Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity(1)
When purified from raw peanut it is a Mr 210 kDa trimeric protein, composed of 63 kDa N-glycosylated subunits which can form multimers of up to Mr 600–700kDa depending on extrac- tion conditions. Ara h 1 is thermostable, only undergoing irreversible denaturation and extensive aggregation after passing through the main endotherm transition occurring above 80C.
N-Ara h 1 (4 mg/mL) in 32.5 mM phosphate buffer containing 100 mM NaCl was heated to 100C alone (heated Ara h 1, H-Ara h 1) or in the presence of 100 mM glucose (glycated Ara h 1, G-Ara h 1) for 15min.
Boiling (H-Ara h 1) resulted in aggregation and hydrolysis of Ara h 1 as indicated by the appearance of lower Mr poly- peptides ranging from 6 to 67kDa and the generally smeared appearance of the gel track. Addition of glucose during boiling (G-Ara h 1) resulted in the formation of high Mr polypeptides of masses 4200 kDa.
Boiling, alone or in the presence of glucose caused a partial loss of secondary structure, as indicated by the loss of the positive maximum at 192–195 nm, and the positive molar ellipticity at 190 nm in the circular dichroism (CD) spectra of H- and G-Ara h 1. Analysis of the secondary structure content indicated that the protein had a much reduced a-helical content of only 5.9% (compared with 22.3% for the native protein) and an increase in b-sheet content from 24.6 to 38.1%, the content of b-turn and unassigned structures remaining unaltered.
H-, G- and R-Ara h 1 were all highly aggregated with Mr of >640000 by gel permeation chromatography and dimensions of in excess of 50nm by fixed angle dynamic light scattering.
Typically, boiling resulted in either a very slight (#2206) or more marked (#66) reduction in IgE-binding capacity of Ara h 1. This was reflected in the increase in IC50 values for H-Ara h 1 compared with N-Ara h 1 of 1.5- to 5800-fold (mean = 715) and 1.8 to 10 000-fold (mean = 1236) for G-Ara h 1.
Similar patterns of reactivity were observed when following b-hexosaminidase release with a humanized RBL cell line passively sensitized with sera from six patients (serum #66). However, the loss of reactivity of the H- and G-Ara h 1 was smaller than that observed by EAST, the EC50 values being increased 6.9- and 7.5-fold, respectively, for H-Ara h 1 and G-Ara h 1 compared with N-Ara h 1.
Stimulation of PBMC cultures from NA controls by Ara h 1 did not induce significant proliferation. Only three of the 12 PA subjects showed detectable numbers of Ki-671 proliferating cells in the Ara h 1 stimulated cultures. The production of IL-5 and IL-13 was significantly enhanced in the culture of PBMC from 5 out of 12 PA subjects upon stimulation with N-Ara h 1.
Boiling for 15min did not completely abrogate IgE reactivity; however, it may be possible to reduce it further through application longer or of higher temperature wet- heating regimes thus supporting the premise that boiling, as opposed to roasting, may reduce the allergenicity of peanut.
Zaborsky, N., Brunner, M., Wallner, M., Himly, M. et al.,Antigen aggregation decides the fate of the allergic immune
response. J. Immunol. 2010, 184, 725–735.
