title: Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes.
abstract: BACKGROUND
  The 2S-albumin Ara h 2 is the most potent peanut allergen and a good predictor of clinical reactivity in allergic children. Posttranslational hydroxylation of proline residues occurs in DPYSP(OH)S motifs, which are repeated 2 or 3 times in different isoforms.
  
  
  OBJECTIVE
  We investigated the effect of proline hydroxylation on IgE binding and the relative contributions of linear and conformational epitopes to Ara h 2 allergenicity.
  
  
  METHODS
  Peptides containing DPYSP(OH)S motifs were synthesized. A recombinant variant of Ara h 2 without DPYSP(OH)S motifs was generated by means of deletion mutagenesis. IgE reactivity of 18 French and 5 American patients with peanut allergy toward synthetic peptides and recombinant allergens was assessed by using IgE-binding inhibition assays and degranulation tests of humanized rat basophilic leukemia cells.
  
  
  RESULTS
  Hydroxyproline-containing peptides exhibited an IgE-binding activity equivalent to that of the unfolded Ara h 2. In contrast, corresponding peptides without hydroxyprolines displayed a very weak IgE-binding capacity. Despite removal of the DPYSP(OH)S motifs, the deletion variant still displayed Ara h 2 conformational epitopes. The IgE-binding capacity of Ara h 2 was then recapitulated with an equimolar mixture of a hydroxylated peptide and the deletion variant. Hydroxylated peptides of 15 and 27 amino acid residues were also able to trigger cell degranulation.
  
  
  CONCLUSIONS
  Sensitization toward linear and conformational epitopes of Ara h 2 is variable among patients with peanut allergy. Optimal IgE binding to linear epitopes of Ara h 2 requires posttranslational hydroxylation of proline residues. The absence of hydroxyprolines could then affect the accuracy of component-resolved diagnostics by using rAra h 2.
authors.affiliation: INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, Jouy-en-Josas, France; CEA, iBiTecS/Service de Pharmacologie et d'Immunoanalyse, Gif-sur-Yvette, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Bernard
authors.affiliation: INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, Jouy-en-Josas, France; CEA, iBiTecS/Service de Pharmacologie et d'Immunoanalyse, Gif-sur-Yvette, France.
authors.forename: Blanche
authors.initials: B
authors.lastname: Guillon
authors.affiliation: INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, Jouy-en-Josas, France; CEA, iBiTecS/Service de Pharmacologie et d'Immunoanalyse, Gif-sur-Yvette, France.
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Drumare
authors.affiliation: Université Paris Descartes-Assistance Publique des Hôpitaux de Paris, Hôpital Necker Enfants Malades, Paris, France.
authors.forename: Evelyne
authors.initials: E
authors.lastname: Paty
authors.affiliation: Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado Denver, Aurora, Colo.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Dreskin
authors.affiliation: INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, Jouy-en-Josas, France; CEA, iBiTecS/Service de Pharmacologie et d'Immunoanalyse, Gif-sur-Yvette, France.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Wal
authors.affiliation: INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, Jouy-en-Josas, France; CEA, iBiTecS/Service de Pharmacologie et d'Immunoanalyse, Gif-sur-Yvette, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Adel-Patient
authors.affiliation: INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, Jouy-en-Josas, France; CEA, iBiTecS/Service de Pharmacologie et d'Immunoanalyse, Gif-sur-Yvette, France. Electronic address: stephane.hazebrouck@cea.fr.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Hazebrouck
mesh_terms: D055516:2S Albumins, Plant / Q000737:chemistry* / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry* / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology*; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline / Q000737:chemistry*; D007073:Immunoglobulin E / Q000276:immunology; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D055516:2S Albumins, Plant; D052179:Antigens, Plant; C500890:Ara h 2 allergen, Arachis hypogaea; D000939:Epitopes; D006023:Glycoproteins; D010455:Peptides; D007073:Immunoglobulin E; D006909:Hydroxyproline
keywords: Food allergy; IgE-binding; conformational and linear epitopes; hydroxyproline; peanut allergen; posttranslational modifications