Crosslinking of peanut allergen Ara h 2 by polyphenol oxidase: digestibility and potential allergenicity assessment.

BACKGROUND: Peanut is one of the eight major food allergens. Its allergen, Ara h 2, can be recognized by over 90% of serum IgE samples from peanut-allergic patients. Therefore, reducing the allergenicity of Ara h 2 is especially important. RESULTS: In the present study, polyphenol oxidase (PPO), a protein cross-linking reaction catalyst that acts on tyrosine residue, was used to modify Ara h 2. After crosslinking, the microstructure, digestibility, IgG binding capability and IgE binding capability of Ara h 2 were analyzed. Cross-linking decreased the potential allergenicity of Ara h 2 by masking the allergen epitope, while the antigenicity of Ara h 2 changed slightly. After crosslinking, the apparent diameter of Ara h 2 was altered from 300 to 1700 nm or 220 nm, indicating that polymerization could either be inter- or intramolecular. Regarding digestibility, crosslinked Ara h 2 was relatively more easily digested by gastric fluid compared with the untreated Ara h 2, but much more difficult in the intestinal fluid. CONCLUSION: The crosslinking reaction catalyzed by PPO, as a non-thermal process, may be beneficial for avoiding food allergy. The reaction could mask allergen epitopes, decreasing the allergenicity of Ara h 2. © 2015 Society of Chemical Industry.

Expression of recombinant Ara h 6 in Pichia pastoris but not in Escherichia coli preserves allergic effector function and allows assessment of specific mutations.

SCOPE: Ara h 6 has recently been recognized as an important peanut allergen. Recombinant allergens have been used for analysis of IgE binding, but have not been used to analyze the allergic effector activity that is more relevant to allergic reactions. METHODS AND RESULTS: Ara h 6 was expressed as a recombinant protein in both Escherichia coli and Pichia pastoris (rAra h 6-E. coli and rAra h 6-Pichia, respectively). Effector activity was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from patients with severe peanut allergy. Compared to native Ara h 6 (nAra h 6), rAra h 6-Pichia had intact effector function whereas rAra h 6-E. coli had significantly reduced function. The lower effector activity in rAra h 6-E. coli compared to nAra h 6 and rAra h 6-Pichia did not appear to be due to differences in posttranslational modifications (analyzed by mass spectrometry and staining for carbohydrates) and may be due to subtle alteration(s) of folding seen on CD analysis and on nonreduced gels. Finally, we introduced point mutations in four important IgE-binding linear epitopes of Ara h 6 and found dramatically reduced allergic effector activity. CONCLUSION: Our studies demonstrate the utility of fully functional rAra h 6-Pichia as a starting point for analysis of specific mutations that adversely affect allergic effector function.