RATIONALE: Epitopes were delineated for allergenic proteins, but studies are required to identify residues mediating IgE binding. In the present study, the in silico approach was used to identify IgE-binding residues of Alt a 13(1-50) fragment and confirmed by experimental approach. METHOD AND RESULTS: IgE-binding epitopes of Alt a 13 mapped computationally were cloned, expressed, purified, and characterized using various immunochemical and biophysical methods. Among four fragments of Alt a 13, Alt a 13(1-50) demonstrated maximum IgE binding with two immunodominant regions and was mutated at these regions. The mutation in first region, Alt a 13(1-50)-K4A_S6F, did not show any change in immunological and biophysical properties of protein. However, mutations in the second region, Alt a 13(1-50)-T21F_N27I, caused reduced IgE binding, histamine release, and low IL-4 release on stimulation of Alternaria alternata positive patients peripheral blood mononuclear cells in vitro. CONCLUSION: This suggests that residues T21 and N27 are important for the secondary structure. In conclusion, Alt a 13(1-50)-T21F_N27I with reduced Th 2 response and intact T-cell proliferation capacity has potential for clinical use.
RATIONALE: Epitopes were delineated for allergenic proteins, but studies are required to identify residues mediating IgE binding. In the present study, the in silico approach was used to identify IgE-binding residues of Alt a 13(1-50) fragment and confirmed by experimental approach. METHOD AND RESULTS: IgE-binding epitopes of Alt a 13 mapped computationally were cloned, expressed, purified, and characterized using various immunochemical and biophysical methods. Among four fragments of Alt a 13, Alt a 13(1-50) demonstrated maximum IgE binding with two immunodominant regions and was mutated at these regions. The mutation in first region, Alt a 13(1-50)-K4A_S6F, did not show any change in immunological and biophysical properties of protein. However, mutations in the second region, Alt a 13(1-50)-T21F_N27I, caused reduced IgE binding, histamine release, and low IL-4 release on stimulation of Alternaria alternata positive patients peripheral blood mononuclear cells in vitro. CONCLUSION: This suggests that residues T21 and N27 are important for the secondary structure. In conclusion, Alt a 13(1-50)-T21F_N27I with reduced Th 2 response and intact T-cell proliferation capacity has potential for clinical use.
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