Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes (1)
Until now, 12 allergens (Ara h 1 to Ara h 13, with Ara h ¾ describing the same protein) have been identified in peanut (Arachis hypogaea). Ara h 1, Ara h 2, and Ara h 3 were initially recognized as the major peanut allergens. Recently, Ara h 2 and Ara h 6 were described as being the most clinically relevant peanut allergens, as observed in vitro with effector cell–based assays and in vivo with skin prick tests. Ara h 2 and Ara h 6 share a compact conformation characterized by 5 a-helical structures and stabilized by a network of 4 conserved disulfide bridges. Ara h 2 and Ara h 6 are 59% homologous, but compared with Ara h 6, 2 insertions of 14 and 26 amino acid residues occur in the Ara h 2 major isoforms Ara h 2.01 and Ara h 2.02. A predominance of IgE recognition of conformational epitopes on Ara h 2, whereas others suggested a higher proportion of IgE binding to linear epitopes.
The peptide 1-21 (RQQWELQGDRRCQSQLERANL) covered the N-terminal part of Ara h 2. The unfolded state of reduced and S-alkylated (r/a) Ara h 2 was confirmed by CD spectra with a single spectrum minimum close to 200 nm instead of the 2 broad minima at 208 and 222 nm, which is typical for the a-helical secondary structures largely present in nAra h 2. This denaturing treatment reduced considerably the IgE-binding capacity of nAra h 2.
For 9 of 18 patients (sera 313, 576, 101, 102, 486, 572, 841, 109, and 907), r/a nAra h 2 retained a significant IgE reactivity, with the concentration inhibiting 50% of IgE binding to labeled allergen ranging from 0.5 to 750 nmol/L. The isoform nAra h 2.02 displayed a slightly but significantly higher IgE-binding capacity than nAra h 2.01. The IgE-binding capacity of the recombinant allergen remained lower than that of the native form. The influence of posttranslational modifications that naturally occur in peanut seeds but not in prokaryotes was then further investigated. Inhibition of IgE binding to nAra h 2 was always more efficient with the 27-residue-long peptide 3POH, with 3 DPYSPOHS motifs, than with the 15-residue-long peptide 2POH, with only 2 DPYSPOHS motifs. Peptide 1-21 did not exhibit any significant IgE-binding capacity for any of the French sera.
The synthetic peptides did not possess any intrinsic ability to induce mediator release and that basophil degranulation was actually dependent on the presence of specific IgE antibodies.
Although domain 1-21 was reported to contain immunodominant IgE-binding epitopes, the corresponding synthetic peptide rarely displayed a significant inhibitory capacity. Ara h 2 digested with trypsin/chymotrypsin displayed a minimal reduction in IgE-binding capacity and allergenicity. Even with an extensive reduction in the size of the IgE-binding peptides and a substantial decrease of IgE-binding capacity, peanut flour hydrolysates still displayed high allergenic potency because of Ara h 2 fragments. Accordingly, any peptide containing more than 1 DPYSPOHS motif, even those of less than 3 kDa, could induce mast cell degranulation and thereby present an allergenic risk.