Effect of Heating and Glycation on the Allergenicity of 2S Albumins (Ara h 2/6) from Peanut (Vissers et al. 2011)

Ara h 2, 6 and 7 are all members of the prolamin superfamily and share a characteristic cysteine skeleton with at least 8 conserved cysteine residues and a three-dimensional structure comprising 5 alpha-helices arranged in a right-handed superhelix. Typically, loss of tertiary structure is followed by reversible unfolding, while loss of secondary structure (70–80C) leads to the formation of new intra/intermolecular interactions, rearrangements of disulfide bonds (80–90C), and formation of aggregates (90–100C). Heating for 90 min at 100C of recombinant refolded Ara h 2 led to a slight increase in its IgE binding capacity. Heating native Ara h 2 for several days at 55C in the presence of different sugars increased its IgE binding capacity compared to protein heated without sugar, which was related to the formation of AGE products. Ara h 2 extracted from heat-processed peanut, such as roasting (140C) was also found to enhance its IgE- binding capacity.

Only three of the 12 PA subjects showed detectable numbers of Ki-67+ proliferating cells (10.5+/-4.8%) in the Ara h 2/6 stimulated cultures compared with medium alone (4.7+/-2.7).

PBMC cultures from NA subjects stimulated with the native and modified Ara h 2/6 did not show any cytokine induction for all five tested cytokines. IL-5 and IL-13 production was significantly and equally enhanced in PBMC cultures from 5 out of 12 PA subjects following stimulation with N-, H- or G-Ara h 2/6. Enhanced production of IL-10 and IFN-c was observed in stimulated PBMC of PA subjects compared to NA subjects but was not significant for all stimuli tested.

Screening of the serum panel showed variable levels of IgE specific to raw peanut extract and N-Ara h 2/6.

Ara h 2/6 from roasted peanut (R-Ara h 2/6) had a similar albeit slightly reduced immunoreactivity to N-Ara h 2/6 from raw peanut. For most patients glycation reduced the IgE reactivity of Ara h 2/6 still further compared with thermal treatment alone, and is consistent with the fact that IC50 values for G-Ara h 2/6 were 1.3 to 73-fold higher than that of N-Ara h 2/6 (mean = 14.5, n = 30).

Heating reduced the biological potency of the Ara h 2/6, but intriguingly histamine release was induced at a lower concentration of heated-glycated than heated Ara h 2/6. A 150 and 130 times increased EC50 value, corresponding to a decreased allergenic activity, was observed for H-Ara h 2/6 and G-Ara h 2/6, respectively, in comparison to N-Ara h 2/6.

2S albumin allergens of peanut, Ara h2 and h6, only begin to unfold following heating at temperatures over 100C, conditions which equate to boiling for extended times (longer than 15 min). IgE responses in peanut-allergic individuals are mainly directed towards the natively folded protein. Hydrolysis and oligomerisation observed after heating with or without glucose, may have led to further linear epitope degradation and masking.