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% a-helix, 10% a-strand, 29% Sturn 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 Caz'-binding motif. The isolated protein displays IgE-binding activity against sera of patients allergic to olivetree 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.Keywords: allergen ; olive pollen ; Olea europaea ; Ole e 3 ; protein characterization.Type-I allergy is a clinical disorder that affects more than 15 % of the human population in developed countries. Olive pollinosis is one of the most important causes of type-I allergy in Mediterranean countries, where the olive tree is widely cultivated [l]. Ole e 1 has been shown to be a major allergen from olive pollen, since the IgE of more than 70% of olive-allergic patients recognize this protein [2-41. In addition to Ole e 1, other allergenic proteins have been detected in saline extracts of the olive pollen 15-71, some of which exhibit low molecular masses. Preliminary studies on these molecules were performed with pools of sera from patients hypersensitive to olive pollen. Lauzurica et al. 121 showed the existence of an IgE-reacting protein of 8 kDa upon SDS/PAGE of the pollen extract. Proteins of 7, 9, 14, 15 and 16 kDa, present in olive pollen, have been immunostained with individual sera from olive allergic patients [8]. However, isolation or molecular characterization was not performed for these allergens.Allergic cross-reactivities have been described among Oleaceae members. such as olive, ash and privet [9-131 as well as between olive-pollen and grass-pollen proteins [8]. In addition to profilin, which is an important and well defined panallergen involved in cross-reactions [ 141, other proteins contained in pollen from different species that have conserved sequences could be responsible for allergic processes in individuals not previously sensitized to a given biological source.In spite of the knowledge of a high number of allergenic structures, little is known about their B-cell and T-cell epitopes and the mechanism of induction of allergy. The protein allergens fre...

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 Ca 2ϩ -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 Ca 2ϩ 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) ϩ 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 Ca 2ϩ -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 Ca 2ϩ -binding proteins from pollen.Keywords : allergen ; Ole e 3; recombinant allergen ; olive-pollen allergy; Ca 2ϩ -binding proteins ; nucleotide sequence.Pollinosis is an IgE-mediated allergic process induced by airborne pollen grains. It frequently depends on the botanical behaviour surrounding the affected individual, since the allergenic pollens are anemophilous and should reach organisms by scattering on the air. Wild grasses Ϫ Lolium perenne, Cynodon dactilon, Phleum pratense Ϫ and woods Ϫ Ambrosia artemisiifolia, Ambrosia vulgaris Ϫ are widespread vegetable sources causing pollinosis. However, some trees such as birch and olive are able to induce dramatic airborne allergies because of seasonal appearance of high-density pollen levels. In fact, allergy to olive-tree (Olea europaea) pollen affects more than 30% of the population in Mediterranean areas during the pollination season [1]. More than 70% of the patients allergic to olive pollen are sensitive to Ole e 1 [2], one major allergen from this pollen [3,4]. Recently, a novel olive-pollen allergen, Ole e 3 (9.2 kDa), has been isolated and partially characterised [5]. Its prevalence accounts for more than 50% of the olive allergic patients, depending on the geographical area, and is thus considered as a major allergen [6]. Partial amino acid sequence data of Ole e 3 revealed the Note. The novel nucleotide sequence reported here has been submitted to the GenBank/EMBL database and is available under accession number AF015810. presence of at least one Ca 2ϩ -binding domain similar to those of calmodulin and parvalbu...

Olive-pollen profilin has been isolated and characterized as a significant allergen. Its molecular properties, such as a molecular mass of 15 kDa; amino-acid composition; and secondary repetitive structure percentages of 15% alpha-helix, 33% beta-strand, 20% beta-turn, and 32% random coil, have been determined. Its allergenic capability, a recognition frequency estimated at 24% of olive-hypersensitive patients, and high cross-reactivity with all the pollen used have been found. The presence of conformation epitopes in the olive profilin, as well as a high structural and immunologic similarity to other pollen sources such as birch and ash, can be established from these studies.

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.z 2000 Federation of European Biochemical Societies.

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.