Hong Li,1# Lixia Zhu,2# Rui-qi Wang,1 Liping Zhu,1 Jing Hu,2 Feng Chen,2,3 Li Ma,4 Rui Tang,1 Siyu Liu,2 Kang Ni,5 Xiaodong Ye,5 Yuzhu Zhang,6 Jing-Lyu Sun,1 Tengchuan Jin2,7

Affiliations:
1 Allergy Department, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China
2 Laboratory of Structural Immunology, the CAS Center for Excellence in Molecular Cell Science, the CAS Key Laboratory of Innate Immunity and Chronic Diseases, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
3 College of Integrated Chinese and Western Medicine, College of Life Science, Anhui University of Chinese Medicine, Hefei, China
4 Cardiovascular Research Institute and Department of Medicine, Morehouse School of Medicine, Atlanta, GA, USA
5 Department of Chemical Physics, University of Science and Technology of China, Hefei, Anhui, China
6 Healthy Processed Foods Research Unit, USDA-ARS, Western Regional Research Center, Albany, CA, USA
7 Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, Anhui, China

#Both authors contributed equally to the work.

Abstract

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.
Key words: Zan b 1.01, Zanthoxylum bungeanum Maxim, 2S albumin, α-amylase inhibitor, pepper allergy, citrus allergy, cashew seed, IgE cross-reactivity