl-Sepharose CL-4B affinity column were used to obtain nOle e 8 from the olive pollen. Inhibition assays by immunoblotting, using rOle e 8-specific rabbit antiserum, were performed to analyze the immunologic equivalence between the natural and the recombinant allergen, as well as to detect its presence in other pollens. Recombinant and natural Ole e 8 resulted immunologically equivalents, since they completely inhibited the IgG binding of the polyclonal antiserum to each other. Ole e 8-like proteins were detected in Oleaceae and Juniperus communis pollen, and might contribute to cross-reactivity processes between taxonomically related pollens.

Background The main allergens from privet and lilac pollens, Lig v 1 and Syr v 1, are proteins homologous to Ole e 1 and have been shown to be involved in cross-reactivity. Objective To overproduce the correctly folded Lig v 1 and Syr v 1 allergens and to study their immunological properties in comparison with those of their natural counterparts. Methods The yeast Pichia pastoris was used as an expression system to produce these recombinant allergens. The proteins were isolated by ion-exchange and size-exclusion chromatographies. Amino acid quantifying, Edman degradation, mass spectrometry and circular dichroism were carried out to obtain molecular properties of the recombinant proteins. Anti-Ole e 1 monoclonal and polyclonal antibodies, as well as sera from patients allergic to olive pollen, were used in immunoblotting and ELISA for immunological characterization. Results Recombinant Lig v 1 and Syr v 1 were secreted at high yield to the extracellular medium of the yeast. The purified proteins displayed the native conformation, as deduced from their spectroscopic properties and binding ability to an IgG monoclonal antibody. The recombinant allergens behaved similarly to their natural counterparts when they were analysed against Ole e I-specific antibodies. IgE and IgG binding properties of lilac and privet allergens to olive allergic sera and Ole e 1-specific antibodies indicated that these molecules share common B-cell epitopes with Ole e 1. Conclusion P. pastoris yeast is an appropriate system for the efficient production of Ole e 1-like allergens, which could be used as analogous allergens and predictors of clinical sensitization.

The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19–Cys90 and Cys22–Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.

The glycoprotein Ole e 1 is a significant aeroallergen from the olive tree (Olea europaea) pollen, with great clinical relevance in the Mediterranean area. To produce a biologically active form of recombinant Ole e 1, heterologous expression in the methylotrophic yeast Pichia pastoris was carried out. A cDNA encoding Ole e 1, fused to a Saccharomyces cerevisiae alpha-mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein secreted into the extracellular medium was purified by ion-exchange and size-exclusion chromatography. The structure of the isolated recombinant Ole e 1 was determined by chemical and spectroscopic techniques, and its immunological properties analysed by blotting and ELISA inhibition with Ole e 1-specific monoclonal antibodies and IgE from sera of allergic patients. The allergen was produced at a yield of 60 mg per litre of culture as a homogeneous glycosylated protein of around 18.5 kDa. Recombinant Ole e 1 appears to be properly folded, as it displays spectroscopic properties (CD and fluorescence) and immunological reactivities (IgG binding to monoclonal antibodies sensitive to denaturation and IgE from sera of allergic patients) indistinguishable from those of the natural protein. This approach gives high-yield production of homogeneous and biologically active allergen, which should be useful for scientific and clinical purposes.

A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen.

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH2-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH2-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X3-Cys-X3-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.

Summary Background An olive allergen-like protein has been detected in privet pollen. This protein could be involved in the allergenic cross-reactivity described for privet and olive tree pollen extracts. Objective Isolation and characterization of natural Lig v 1. Cloning and expression of its cDNA in order to assess its structural similarity with the olive allergen. Methods Current chromatographic methods were used to isolate the privet counterpart of Ole e 1. A pool of sera from subjects allergic to olive tree pollen was used to immunodetect the protein in the elution profiles. Ole e 1-specific polyclonal antibody and allergic sera were used in immunoblotting assays of the isolated protein, Polymerase chain reaction amplification of the first strand cDNA synthesized from the privet pollen total RNA was carried out to prepare a full-length fragment encoding Lig v 1. After nucleotide sequencing, expression of one clone was performed in Escherichia coli, under the form of a fusion protein with glutathione S-transferase. The IgE binding capability of the recombinant protein was also analysed. Results The major allergen from privet pollen, Lig v 1, was purified to homogeneity by two gel filtration chromatographies and one reverse-phase high-performance liquid chromatography. Its amino acid composition and N-terminal amino acid sequence were determined. Two different clones encoding Lig v 1 were sequenced. Strong sequence similarity between Lig v 1 and Ole e 1 was observed, the identity being 85 and 96%. One of the sequenced clones was expressed and the recombinant product exhibited IgG and IgE binding activities against both anti-Ole e 1 polyclonal antibodies and olive-allergic sera. Conclusion Privet pollen contains a protein structurally and immunologically related to the major allergen of ohve pollen. The similarity exhibited by these proteins could explain the cross-reactivity observed between the two pollen extracts. Since these allergens are highly polymorphic, the expression of an immunologically active recombinant Lig V 1 will permit the preparation of well defined molecules for both research and chnical purposes.

An olive allergen-like protein has been isolated from lilac (Syringa vulgaris) pollen extract. The protein can be considered as an allergen since is recognized by IgE from olive hypersensitive human sera, and has been called Syr v I (IUIS nomenclature). This protein consists of a glycosylated polypeptide of 20 kDa, which has an amino acid composition, spectroscopic properties, and an N-terminal sequence similar to the major allergen from olive pollen, Ole e I. The lilac allergen is recognized by rabbit polyclonal antisera raised against olive allergen as well as by an Ole e I-specific monoclonal antibody. Using a polymerase chain reaction strategy, based on the similarities observed between these olive and lilac proteins, three cDNA clones encoding Syr v I have been isolated and sequenced. These clones code for a polymorphic protein of 145 residues with a derived molecular mass of about 16,400Da, which contains a potential N-glycosylation site. Comparison of the deduced amino acid sequences of these Syr v I isoforms to each other revealed identities of 90-97%. Moreover, these sequences showed a high degree of similarity (85.5-89.6% identity) with Ole e I. The structural and immunological characterization of Syr v I justify the cross-reactions observed between olive and lilac pollen extracts. The molecular cloning of Syr v I is relevant for the epitope mapping in Oleaceae allergens, and may contribute to an improvement in the design of reagents for diagnosis and therapy of IgE-dependent allergic reactions.

The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which have been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.