title: Isolation and characterization of relevant allergens from boiled lentils.
abstract: BACKGROUND
  Lentils seem to be the most common legume implicated in pediatric 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 proteins from boiled lentil extracts.
  
  
  METHODS
  IgE-binding proteins from crude and boiled lentil extracts were detected with a pool of sera from patients with lentil allergy. Allergens were isolated by gel-filtration chromatography followed by cation- and anion-exchange chromatography or by reverse-phase HPLC. Their characterization included N-terminal amino acid sequencing, complex asparagine-linked glycan detection, 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 bands 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 of 12 kd, protein L3, were purified. N-terminal sequencing indicated that all 3 were related and allowed their identification as gamma-vicilin subunits. Protein L1 was recognized by 68% of the individual sera tested and inhibited 64% of the IgE binding by commercial lentil CAPs. A second type of allergen of 66 kd, named protein H, was also isolated and identified as a seed-specific biotinylated protein. Protein H reacted with 41% of the individual sera and produced 45% inhibition in CAP inhibition assays.
  
  
  CONCLUSIONS
  Two different types of allergens have been identified in boiled lentils. Those of 12 to 16 kd, called Len c 1, correspond to gamma-vicilin subunits, and those of 66 kd, designated Len c 2, correspond to seed-specific biotinylated protein. Homology with proteins from other legume species can explain potential cross-reactions among these foods.
authors.affiliation: Unidad de Bioquímica, Departamento de Biotecnología, E.T.S. Ingenieros Agrónomos, Madrid, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Sánchez-Monge
authors.forename: C Y
authors.initials: CY
authors.lastname: Pascual
authors.forename: A
authors.initials: A
authors.lastname: Díaz-Perales
authors.forename: J
authors.initials: J
authors.lastname: Fernández-Crespo
authors.forename: M
authors.initials: M
authors.lastname: Martín-Esteban
authors.forename: G
authors.initials: G
authors.lastname: Salcedo
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D002648:Child; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007887:Fabaceae / Q000276:immunology*; D006361:Heating; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000032:analysis / Q000276:immunology*; D010946:Plants, Medicinal; D055314:Seed Storage Proteins; D020539:Sequence Analysis, Protein / Q000379:methods
chemical_list: D000485:Allergens; D010936:Plant Extracts; D010940:Plant Proteins; D055314:Seed Storage Proteins; D007073:Immunoglobulin E; C015575:vicilin protein, plant