Tree nuts are a widely consumed food and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, macadamia, and Brazil nut. Although enjoyed safely by most individuals, allergic reactions are common and ~0.6% of the US population is allergic to one or more tree nuts. An important IgE-reactive protein in almonds is prunin (Pru du 6), a legumin, which represents a major component of the seed and has been identified as an important allergen. Two prunin isoforms have been identified: prunin 1 (Pru du 6.01) and prunin 2 (Pru du 6.02). IgE reactivity to recombinant Pru du 6.01 and Pru du 6.02 was found in the sera of 9 of 18 (50%) and 5 of 18 (28%) patients, respectively. To test for epitope stability, the recombinant proteins (rPru du 6) were treated with various denaturants. Immunodot blotting assays revealed the presence of both stable (sequential) and unstable (conformational) rPru du 6 epitopes. The sequential IgE binding epitopes on Pru du 6 were identified by solid-phase overlapping peptide analysis (SPOTs assay). Six IgE-binding sequential epitope-bearing peptides were found on Pru du 6.01 and eight on Pru du 6.02 using almond-allergic sera. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. The sequential epitopes targeted for several mAbs (4G2, aa 353LQQERQQ359 ; 2B4 and 5D1, aa 120QQGRQQ125) were identified by SPOTs assay. The 5D1 and 2B4 epitopes were found to directly overlap with an IgE binding epitope on Pru du 6.01. Immunoblots, immunodot blots, and inhibition immunoblots indicate that Pru du 6.01 is the predominate isoform in the nut. To investigate conformational epitopes on prunin (Pru du 6), phage display technology was used to identify IgE-binding epitopes. Total IgE was purified from an almond-allergic patient by affinity chromatography and used to capture phage displaying 12 aa-long peptides that mimic prunin epitopes. The phage sequences were analyzed using the Pepitope program and three conformational epitopes were identified (epitope 1, E49,N78, L80, L82, P83, L244, A245, N290, R312, L314, G316, N322, I325, Q326; epitope 2, V104, F105, H190, Q191, T193, P205, A206, G207, V208, V327, R328, G329, N330, L331, D332, F333; epitope 3, H227,S229, S230, D231, H232, F411, W431, V433, N434, H436, V451, Q473, N474, H475, G476, T493, N496, A497, F498, L502). One of these epitopes partially overlaps a sequential epitope previously identified by the SPOTs assay. Conformational epitope mapping studies were also performed using a murine monoclonal antibody (mAb) 4C10. This mAb reacts exclusively with non-reduced native prunin in immunoblotting assays, indicating that 4C10 binds to a conformational epitope expressed on prunin that is dependent on the large and small subunit association. Inhibition ELISA assays found that human IgE and IgG binding epitopes (sequential and/or conformational) overlap or sterically hinder 4C10 binding to prunin. To identify the epitope, hydrogen/deuterium exchange (HDX) monitored by 14.5 T Fourier transform ion cyclotron resonance mass spectrometry was performed on prunin and the 4C10-prunin complex. Comparison of deuterium uptake between the free vs. mAb-bound prunin identified several epitope candidate peptides that differ in deuterium uptake, suggesting that these peptides are part of the 4C10 epitope. Analyses of chimeric molecules using the homologous soybean allergen, Gly m 6, and alanine mutants further localized the epitope to three discontinuous strands (aa 21-45, 320-328, and 460-465). These data demonstrates that HDX-MS is a useful technique to aid in the identification of unknown conformational epitopes on native tree nut allergens.
