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PROTEOMIC ANALYSIS OF ALLERGENS FROM METARHIZIUM ANISOPLIAE
Donohue, M. J., J A. Shoemaker, M J. Selgrade, M. W. Ward, AND L B. Copeland. PROTEOMIC ANALYSIS OF ALLERGENS FROM METARHIZIUM ANISOPLIAE. Presented at 51st ASMS Conference, Montreal, Canada, June 8-12, 2003.
To understand children's risks from exposure to molds in their environment and to explore risk management options for mitigating those risks.
The goal of this project is the identification and characterization of allergens from the fungus Metarhizium anisopliae, using mass spectrometry (MS). The US EPA, under the "Children at Risk" program, is currently addressing the problem of indoor fungal bioaerosol contamination. One of the research objectives is to develop a basic understanding of IgE inducing proteins from fungi, using advanced proteomics. The fungus M. anisopliae has been used as a bio-pesticide for insect control since the 1800's. Recent studies have shown that exposure to this microorganism can cause an immediate hypersensitivity or Type I allergic responses in mice1. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were used to analyze the fungal peptides and proteins isolated by 2-D gel electrophoresis and immuno-blot analysis. The MS data on molecular weight, peptide profiles, and amino acid sequence were used to mine databases for potential sequence or domain homology to known allergens. This information will provide better characterization of the M. anisopliae proteins that induce IgE responses, eventually paving the way to a better understanding as well as the prevention and/or treatment of protein allergies.
The fungal extract preparation was preformed following a modified method described by Stankus and O'Neil2. Metarhizium anisopliae was grown under three different preparations. The proteins were extracted, and assayed for total protein concentration. Equal protein concentrations of each growth condition were combined to create the M. anisopliae crude antigen (MACA) preparation.
2D gel Electrophoresis and Immuno-blot Analysis
Total protein extracts of M. anisopliae were separated using 2-D gel electrophoresis. Allergenic proteins were identified by immuno-blot analysis, using hyperimmune mouse serum against M. anisopliae extract as the primary antibody and anti-mouse IgE as the secondary antibody. Protein spots identified as inducing IgE were excised and in-gel digested. The peptides were extracted and analyzed by MALDI-TOF-MS and ESI-MS/MS. Peptide sequences were analyzed using the MASCOT data mining algorithm.
All MALDI analyses were acquired on a Bruker Biflex III. Samples were diluted 1:1 in matrix (a -HCCA, 10mg/ml; ACN 50% (v/v); TFA 0.1% (v/v). One microliter of this solution was spotted onto the MALDI target.
All nanospray ESI/MS/MS data were acquired using an Applied Biosystem QSTAR XL equipped with a Proxeon nano-ESI source. Low-energy collision induced dissociation (CID) was preformed using nitrogen as the collision gas.
Results and Discussion
Eight proteins were identified as allergenic proteins, based on IgE reactivity in the immuno-blot assay. Five of the eight proteins had apparent molecular weights greater than 75 kDa. All the eight proteins have acidic pIs in the range between 4.8 and 5.5. At this time, all eight spots have been excised from the Coomassie stained gel. Protein fingerprints have been obtained for Spots 1, 2, and 5. The mass values have been used to mine the non-redundant databases to identify these allergenic proteins. Based on database mining, the proteins have been determined to be novel and are currently being sequenced by ESI-MS/MS.
In this study, we have clearly demonstrated that exposures to M. anisopliae can induce an allergenic response by the identification of eight proteins that are reactive to IgE antibodies. Three of the identified proteins appear to be novel due to the lack of significant matches when mining the non-redundant databases. Efforts are ongoing to verify that the proteins are novel by amino acid sequencing the observed peptides.
1. Ward, M.D.; Sailstad, D.M.; Selgrade MJ.K. Toxicol. Sci. 1998, 4,195-203.
2. Stankus, R.P.; O'Neil, C.E. J. Allergy Clin. Immunol. 1988, 81, 563-570.
Record Details:Record Type: DOCUMENT (PRESENTATION/ABSTRACT)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL EXPOSURE RESEARCH LABORATORY
MICROBIOLOGICAL AND CHEMICAL EXPOSURE ASSESSMENT DIVISION
CHEMICAL EXPOSURE RESEARCH BRANCH