Identification, Characterization, and Epitope Mapping of Tree Nut Allergens

Recent years have seen a dramatic worldwide increase in allergies and asthma that can now be described as reaching epidemic proportions. Food allergies affect approximately 2% of the adult population and up to 6% of the pediatric population. Tree nut allergies, in particular, affect about 0.5% of the US population. Five commonly consumed tree nuts in the US are almonds, walnuts, cashews, pistachios, and pecans, all of which are allergenic to a subset of the consuming population. Among the proteins thus far associated with tree nut allergies include the seed storage proteins belonging to the 2S albumin and 11S globulin gene families. Complementary DNA expression libraries were created from English walnut and cashew nut embryos. To identify the 2S albumin and 11S globulin genes in both nuts, the libraries were used either directly as targets for degenerative primers in PCR 'fishing' experiments or transferred to nitrocellulose membranes and screened with nut allergenic patient sera. Once the genes were identified and amplified, they were modified with restriction enzymes, ligated into an expression vector, and expressed as fusion proteins. These purified fusion proteins were used to screen for walnut and cashew nut-allergic patient IgE-reactivity in direct immunoblots and to identify the native counterparts of these proteins in crude nut extracts via inhibition immunoblots. Synthetic overlapping peptide libraries were created using the SPOTs technology and the linear IgE-binding epitopes of each allergen subsequently determined by screening with pooled patient sera. For the walnut 2S albumin and cashew 11S globulin the most reactive epitopes were examined further and amino acids critical or influential for IgE binding identified. Some reactive peptides appeared to be conserved in sequence position and composition among the 2S albumins and 11S globulins of the walnut, cashew, and other previously characterized plant allergens when analyzed in comparative epitope maps. Structural motifs that might explain the conservation of these antibody-binding regions among the proteins were identified through homology modeling experiments. Largely unexplored in terms of allergen epitope characterization are those of a conformational nature, comprised of amino acids distant in the proteins primary structure but adjacent once the protein folds. A chimeric molecule-based strategy for identifying the existence and proposed location of a conformational IgE-binding epitope was successfully carried out for the cashew 11S globulin.