Identification Of The Ara H 2 Tcell Epitopes

Twenty-nine overlapping peptides representing the amino acid sequence of the Ara h 2 protein were synthesized to determine which regions were capable of stimulating CPE-specific T-cell lines to proliferate. Each peptide was 20 amino acids long and was offset from the previous peptide by 5 amino acids. In this manner, the entire length of the Ara h 2 protein could be studied in large overlapping fragments. These peptides were used in a T-cell proliferation assay to identify which Ara h 2 fragments contained T-cell epitopes. CPE-specific T-cell lines that displayed a high SI when stimulated with purified Ara h 2 protein were used to map T-cell epitopes. Experiments were performed with 17 CPE-specific T-cell lines that showed high SIs when stimulated with the intact Ara h 2 protein. Data from all T-cell lines tested were compiled and shown as an average SI for each peptide (Fig. 5). The five T-cell epitopes identified in this manner were centered on amino acid residues 19-33, 39-58, 84-93, 99-109, and 135-148 which correspond to peptides number 908, 913, 924. 926 and 931 in

mean.

mean.

1.5 nnn hn hhnnh 0

904 906 908 910 912 914 916 918 920 922 924 926 928 930 932 Peptide Number

Figure 5. Proliferation of T cells isolated from peanut allergic individuals in response to Ara h 2 derived peptides. T-cell proliferation was measured by 3H-thymidine incorporation in the DNA of proliferating cells and reported here as stimulation index (SI, y-axis). The mean SI of the T cells of all 17 allergic individuals is shown here.

the figure. Most T-cell lines (~70%) were stimulated to proliferate by multiple Ara h 2 regions. In addition, there were no common amino acid sequence motifs shared by all the epitopes.

To understand food hypersensitivity reactions it is important to identify and characterize the protein allergens that are responsible for the immunologic disorder as well as the immunological responses. What is so different about the protein allergens that causes them to be recognized as pathogenic moieties by the immune system? Why do peanut allergies persist throughout the life of an individual while allergies to similar proteins in other legumes such as soy are outgrown early in life? Various proteins involved in hypersensitivity reactions to peanut have been identified (8, 9, 10, 11, 12), three of which have been cloned and characterized (Ara h 1, Ara h 2 and Ara h 3, 10, 11, and 12 respectively). Identification, isolation of the native Ara h 2 and characterization

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