2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify and compare the structural, chemical, functional, and immunological characteristics of peanut with homologous; less allergenic legume (green pea and soy) and tree nut allergens in raw and processed forms towards delineating the clinically-relevant antibody-allergen interactions.
Objective 2: Clone, express, and purify the major peanut and select tree nut and legume allergens, and fragments thereof, in recombinant form to further delineate clinically-relevant antibody-allergen interactions. Objective 3: Assess the role of processing-induced chemical or structural modifications on the individual allergens by systematically altering amino acids thought to be important in clinically-symptomatic allergic reactions. Objective 4: Develop computer models and/or determine NMR/crystal structures of native and recombinant peanut and select tree nut and legume allergens in raw and processed forms. Objective 5: Combine the structural information obtained with the empirical knowledge from Objectives 1, 2, and 3 to identify clinically-relevant allergen-antibody interactions in peanut and tree nut allergy. Objective 6: Develop processing technologies for peanut products with reduced allergenic properties. Objective 7: Develop and improve immunoassays for detection of peanut, select tree nut, and soy allergen residues before and after processing (i.e. roasting, baking into cookies, etc.). Objective 8: After establishing standardized protocols for determining threshold doses for peanut, select tree nut, and soy allergens, determine threshold (minimal eliciting) dose of reactivity for processed forms of peanut allergens and develop computational and statistical models to estimate population thresholds.
1b.Approach (from AD-416):
Specifically, peanuts and tree nuts will be subjected to thermal processing (i.e., roasting). New allergens or changes in allergenic properties of existing allergens due to the thermal processing will be identified by immunoassays, using serum (containing IgE antibodies) from peanut and/or tree nut allergic individuals. Proteins found to be immunologically altered by thermal processing will be purified by conventional chromatography and analyzed for alterations in size, structure, digestibility, and binding to various antibodies, including anti-Maillard reaction products and specific anti-allergen antibodies. The specific amino acid residues, thought to be modified during different processing events and to contribute to altered allergenic properties (i.e. IgE binding), will be identified. These amino acids will be identified by cloning and expression of select recombinant major allergens of peanut and tree nuts in E. coli followed by site directed mutagenesis, simulated processing and immunological analysis of previously identified IgE binding sites, specifically, sites thought to be modified by processing. Understanding the molecular basis of processing-induced alterations of allergens will guide development of processing technologies towards reduced allergenicity of nuts. This knowledge will also contribute to the development of better labeling practices and detection tools for industry and regulatory agencies resulting in better protection of consumers.
The effect of D-amino acids versus L-amino acids on the binding of immunoglobulin E (IgE) antibodies to peanut allergens was determined. Results showed that D-aspartic acid in combination with D-glutamic acid inhibited IgE binding, and exhibited a stronger inhibition effect than the L amino acids. Identification of specific amino acids that contribute to IgE binding is objective 4 of the project. Also, the penetration of whole peanut kernel by pulsed ultraviolet (PUV) light and the effects on the allergenic capacity of peanut kernel (a collaborative study) was assessed. Results showed that PUV light penetrated peanut kernel and reduced allergenic potential of peanut. High pressure (HP) and PUV were used to treat almond extracts. While HP had little effect, PUV caused a reduction in the IgE binding capacity of almond extracts. Peanut allergens, heated in the presence of oleic acids became cross-linked to the oleic acid, which in turn reduced IgE binding to these allergens. Objective 3 and 6, which involve exploring and developing processing technologies in order to reduce the allergenic potential of peanuts were addressed with these experiments.
To meet the goals of Objective 2, the major cashew allergens Ana o 3 (2S albumin) was purified from cashew, and expressed in E. coli and the recombinant protein purified. An allergen from peanut Ara h 8, from peanut, and rJug r 1, an allergen from walnut; were cloned, expressed, and purified from E. coli and native Jug r 1 was purified from walnut. Both native and recombinant forms of the allergens were used for structural, immunological, and biological studies. To address Objective 3 and 4, the specific chemical modifications to the peanut allergen Ara h 1 and Ara h 2 were identified in a simulated roasting model to determine immunologic and allergic consequences of roasting and to explore methods to reduce the allergic potential. A fragment of Ara h 1 was cloned, expressed, and purified to assist in these immunological evaluations (Objective 4). Structural models were developed for one cashew, two peanut and one walnut allergens to meet objective 5.
Structural and empirical information was combined to help identify clinically relevant allergen-antibody interactions in peanut and tree nut allergy as stated in Objective 5. A peptide spot assay was optimized to screen peanut and tree nut allergic sera for binding to allergenic peptides (epitopes). A computational prediction method was enhanced to predict immunoglobulin E (IgE) binding sites (or peptide epitopes) that could be cross reactive among peanuts and tree nut allergens. The epitope identification study will assist in determining more IgE binding sites in peanuts and tree nuts, which is important for designing better, detection, diagnostic and therapeutic tools, as well as processing methods to reduce allergenic potential in nuts. Also, we have identified epitopes that cross-react among the different peanut allergens, which may be the reason that peanuts are so allergenic.
Identification of roasting-induced modifications to peanut allergens contribute to enhanced allergicity. Roasting has been shown to enhance the allergenic potency of Ara h 1 and other peanut proteins. Recombinant Ara h 1 was cloned, expressed in E. coli and purified by ARS scientists at the Southern Regional Research Center (SRRC) in New Orleans, LA. The allergen was then subjected to a simulated roasting system which causes specific chemical modifications to the allergens and it was determined that these modifications contribute to enhanced recognition by immune cells in collaboration with National Institute of Environmental and Health Science (NIEHS). Understanding what modifications contribute to enhance immunoglobulin E binding and immunological reactions to peanut will enable us to design better tools to reduce the allergenic potential.
Improvement of computational tools to predict cross-reactive binding sites among peanuts and tree nuts. Individuals with peanut or tree nut allergy are often allergic to multiple nuts, but we are unable to predict which nuts based on existing diagnostic tests, with the exception of a food challenge (feeding the food to the patient). A computational prediction method developed to predict clinically-relevant immunoglobulin E (IgE) binding sites (or peptide epitopes) among peanuts and tree nuts is being improved with wet-lab experimentation using clinically well-characterized nut allergic patient sera by ARS scientists at the Southern Regional Research Center (SRRC) in New Orleans, LA. This method allows previously unknown epitopes to be identified in walnut and other nuts based on known epitopes of peanut allergens. This finding is important for designing better detection, diagnostic and therapeutic tools as well as processing methods to reduce allergenic potential in nuts.
False positive detection of peanut residue in liquid caramel coloring using commercial enzyme linked immunsorbent assay (ELISA) kits. Scientists at University of Nebraska, Food Science Department determined that ELISA methods are not reliable for the detection of peanut in class IV caramel ingredients and their use is not recommended with this matrix.
Gene expression of pecan nut allergens peaks at dough stage during nut development. The expression of pecan allergens was characterized during pecan nut development. ARS scientists at Southern Regional Research Center (SRRC) in New Orleans, LA, in cooperation with Louisiana State University (LSU) scientists found that transcript levels of the Car i 1, Car i 4, and pecan 7S vicilin genes in the ‘Desirable’ and ‘Sumner’ cultivars peaked during the growing season in September, during the dough stage. Both cultivars had similar expression patterns and levels of transcripts during nut development. Understanding the timing of allergen gene transcription could assist plant breeders to develop cultivars with lower levels of allergens.
Testing methods to reduce the allergenic properties of peanuts. Peanut allergy is very severe and rarely outgrown. Previously it was thought that pulsed ultraviolet (PUV) light cannot penetrate a whole peanut kernel. ARS scientist at the Southern Regional Research Center (SRRC) in New Orleans, LA, recently proved that it could. By doing so, the energy from the PUV light transformed the inside peanut allergens into less allergenic proteins, and thus potentially produced a less allergenic peanut kernel. The research could lead to development of less allergenic peanut-based products and beverages.
Li,Y., Yang, W., Chung, S., Chen, H., Teixeira, A.A., Gregory, J.F., Welt, B.A., Shriver, S. 2013. Effect of Pulsed Ultraviolet Light and High Hydrostatic Pressure on the Antigenicity of Almond Protein Extracts. Food and Bioprocess Technology. 6(2):431-440.
Mattison, C.P., Tarver, M.R., Florane, C.B., Graham, C.J. 2013. Temporal expression of pecan allergens during nut development. Journal of Horticultural Science and Biotechnology. 88-2:173-178.
Cabanillas, B., Cheng, H., Grimm, C.C., Hurlburt, B.K., Rodriguez, J., Crespo, J.F., Maleki, S.J. 2012. Pine nut allergy: clinical features and major allergens characterization. Allergy. 56:1884-1893.
Nesbit, J.B., Hurlburt, B.K., Schein, C., Cheng, H., Wei, H., Maleki, S.J. 2012. Ara h 1 structure is retained after roasting and is important for enhanced binding to IgE. Molecular Nutrition and Food Research. 56:1-9.