Effect of Processing on the Structure and Allergenicity of Peanut Allergen Ara h 2 Roasted in a Matrix

Abstract

Peanut allergy is the leading pediatric food allergy. Many attempts have been made to reduce its allergenicity by processing. After roasting, Ara h 2 and its derivatives in the matrix were isolated by immunoaffinity chromatography (IAC). The structure and allergenicity of Ara h 2 were analyzed by circular dichroism, mass spectrometry (MS), western blotting, the enzymelinked immunoassay, and cell modeling. Our results showed that a large portion of Ara h 2 was fragmented and cross-linked. Ara h 2 monomers accounted for only 13% of the total proteins after IAC purification. In addition, the structure of Ara h 2 changed after roasting. In addition to methylation and oxidation modification, the disulfide bonds of Ara h 2 were found to be rearranged after roasting. In the conformational structure of Ara h 2, the content of the α-helix decreased from 27.1 to 21.6% after roasting, while the content of the random coil increased from 29.1 to 34.3%. Six cleavage sites of trypsin were exposed, while three were covered. In terms of allergenicity, most of the cross-linking products were not recognized by patients' sera. Only one faint band around 40 kDa was observed in our blotting. For Ara h 2 monomers, roasting enhanced their IgE binding capacity and ability to stimulate the degranulation of basophils. The potential allergenicity increase of Ara h 2 monomers did not reflect the allergenicity change of Ara h 2 in the matrix due to the amount and property of its derivatives after roasting.

Effect of Structural Targeted Modifications on the Potential Allergenicity of Peanut Allergen Ara h 2

“…Carrying the six-histidine tag, wild-type Ara h 2 (wAra h 2) and the mutants were purified by Ni-ion affinity gel columns (Sangon Biotech). Finally, wAra h 2 and its mutants were verified by Western blotting (WB) 4 and mass spectrometry. 3 Structure Characterization and Simulation.…”

“…3 Structure Characterization and Simulation. The conditions were based on the method of Chang et al 4 A MOS-450 spectropolarimeter (French Bio-Logic SAS, Claix, France) was used for the analysis of secondary structures by circular dichroism (CD) spectroscopy. The concentration of Ara h 2 was 0.1 mg/mL (dissolved in PBS) for the far-ultraviolet region ranging from 190 to 240 nm.…”

“…Total peanut protein was extracted by defatting fresh peanuts (Ganhua 7, Nanchang, China) and purified using a homemade immunoaffinity chromatography (IAC) column to obtain natural Ara h 2 (nAra h 2) . The pET-30a vectors with Ara h 2 and mutated sequences were transformed into Escherichia coli BL21­(DE3) .…”

“…3 In terms of the allergenicity of peanut that resides in the proteinaceous fraction of peanut kernels, 17 allergens have been described, named Ara h 1−Ara h 18 (Ara h 4 is regarded as the isoform of Ara h 3.02). 4 Ara h 2, one of the most potent components of peanut allergenicity, is recognized by the sera of >90% of peanut allergic patients, and it contains five α-helices and four pairs of disulfide bonds. 5 Peanut allergens trigger allergy by their localized structures, and these structures, which could be bound by IgE, are called epitopes.…”

“…Therefore, more attention has been focused on peanuts . In terms of the allergenicity of peanut that resides in the proteinaceous fraction of peanut kernels, 17 allergens have been described, named Ara h 1–Ara h 18 (Ara h 4 is regarded as the isoform of Ara h 3.02) . Ara h 2, one of the most potent components of peanut allergenicity, is recognized by the sera of >90% of peanut allergic patients, and it contains five α-helices and four pairs of disulfide bonds …”