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Trypsin resistance of the major peanut allergen A ra h 6 and allergenicity of the digestion products are abolished after selective disruption of disulfide bonds
Author(s) -
Hazebrouck Stéphane,
Guillon Blanche,
Drumare MarieFrançoise,
Paty Evelyne,
Wal JeanMichel,
Bernard Hervé
Publication year - 2012
Publication title -
molecular nutrition and food research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.495
H-Index - 131
eISSN - 1613-4133
pISSN - 1613-4125
DOI - 10.1002/mnfr.201100614
Subject(s) - trypsin , chemistry , allergen , proteolysis , digestion (alchemy) , potency , degranulation , protease , in vitro , biochemistry , peanut allergy , immunoglobulin e , enzyme , immunology , allergy , biology , antibody , receptor , chromatography
Scope 2S‐albumins A ra h 2 and A ra 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 A ra h 6 overall structure and their influence on the trypsin resistance and on the allergenicity of the digestion products was investigated. Methods and results Progressive disruption of each DB was performed by site‐directed mutagenesis. Successful refolding of A ra h 6 variants was confirmed by circular dichroism. T rypsin resistance, I g E ‐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 C ys 84 ‐ C ys 124 . Additional disruption of DB at C ys 14 ‐ C ys 71 or at C ys 73 ‐ C ys 115 rendered A rg 16/20 or A rg 114 susceptible to trypsinolysis, respectively, but affected principally the I g E ‐binding capacity of A ra h 6. DB disruption at C ys 26 ‐ C ys 58 or at C ys 59 ‐ C ys 107 led to an extensive proteolytic degradation and a complete loss of allergenic potency of the digestion products. Conclusion Selective disruption of the DB stabilizing the protease‐resistant core of A ra h 6 eliminated the IgE‐binding capacity of the trypsin‐degradation products and their ability to trigger mast cell degranulation.