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Engineered Alt a 13 Fragment of Alternaria alternata Abrogated IgE Binding without Affecting T-cell Stimulation

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Abstract

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 131–50 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 131–50 demonstrated maximum IgE binding with two immunodominant regions and was mutated at these regions. The mutation in first region, Alt a 131–50-K4A_S6F, did not show any change in immunological and biophysical properties of protein. However, mutations in the second region, Alt a 131–50-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 131–50-T21F_N27I with reduced Th 2 response and intact T-cell proliferation capacity has potential for clinical use.

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Acknowledgements

Jay Shankar is an SRF from Council for Scientific and Industrial Research, New Delhi. Thanks are due to Sh. Dinesh Chandra, V.P.Chest Institute, Delhi for help in skin tests on patients. We sincerely thank the Council for Scientific and Industrial Research for the financial support for the study.

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Authors and Affiliations

  1. Institute of Genomics and Integrative Biology, Mall Road, Delhi, 110007, India

    Jay Shankar, Bhanu P. Singh & Naveen Arora

  2. Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, 110007, India

    Jay Shankar

  3. V. P. Chest Institute, University of Delhi, Delhi, 110007, India

    Shailendra N. Gaur

  4. Room 509, Allergy and Immunology Section, Institute of Genomics and Integrative Biology, Mall Road (near Jubilee hall), Delhi, 110007, India

    Naveen Arora

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  1. Jay Shankar
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Correspondence to Naveen Arora.

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Shankar, J., Singh, B.P., Gaur, S.N. et al. Engineered Alt a 13 Fragment of Alternaria alternata Abrogated IgE Binding without Affecting T-cell Stimulation. J Clin Immunol 29, 63–70 (2009). https://doi.org/10.1007/s10875-008-9224-1

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