Immunological Analysis of Allergenic Tree Nut Proteins

Allergies are a growing problem in industrialized countries, with food allergies affecting 6% of children and 3-4% of adults. Tree nuts are a common cause of food-induced allergy and include walnut, cashew, almond, hazelnut, pistachio, pecan, chestnut, and Brazil nut. Tree nut allergy, in particular, affects 0.5% of the US population. Unlike other food allergies, tree nut allergy persists throughout life and is known to cause severe allergic reactions. Therefore, it is critical to identify allergenic proteins in tree nuts that are directly involved in the allergic response and study these proteins on both the biochemical and immunological level. To identify allergenic proteins in pistachio and almond, polymerase chain reaction (PCR) was preformed using degenerate or gene specific primers, designed to amplify likely tree nut allergens in complementary cDNA libraries. The coding genes for the 7S vicilin from pistachio and lipid transfer protein (LTP) from almond were identified and were ligated into an expression vector to be expressed as fusion proteins. The generated fusion proteins were purified and used to screen almond- or pistachio-allergic individuals for IgE reactivity using immunoblot, ELISA, and dot blot assays. IgE reactivity to the pistachio 7S was found in 7 of 19 (37%) patients tested, and the allergen was designated Pis v 3. Inhibition immunoblots using the 7S fusion protein lead to the identification of the native protein in the crude nut extract. It is not uncommon for food-allergic individuals to react to more than one allergen, and tree nut allergic individuals are often sensitized to multiple nuts. In particular, patients allergic to cashew frequently report allergy to pistachio as well, which is likely a result of cross-reactivity between the two closely related tree nuts. Analysis of the 7S vicilin pistachio allergen revealed that it was highly homologous to the 7S vicilin allergen, Ana o 1, from cashew. Cross-reactivity between these two allergens was investigated by inhibition dot- and immunoblot assays using serum IgE from pistachio and/or cashew-allergic individuals. The seven patients with IgE that recognized Pis v 3 also recognized Ana o 1. Similar results were obtained using a panel of murine anti-rAna o 1 monoclonal antibodies (MAbs), as six of nine (67%) MAbs tested showed reactivity to Pis v 3 on dot-blots. The data does not identify the primary sensitizing agent but suggests that IgE reactivity to Pis v 3 and Ana o 1 is focused on the most conserved regions of the proteins. The results demonstrate that Pis v 3 is a likely contributor to the observed co-sensitivity to pistachio and cashew in some patients. Plant LTPs have been identified as allergens in a variety of fruits, vegetables, and nuts, and several studies have demonstrated cross-reactivity among LTPs. Immunoblotting using a rabbit polyclonal antibody (pAb) raised against peach LTP identified native LTP in the crude nut extract. IgE reactivity to almond LTP was found in 5 of 25 (20%) patients tested and designated Pru du 3. LTP is found in low abundance in the seed, therefore, an enriched almond extract was used in immunoblotting assays with LTP-reactive patients. Of the five LTP-reactive patients, only two recognized native LTP in the enriched nut extract. The lack of IgE reactivity by immunoblot suggests that some patients recognize primarily conformational epitopes on LTP that are destroyed under the denaturing conditions of immunoblotting. Subsequent dot blot assays confirm this hypothesis as IgE reactivity was lost when LTP was treated with common reducing reagents used in immunoblotting. Overall, the results demonstrate that LTP is an allergenic protein in almond and IgE antibodies in allergic individuals are directed against both conformational and linear epitopes.