Identification and characterization of the major allergen of green bean ( Phaseolus vulgaris) as a non-specific lipid transfer protein (Pha v 3)

Abstract

Background Green bean (GB) has been reported to cause allergic reactions after ingestion, contact or inhalation of particles deriving from processing or cooking. Up-to-date no food allergens have been fully characterized in GB. Objective To characterize the GB major allergen(s) on a molecular level and to verify the involvement of non-specific lipid transfer proteins (nsLTPs) in GB allergy. Methods We recruited 10 Spanish patients reporting adverse reactions to GB. Skin prick tests, specific IgE detection and oral provocation were performed. Two nsLTP cDNAs were cloned from GB and over-expressed in Pichia pastoris. The recombinant LTPs (rLTPs) were characterized by circular dichroism spectroscopy and IgE-binding assays (immunoblotting and ELISA) with the patients’ sera. Three natural LTPs (nLTPs) were further purified from GB fruit by chromatography. In vitro histamine release test was applied to compare the allergenic potency of rLTPs and nLTPs. Results Oral provocation test confirmed GB allergy. A 10 kDa protein in GB extract was recognized by 80% of the sera and identified as nsLTP. The two rLTPs (named LTP1a and LTP1b), share 61.3% aa identity and present the typical nsLTP-like secondary structure. The IgE-binding and histamine release assays provided evidence that rLTPs and nLTPs possess different allergenic potency. Conclusions nsLTP (Pha v 3) is the major allergen in GB and constitute a potential risk for patients affected by LTP-syndrome. GB encodes for several LTPs with different immune reactivity.