2013 Annual Report
1a.Objectives (from AD-416):
Protein plays an important role in human nutrition. However, most food allergens are proteins and food allergies have been adversely affecting an increasing number of patients. A recent study by the Centers for Disease Control and Prevention indicated that the number of American children suffering from food allergies increased 18% in just 10 years (1997-2007). Because the understanding of this potentially life-threatening IgE-mediated hypersensitivity disease is still very limited, the recommended care for individuals with known anaphylactic reactions to foods is avoidance, recognition of early symptoms of an allergic reaction, and prompt use of injected epinephrine in case of accidental ingestion. Steps to prevent severe food allergic reactions include precautions in the lunch rooms at schools and proper labeling of packaged food. Thus, the food allergy problem affects not only allergic patients but also the general public, and its negative impacts on food distribution and on the agricultural and food industries are becoming more and more serious. This study aims to increase food safety and sustainable use of agricultural crops, including tree nuts, milk and tomatoes.
The overall goal of this project is to improve basic scientific understanding of allergenicity through characterization of food allergens and to study effects of conventional and novel food processes on allergenicity. This project will concentrate on several tree nuts (almonds, pecans and peanuts) that cause severe persistent allergies, on milk which causes the most common childhood allergy, and on tomatoes that cause allergies that have received less attention.
The following are specific objectives of the proposed plan:
Objective 1: Characterize allergens from different foods to obtain information regarding their relative potency.
Objective 2: Determine the relative importance of linear and conformational epitopes in different food allergens for binding to immunoglobulin E.
Objective 3: Investigate the effect of different food processing technologies on the allergenicity of allergens in milk, tomatoes, almonds, and pecans.
1b.Approach (from AD-416):
Food allergen researchers either concentrate on studying a single allergen or a few selected allergens from a food. In this project, all known allergens from a chosen food will be purified and their relative potency will be assayed by IgE binding using patient sera with Western blot and/or ELISA. If a food belongs to one of the eight major food allergen sources (e.g. almonds considered as a tree nut) but only few allergens has been identified in it, potential allergens will also be purified based on knowledge of protein families and information about allergens in other foods to facilitate the identification of new allergens and better understanding of the allergenicity of food proteins. The relative importance of linear and conformational epitopes in different food allergens will be studied by competition ELISA using patient sera, native allergens, and allergen fragments. Nuts, milk, and fruit will be subjected to novel and conventional food processing methods, including jet cooking, extrusion, and pulsed UV. The effects of food processing on the allergenicity of food allergens will be assessed by Western blot and ELISA and by cell based methods, such as basophil activation tests in order to obtain information that can be used to understand the use of and guide the development of food processing technologies for reducing allergenic potential of food proteins.
Under Subobjective 1.1, plasmids for expressing almond allergens have been constructed. Under Subobjective 1.2, we have made significant progress in cloning pecan allergens and potential allergens.
Under Subobjective 2.1, we made significant progress in expressing and purifying fragments of peanut allergen Ara h 5. These reagents will be used for assessing the relative importance of linear and conformational IgE epitopes in peanut allergy.
Under Subobjective 2.2, we made significant progress in cloning fragments of cow’s milk allergen Bos d 5 and in purifying and characterizing the fragments.
Under Subobjective 3.1, we have expressed recombinant milk allergens with a fusion partner to enhance their solubility during expression. The recombinant allergens were purified and the fusion partner was removed with the protease.
Under Subobjective 3.2, we have expressed almond and pecan allergens and one of the pecan allergens were purified and crystallized for structural characterization.
Structural characterization of peanut allergen Ara h 5. Peanut allergen Ara h 5 is a member of the profilin family. This family of proteins includes a great number of pollen allergens food allergens and are considered pan-allergens an a big contributing factor of patients’ cross-reactivity to different foods and pollen. Structural characterization of profilin allergens is needed for understanding the allergenicity of food allergens and their cross-reactivities. ARS Researchers at Albany, California, and their collaborators solved the structure of peanut allergen Ara h 5 at a very high resolution (0.11 nanometers). The new result indicated that experimental structural information is required to avoid over-interpretation of homology-model-based prediction of immunoglobulin E epitopes. The structure of Ara h 5 and other profilin type of food allergens will facilitate the understanding of the allergenicity of food allergens and their cross-reactivity.
Jin, T., Guo, F., Wang, Y., Zhang, Y. 2013. High resolution crystal structure of human Dim2/TXNL4B. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 69(Pt 3):223-227. DOI: 10.1107/S1744309113000973.
Wang, Y., Fu, T., Howard, A., Kothary, M.H., Mchugh, T.H., Zhang, Y. 2013. Crystal structure of peanut (Arachis hypogaea) allergen Ara h 5. Journal of Agricultural and Food Chemistry. 61:1573-1578.