Hypoallergenic mutants of Ana o 2, a major cashew allergen & identification and characterization of tree nut allergens

Allergic diseases are a heterogeneous group of type I IgE-mediated hypersensitivity reactions affecting more than 25% of the world's population of developed countries becoming a major clinical and public health issue. According to the American Academy of Allergy, Asthma and Immunology (AAAAI), allergies affect as many as 40-50 million people in the United States, making it the sixth most common cause of chronic illness. Recent studies have estimated that food allergies occur in 6 - 8% of children under 3 years of age and in 4% of adults in the US population. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) proposed that milk, shellfish, egg, fish, peanut, soybean, tree nuts and wheat are eight major sources of food allergens and the causes of most food allergies. Peanut, tree nuts, and seafood allergies predominate in adults, whereas milk and egg allergies are important in children.;Tree nut allergies, in particular, affect about 0.5% of the US population. The commonly consumed tree nuts in the US include walnuts, almonds, cashews, pistachios, and pecans, all of which are allergenic to predisposed individuals of the consuming population. The major allergenic proteins associated with tree nut allergies include the seed storage proteins belonging to the 7S and 11S globulin, and 2S albumin gene families.;Complementary DNA expression libraries were created from English walnut, cashew and almond. To identify the almond profilin, almond and walnut 60S ribosomal protein P2, 7S and 11S globulin genes in cashew and pistachio nuts, the libraries were used either directly as targets for degenerate primers in PCR 'fishing' experiments or transferred to nitrocellulose membranes and screened with tree nut allergenic patient sera. Upon identification and amplification of the respective coding genes, 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 tree nut-allergic patient IgE-reactivity in direct immunoblots and to identify the native counterparts of these proteins in crude nut extracts via inhibition immunoblots and/or inhibition ELISA.;Information on the linear epitopes and homology modeling of 7S and 11S globulins from cashew and pistachio would provide structural insight into the issue of serological and clinical cross-reactivity between these two nuts from the Anacardiaceae family.;The 11S globulins represent a family of most abundant seed storage proteins, and have been shown to be the most allergenic (i.e., react with high percentage of patients sera) of the seed storage proteins (allergenic in cashew, pistachio, almond, walnut, Brazil nut, and hazelnut). Linear IgE-reactive epitopes on the primary amino acid sequence of major cashew allergen and protein---11S globulin, Ana o 2 were located by overlapping synthetic peptide analysis using cashew allergic sera. Additionally, alanine scanning mutagenesis of the immunodominant IgE reactive linear epitopes was used in the identification of key amino acid residues critical for IgE binding. The identified critical amino acids would serve as targets to create hypoallergenic variants of Ana o 2, by introducing point mutations of those critical amino acids using site-directed mutagenesis in the Ana o 2 cDNA and unaltering the overall structural fold of the molecule. The biological potential of the hypoallergenic engineered Ana o 2 mutants would be analyzed using an ex vivo basophil activation assay. Thus, by reducing the anaphylactic potential of Ana o 2 while preserving T cell epitopes, we could potentially use these hypoallergens for allergen specific immunotherapy in cashew sensitized individuals.