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THE IDENTIFICATION AND CHARACTERIZATION OF AN IGE-INDUCING PROTEIN IN METARHIZIUM ANISOPLIAE EXTRACT
Citation:
Ward, MDW, L B. Copeland, M. J. Donohue, AND J A. Shoemaker. THE IDENTIFICATION AND CHARACTERIZATION OF AN IGE-INDUCING PROTEIN IN METARHIZIUM ANISOPLIAE EXTRACT. Presented at Society of Toxicology, Baltimore, MD, March 21-25, 2004.
Description:
The Identification and Characterization of an IgE-Inducing Protein in Metarhizium anisopliae Extract
Marsha D.W. Ward1, Lisa B. Copeland1, Maura J. Donahue2, and Jody A. Shoemaker3
1ORD, NHEERL, US EPA, RTP, NC; 2Oak Ridge Institute for Science and Education, Cincinnati, OH; 3ORD, NERL, US EPA, Cincinnati, OH.
BALB/c mice exposed by involuntary aspiration to Metarhizium anisopliae extract (MACA), a microbial pesticide, have shown responses characteristic of human allergic lung disease. The current study was undertaken to identify and characterize fungal allergens from MACA. We have identified 4 protein bands on SDS-PAGE Western blots that bind IgE in hyperimmune serum. These proteins have an apparent molecular weight of 127.6 kDa, 114.7 kDa, 89.7 kDa, and 52.6 kDa, respectively. Immunoblots of 2-dimensional (2-D) gels shows 8 protein spots with acidic pIs ranging between pH 4.8 and pH 5.5. These protein spots were collected following alignment of 2-D gel Western blot autoradiographs with Coomassie blue stained 2-D gels. Subsequently, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS) were used to analyze these isolated fungal peptides and proteins. 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 using the MASCOT database-mining algorithm. Database mining results showed a high homology between one of the MACA proteins and catalase. (Catalase is an enzyme that converts H2O2 to water and O2 -probably as a defense mechanism.) A subsequent immunoblot, using rabbit polyclonal antibodies against bovine liver catalase confirmed one of the proteins as catalase. Identification, characterization, and purification of the IgE-inducing proteins in this fungal extract will provide a tool for assessing relative potency by comparison to better characterized allergens such as house dust mite. These newly characterized fungal proteins will be added to the database of allergens, which collectively can be used to identify other structures as potential allergens. (This abstract does not reflect EPA policy.)