Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach

Authors

  • P. Gautam,

    1. Molecular Biochemistry and Diagnostics Division, Institute of Genomics and Integrative Biology, Delhi, India,
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    • 1Both the authors contributed equally.

  • C. S. Sundaram,

    1. Proteomics Research Facility, Centre for Cellular and Molecular Biology, Hyderabad, India
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    • 1Both the authors contributed equally.

  • T. Madan,

    1. Molecular Biochemistry and Diagnostics Division, Institute of Genomics and Integrative Biology, Delhi, India,
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    • 2Present address: Department of Immunology, National Institute for Research in Reproductive Health, Mumbai, India.

  • W. N. Gade,

    1. Molecular Biochemistry and Diagnostics Division, Institute of Genomics and Integrative Biology, Delhi, India,
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    • 3Present address: Department of Biotechnology, University of Pune, Pune, India.

  • A. Shah,

    1. Department of Respiratory Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
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  • R. Sirdeshmukh,

    1. Proteomics Research Facility, Centre for Cellular and Molecular Biology, Hyderabad, India
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  • P. U. Sarma

    1. Molecular Biochemistry and Diagnostics Division, Institute of Genomics and Integrative Biology, Delhi, India,
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    • 4Present address: Department of Plant Pathology, Indian Agricultural Research Institute, Pusa, Delhi, India.


Correspondence:
Dr Taruna Madan, Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, India.
E-mail: taruna_m@hotmail.com

Summary

Background Approximately 20% of the world's asthmatics are suffering from Aspergillus fumigatus (Afu)-induced allergies. The characterization of specific IgE-inducing allergens in allergic aspergillosis patients is fundamental for clinical diagnosis and for immunotherapy.

Methods Immunoproteomics combined with mass spectrometric analysis was used to identify proteins of third-week culture filtrate (3wcf) potentially responsible for Afu-specific IgE immunoreactivity, using pooled sera from Afu-sensitized asthmatics. Their allergenic potential was also tested against patients with allergic bronchopulmonary aspergillosis (ABPA), by two-dimensional (2-D) gel electrophoresis immunoblotting of 3wcf proteins with individual sera from such patients. This helped us to establish a set of candidate allergens, which could be explored further for diagnostic application in allergic aspergillosis asthmatics including ABPA.

Results Peptide mass fingerprint using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and/or de novo sequencing by MS/MS analysis of the protein spots from 2-D gels led to the identification of a total of 16 allergens of Afu. Eleven of them are being reported as allergens for the first time and five had been reported earlier. Putative isoforms of the proteins Asp f 13 and chitosanase have been observed for the first time. When studied for reactivity of these proteins among patients with ABPA using their individual sera, these patients exhibited sensitization although the pattern was varying. Taken together, these proteins could thus be considered as potential allergens even among patients with ABPA. Three of these proteins viz. the hypothetical protein (# spot no. 5), extracellular arabinase (# spot no. 6) and chitosanase (# spot no. 11) could be major allergens with specific IgE immunoreactivity with six out of eight patients' sera.

Conclusions The immunoproteomic approach applied to the analysis of culture filtrate proteins resulted in the identification of several candidate allergens, many of them novel, contributing to the catalogue of Afu allergenic proteins, which would facilitate improved serodiagnosis for allergic aspergillosis. In addition, the immunoreactivity of these proteins observed among the patients with ABPA may be potentially useful for its serodiagnosis and opens up further opportunities for the development of personalized immunotherapeutics for patients with ABPA.

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