Pomegranate LTP isoforms identified by a new proteomic approach show different immunological properties

Background: Plant Lipid Transfer Proteins (LTPs) belong to a class of high conserved proteins extensively studied in the last decade. These proteins are recognized as food allergens capable of eliciting severe systemic symptoms, especially among the Mediterranean population. Several polypeptides belonging to this family have been characterized so far. For some LTPs different isoforms have been identified in a given plant. The sequence information was mainly derived from the cDNA, by contrast, the investigation of isoforms diversity is frequently not addressed. Allergen isoforms often exhibit different IgE binding capacities, hence contributing differently to the whole allergenic potencies (e.g. Bet v 1 and Mal d 1). Recently two different LTP isoforms have been isolated from pomegranate (PG) and characterized by mass spectrometry (MS). Here we applied a new proteomic approach based on acid-urea electrophoresis (AU-PAGE) followed by SDS-PAGE for the separation of LTP isoforms from PG and peach. Methods: Proteins from PG juice and peach peel were separated first by the AU-PAGE. The lanes were cut, equilibrated in SDS containing buffer, and subjected to 15% SDS-PAGE. The separated proteins were transferred to PVDF membrane and assayed with a polyclonal antibody raised against Pru p 3 (PAB). IgE immunoblotting assays were carried out using sera from two groups of patients: 1) allergic to pomegranate, 2) allergic to peach (sensitised to Pru p 3). For identification the proteins were subjected to MS. Results: By this approach up to 10 spots with an apparent MW of 9–12 kDa (depend- ing on the redox conditions) were separated for PG. Five of these proteins were recognized by the PAB and by the patient’s sera. IgE immunoblotting experiments clearly showed that PG LTP isoforms are differentially recognized. In contrast, 2D analysis of peach revealed only a single predominant9kDa spot recognized by the PAB and by the patient’s sera. Conclusions: Up to 5 LTP isoforms have been separated from PG by the proteomic approach. The power of this technique is demonstrated by the identification of a large number of IgE reactive LTP isoforms never described for other organisms. The immunological study of the identified isoforms indicates that they possess different IgE epitopes which might contribute differently to the whole allergenic potency of pomegranate.