Identification of a polygalacturonase as a major allergen (Pla a 2) from Platanus acerifolia pollen

Background: Planetree pollen allergy is a clinical disorder affecting human populations in cities of the United States and Western Europe, but little is known about its relevant allergens. Objective: We sought to purify, characterize, and clone the 43-kd allergen from Platanus acerifolia. Methods: P acerifolia pollen extract was fractionated by using ion-exchange and gel-permeation chromatography. Analyses were carried out by using ELISA, SDS-PAGE, isoelectrofocusing, and immunoblotting. Partial amino acid sequence was obtained by means of Edman sequencing of cyanogen bromide-digested peptides. Specific cDNA was cloned by using reverse transcription, followed by PCR, with amino acid sequences from peptides of the allergen. Results: The allergen isolated from P acerifolia pollen, Pla a 2, is a glycoprotein with an observed molecular mass of 43 kd and an isoelectric point value of 9.3. It is involved in the allergic responses of 84% of patients with planetree-induced pollinosis and represented 52% of the total IgE-binding capacity of the P acerifolia extract. Pla a 2 displays polygalacturonase (PG) activity, being the first PG with functional enzyme activity from an angiosperm plant pollen described as an allergen. The cDNA allergen sequence codified for a 372-residue protein with 56% and 42% sequence identity to PGs from pollen and fruits, respectively. Western blot analysis showed that Pla a 2 is present in pollen and stems and has IgG cross-reactivity with a PG from tomato and pectate lyases from Cupressaceae pollen. Conclusion: Pla a 2, a major allergen of P acerifolia pollen with PG activity has been purified, characterized, and cloned.