Isolation and characterization of relevant allergens from boiled lentils

Background: Lentils seem to be the most common legume implicated in pediatr ic allergic patients in the Mediterranean area, However, no lentil allergen has been isolated and characterized. Objective: We sought to purify and characterize relevant IgE-binding protei ns From boiled lentil extracts. Methods: IgE-binding proteins from crude and boiled lentil extracts were de tected with a pool of sera from patients with lentil allergy, Allergens wer e isolated by gel-filtration chromatography followed by cation- and anion-e xchange chromatography or by reverse-phase HPLC. Their characterization inc luded N-terminal amino acid sequencing, complex asparagine-linked glycan de tection, specific IgE immunodetection with 22 individual sera from allergic patients, and immunoblot and CAP inhibition assays. Results: Heat treatment of lentils produced substantial changes in the SDS- PAGE patterns of whole extracts, mainly a strong increase of 12- to 16-kd b ands and a decrease of 25- to 45-kd components, Major IgE-binding proteins from the boiled lentil extract were located in the 12- to 16-kd and 45- to 70-kd ranges. Two allergens of 16 kd, proteins L1 and L2, and another one o f 12 kd, protein L3, were purified, N-terminal sequencing indicated that al l 3 were related and allowed their identification as gamma -vicilin subunit s, Protein L1 was recognized by 68% of the individual sera tested and inhib ited 64% of the IgE binding by commercial lentil CAPs, A second type of all ergen of 66 kd, named protein H, was also isolated and identified as a seed -specific biotinylated protein. Protein H reacted with 41% of the individua l sera and produced 45% inhibition in CAP inhibition assays. Conclusions: Two different types of allergens have been identified in boile d lentils. Those of 12 to 16 kd, called Len c 1, correspond to gamma -vicil in subunits, and those of 65 kd, designated Len c 2, correspond to seed-spe cific biotinylated protein. Homology with proteins from other legume specie s can explain potential cross-reactions among these foods.