Identification of two pistachio allergens, Pis v 1 and Pis v 2, belonging to the 2S albumin and 11S globulin family

Abstract

IgE-mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins. We attempted to identify pistachio allergens and to clone the encoding genes. Pistachio proteins were extracted and separated by SDS-PAGE. Immunolabelling was performed with sera from 28 pistachio-allergic individuals. Proteins of interest were further analysed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS). In parallel, a cDNA library was generated from immature pistachios and screened with primers designed on the basis of internal sequences and peptide spectra. Full-length cDNA clones were isolated from the library and sequenced. Recombinant proteins were expressed and tested with sera from pistachio-allergic patients. Nineteen out of 28 patients (68%) showed IgE binding to a 7 kDa protein fraction, while 14 (50%) showed specific IgE to a 32 kDa protein fraction. Analysis by Edman sequencing and MS/MS revealed that these proteins were homologue to the cashew nut allergens Ana o 3 and Ana o 2, respectively. Screening of the pistachio cDNA library resulted in isolation of novel protein cDNAs. Open-reading frame translation provided the complete amino acid sequences of two new allergenic pistachio proteins. Recombinant proteins were recognized by six out of six selected patients. Therefore, these new allergens were named Pis v 1 and Pis v 2 by the Allergen Nomenclature Subcommittee. Novel allergens in pistachio, Pis v 1 and Pis v 2, which belong to 2S albumin and 11S globulin family, respectively, were isolated and the genes encoding these allergens were identified.