Abstract
Olive pollen allergy is a clinical disorder affecting the human population of Mediterranean areas. A novel major allergen, Ole e 9, has been isolated from olive pollen by gel permeation, hydrophobic affinity, and reverse-phase high performance liquid chromatographies. It is involved in the allergic responses of 65% of patients suffering olive pollinosis. Ole e 9 (molecular mass of 46.4 kDa) displays 1,3-beta-endoglucanase activity (38.9 +/- 5.6 mg of glucose released/min x micromol of protein at pH 4.5-6.0 using laminarin as substrate). It is the first 1,3-beta-glucanase, a member of the "pathogenesis-related" protein family, detected in pollen tissue. Seven tryptic peptides of the allergen were sequenced by Edman degradation and used for designing primers to clone the cDNA codifying the protein. Specific cDNA for Ole e 9 was synthesized from total RNA and amplified using the polymerase chain reaction. The allergen sequence showed an open reading frame of 460 amino acids comprising a putative signal peptide of 26 residues. It shows 39, 33, and 32% sequence identity including the catalytic residues when compared with 1,3-beta-glucanases from wheat, willow, and Arabidopsis thaliana, respectively. Northern blot analysis showed that Ole e 9 transcript is specifically expressed in the pollen tissue, and highly conserved counterparts were only detected in taxonomically related pollens.
Highlights
Olive pollen allergy is a clinical disorder affecting the human population of Mediterranean areas
IgE reactive proteins of 42, 45– 47, 60 – 65, and 70 kDa have been detected in the pollen extract (12, 14 –17), and some of them seem likely to be of high clinical significance close to that of Ole e 1, the main allergen from olive pollen, to which Ͼ70% olive allergic patients are sensitive as deduced from the prevalence observed in immunoblotting assays [6, 15, 16]
Based on these preliminary prevalence data, this allergen would be in addition to Ole e 1 the most important allergen from olive pollen
Summary
Isolation of Olive Pollen Allergen—Olive tree pollen was obtained from Biopol (Spokane, WA) lot No 97-0524 containing 0.17% foreign pollen and 0.68% other plant parts. The membranes were incubated with a pool of sera from patients allergic to olive pollen (diluted 1:10) followed by reaction with mouse anti-human IgE (diluted 1:5000) kindly donated by Dr M. A 701-base pair fragment, generated from olive pollen cDNA by PCR with OL9C and OL9A primers, was radiolabeled with a commercial random-primed kit (Amersham Pharmacia Biotech) containing [␣-32P]dCTP (6000 Ci/mmol, Amersham Pharmacia Biotech). This probe was denatured and hybridized to the immobilized RNA in prehybridization solution for 16 h at 42 °C. The hybridization conditions and development of signals were performed following the experimental procedure described previously [7]
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