title: Trypsin resistance of the major peanut allergen Ara h 6 and allergenicity of the digestion products are abolished after selective disruption of disulfide bonds.
abstract: METHODS
  2S-albumins Ara h 2 and Ara h 6 are the most widely recognized and potent allergens for peanut-allergic patients. These allergens are particularly resistant to proteolysis and the digestion products generally retain significant allergenicity. Five disulfide bridges (DB) stabilize Ara h 6 overall structure and their influence on the trypsin resistance and on the allergenicity of the digestion products was investigated.
  
  
  RESULTS
  Progressive disruption of each DB was performed by site-directed mutagenesis. Successful refolding of Ara h 6 variants was confirmed by circular dichroism. Trypsin resistance, IgE-binding capacity and allergenic potency, as assessed by in vitro mediator release assay with sera from peanut-allergic patients, was not affected by the deletion of the C-terminal DB at Cys(84) -Cys(124) . Additional disruption of DB at Cys(14) -Cys(71)  or at Cys(73) -Cys(115)  rendered Arg(16/20)  or Arg(114)  susceptible to trypsinolysis, respectively, but affected principally the IgE-binding capacity of Ara h 6. DB disruption at Cys(26) -Cys(58)  or at Cys(59) -Cys(107)  led to an extensive proteolytic degradation and a complete loss of allergenic potency of the digestion products.
  
  
  CONCLUSIONS
  Selective disruption of the DB stabilizing the protease-resistant core of Ara h 6 eliminated the IgE-binding capacity of the trypsin-degradation products and their ability to trigger mast cell degranulation.
authors.affiliation: INRA, UR 496, Unité d'Immuno-Allergie Alimentaire, CEA/iBiTeC-S/SPI, CEA de Saclay, Gif-sur-Yvette, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Hazebrouck
authors.forename: Blanche
authors.initials: B
authors.lastname: Guillon
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Drumare
authors.forename: Evelyne
authors.initials: E
authors.lastname: Paty
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Wal
authors.forename: Hervé
authors.initials: H
authors.lastname: Bernard
mesh_terms: D055516:2S Albumins, Plant / Q000276:immunology* / Q000378:metabolism*; D000485:Allergens / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000276:immunology* / Q000378:metabolism*; D002648:Child; D002675:Child, Preschool; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007223:Infant; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021183:Peanut Hypersensitivity / Q000276:immunology* / Q000503:physiopathology; D059748:Proteolysis; D014357:Trypsin / Q000378:metabolism*
chemical_list: D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C492737:Ara h 6 allergen, Arachis hypogaea; D007073:Immunoglobulin E; D014357:Trypsin