title: Len c 1, a major allergen and vicilin from lentil seeds: protein isolation and cDNA cloning.
abstract: BACKGROUND
  Lentils are among the main plant foods causing allergic reactions in pediatric patients in the Mediterranean area and in many Asian communities. However, very few reports have been devoted to identifying lentil allergens. Seed storage proteins of the vicilin family have been characterized as major allergens in several seed legumes and tree nuts.
  
  
  OBJECTIVE
  We sought to evaluate the role of lentil vicilins as food allergens.
  
  
  METHODS
  A serum pool and individual sera from 22 patients with lentil allergy were used in different IgE-binding assays. Mature lentil vicilin was isolated by means of cation-exchange chromatography, followed by reverse-phase HPLC, and characterized by means of N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) analysis, complex asparagine-linked glycan detection, specific IgE immunodetection with individual sera, and ELISA inhibition assays. Complete cDNAs encoding lentil vicilin variants were isolated by means of PCR with primers based on the amino acid sequence of the allergen.
  
  
  RESULTS
  A major IgE-binding component of approximately 50 kd was detected in lentil extracts. This component was isolated and characterized, showing a single N-terminal amino acid sequence homologous to those of legume vicilins and a broad peak (maximum at 48613 d) in MALDI analysis. The purified allergen was recognized by 77% (17/22) of the individual sera from patients with lentil allergy and reached up to 65% inhibition of the IgE binding to the crude lentil extract. The allergen showed 3 isoforms varying in their degree of N-glycosylation. Two cDNA clones encoding different allergen variants were isolated. The amino acid sequences deduced from both clones (415 and 418 residues; 47.4 and 47.8 kd) showed greater than 50% identity with major peanut (Ara h 1) and soybean (conglutinin subunits) allergens belonging to the vicilin family. Furthermore, these sequences included those of the previously characterized lentil allergen Len c 1.02 (108 amino acid residues of the C-terminal domain) and those of a novel lentil IgE-binding protein of 26 kd.
  
  
  CONCLUSIONS
  The mature 48-kd lentil vicilin, designated Len c 1.01, is a major allergen. Two of its processing fragments, corresponding to subunits of 12 to 16 kd (previously named Len c 1) and 26 kd, are also relevant lentil IgE-binding proteins. The sequence homology of Len c 1.01 to those of major allergens from peanut, soybean, walnut, and cashew can help to investigate potential cross-reactions among these plant foods.
authors.affiliation: Departamento de Biotecnología, Ingenieros Agrónomos, UPM, Madrid, Spain.
authors.forename: Gema
authors.initials: G
authors.lastname: López-Torrejón
authors.forename: Gabriel
authors.initials: G
authors.lastname: Salcedo
authors.forename: Manuel
authors.initials: M
authors.lastname: Martín-Esteban
authors.forename: Araceli
authors.initials: A
authors.lastname: Díaz-Perales
authors.forename: Cristina Y
authors.initials: CY
authors.lastname: Pascual
authors.forename: Rosa
authors.initials: R
authors.lastname: Sánchez-Monge
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005512:Food Hypersensitivity / Q000209:etiology*; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D027806:Lens Plant / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D055314:Seed Storage Proteins; D012639:Seeds / Q000737:chemistry*; D017422:Sequence Analysis, DNA
chemical_list: D000485:Allergens; D018076:DNA, Complementary; D010940:Plant Proteins; D055314:Seed Storage Proteins; D007073:Immunoglobulin E; C015575:vicilin protein, plant