Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa).

SCOPE: Today, about 2-8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. METHODS AND RESULTS: Sera from 42 Spanish lettuce-allergic patients were obtained from patients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. CONCLUSION: Two new major lettuce allergens-a thaumatin-like protein and an aspartyl protease-have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

Molecular basis of allergen cross-reactivity: non-specific lipid transfer proteins from wheat flour and peach fruit as models.

Peach non-specific lipid transfer protein (Pru p 3; nsLTP) has been characterized as the major food allergen in the adult Mediterranean population. Its wheat homologous protein, Tri a 14 has a relevant inhalant allergen in occupational baker's asthma. Different sensitization patterns to these allergens have been found in patients with this latter disorder. The objective of the present study was to characterize IgE epitopes of Tri a 14 and to compare them with those of Pru p 3 using three complementary strategies: the analysis of IgE-binding capacity of decapeptides bound to membrane, the identification of mimotopes using a phage display random peptide library, and the analysis of the surface electrostatic potential of both allergens. Thus, synthetic overlapping decapeptides, covering the Pru p 3 and Tri a 14 amino acid sequences, were used to identify sequential regions involved in recognition of IgE from baker's asthma patients sensitized to both nsLTPs. A phage display library was screened with total IgE from the same patients, and positive clones sequentially selected using the purified allergens, allowed to identify mimotopes (conformational epitopes) of Tri a 14 and Pru p 3. Both sequential regions and mimotopes were localized in the corresponding 3D molecular surface and their electrostatic properties were analyzed. Common sequential regions with strong IgE-binding capacity (residues 31-40 and 71-80) were identified in Tri a 14 and Pru p 3, whereas regions Tri a 14(51-60) and Pru p 3(11-20) were found specific of each allergen. A major conformational epitope (mimotope), L34H35N36R39S40S42D43G74V75L77P78Y79T80, which comprised the two common sequential epitopes, was located in Tri a 14, and a very similar one in Pru p 3. However, differences were detected on the surface electrostatic potential of both mimotopes: a first part (around residues 31-45) showed similar positive features in both allergens, whereas a second part (around residues 74-80) was markedly negative in Tri a 14 but neutral-positive in Pru p 3. Tri a 14 and Pru p 3 have a similar conformational region involved in IgE-binding, although their electrostatic features are different. Additionally, common and specific sequential IgE-binding regions were mapped in both allergens. These findings could be instrumental in understanding the cross-reactivity and specificity of sensitization to both homologous allergens.