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Fractionation and identification of the allergic proteins in Aspergillus species
M Falahati1, S Ghanbari1, M Ebrahimi1
1Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Background And Purpose:
Allergy is an undesired immune response to non-pathogenic agents. However, some opportunistic microorganisms such as fungi can also cause allergy. Among those fungi, hyphae form of Aspergillus strains including A. fumigatus, A. flavus, and A. niger could be mentioned. In this study, we aimed to separate allergic proteins from Aspergillus strains and determine their identity.
Materials And Methods:
Standard species of Aspergillus strains were cultivated in optimized conditions and the mycelium was separated by centrifugation. The fungal cells were lysed through physical methods such as freeze-thawing and grinding to prepare a suitable protein extract. The protein concentration was measured by Bradford method and the electrophoretic pattern of the extract was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were fractionated by ammonium sulfate precipitation and anion exchange chromatography using fast protein liquid chromatography (FPLC) system. The IgE immunoreactivity of the sensitized patients and controls was studied using the fractionated proteins by enzyme-linked immunosorbent assay (ELISA). Following SDS-PAGE, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes and the strips were blotted with allergic patients' and controls' sera. The immunoreactive bands were excised from colloidal coomassie-stained SDS-PAGE gels and studied by mass spectroscopy methods.
Results:
Among the studied species, A. fumigatus showed stronger IgE reactivity and more IgE reactive protein bands than others did. The proteins with higher molecular weights showed stronger immunoreactivity in Western blotting. Receiver operating characteristic curve analysis demonstrated a correlation between the results of the applied ELISA methods. One of the most prominent IgE-reactive proteins was confirmed to be 45 kDa mycelia catalase.
Conclusion:
Our findings confirmed that high molecular weight proteins might play a major role in allergy and IgE reactivity to Aspergillus species. Moreover, the results showed that precipitation and chromatographic methods are applicable for fractionation of fungal proteins such as mycelial catalase.
Insights
This study identifies high molecular weight proteins in Aspergillus strains as key allergens. Mycelial catalase was confirmed as a significant IgE-binding protein, important for understanding fungal allergy.
Area of Science:
- Mycology
- Immunology
- Biochemistry
Background:
- Allergies arise from immune responses to non-pathogenic agents, including fungi like Aspergillus.
- Specific Aspergillus strains, such as A. fumigatus, A. flavus, and A. niger, are known to cause allergic reactions.
Purpose of the Study:
- To isolate and identify allergenic proteins from Aspergillus strains.
- To investigate the IgE reactivity of these fungal proteins in allergic patients.
Main Methods:
- Cultivation of Aspergillus strains and extraction of mycelial proteins.
- Fractionation of proteins using ammonium sulfate precipitation and anion exchange chromatography.
- Analysis of IgE immunoreactivity via ELISA and Western blotting, followed by mass spectrometry.
Main Results:
- Aspergillus fumigatus exhibited stronger IgE reactivity compared to other tested species.
- Higher molecular weight proteins demonstrated increased immunoreactivity.
- A 45 kDa mycelial catalase was identified as a prominent IgE-binding protein.
Conclusions:
- High molecular weight proteins are significant contributors to IgE reactivity in Aspergillus allergies.
- Precipitation and chromatographic techniques are effective for isolating fungal allergens like mycelial catalase.
