Cloning of the minor allergen Api g 4 profilin from celery (Apium graveolens) and its cross-reactivity with birch pollen profilin Bet v 2

Background Profilin is a panallergen that is recognized by IgE from about 2 0% of birch pollen- and plant food-allergic patients. A subgroup of celery- allergic patients shows IgE-reactivity with this minor allergen. To investi gate the IgE-binding potential and crossreactivity of celery profilin at th e molecular level, this study was aimed at the cloning and immunological ch aracterization of this allergen. Objectives Cloning, expression and purification of profilin from celery tub er to characterize its immunological properties and its cross-reactivity wi th birch pollen profilin. Methods Cloning of celery profilin was performed by polymerase chain reacti on using degenerated primers and a 5'RACE method for the identification of the unknown 5'-end of the cDNA. Expression was carried out in Escherichia c oli BL21 (DE3) using a modified vector pET30a. The recombinant profilin was purified by affinity chromatography on poly L-proline coupled to sepharose . Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. Results The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence o f the corresponding protein showed high identity with other plant profilins (71-82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was si milar to that of natural protein. Seven of 17 celery-allergic patients test ed presented specific IgE-antibodies to the recombinant protein tested by i mmunoblotting. Inhibition experiments showed high cross-reactivity of IgE w ith both profilins from celery and birch pollen. Moreover, the biological a ctivity of recombinant celery profilin was demonstrated by a histamine rele ase assay. Conclusions Celery profilin is an important allergenic compound in celery a nd shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin A pi g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore addit ionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.