A new pepper allergen Zan b 1.01 of 2S albumins: Identification, cloning, characterization, and cross-reactivity

Background: Zanthoxylum bungeanum (Sichuan pepper; 花椒 in Chinese) is used as a spice worldwide and is a potentially life-threatening allergenic food source, as first reported by our team in 2005.However, its allergen components are unknown. Objective:We aim to identify and characterize its major allergen and determine its cross-reactivities with citrus seeds, pistachios, and cashew seeds.Methods: Ionic exchange and molecular exclusion chromatography were used to isolate the protein components from Sichuan pepper seed.A protein fraction was characterized by SDS-PAGE, analytical ultracentrifugation, mass spectrometry, and circular dichroism spectroscopy.The coding region of it was amplified from the genome.ELISA and competitive ELISA assays were used to investigate the allergenicity and cross-reactivity of allergens.Results: This protein allergen was around 14 kDa.It was a 2S albumin similar to an α-Amylase inhibitor (AAI) domain-containing protein of Citrus sinensis.Circular dichroism spectroscopy showed its thermal stability was high.A 303 bps DNA sequence of the AAI domain was cloned from the genome of the Sichuan pepper.Competitive ELISA assays showed positive cross-reactivities between this allergen and citrus seeds, pistachios, and cashew seeds. Conclusions:A major allergen of around 14 kDa from Sichuan pepper seed was confirmed, which belongs to the 2S albumin of plant seed storage proteins.Based on the nomenclature of the IUIS Subcommittee for Allergen Nomenclature, this allergen is designated as Zan b 1.01.The cross-reactivities were demonstrated between Zan b 1.01 and citrus seeds, pistachios, and cashew seeds.