Cloning, Production, Characterization and IgE Cross-Reactivity of Different Manganese Superoxide Dismutases in Individuals Sensitized to <i>Aspergillus fumigatus</i>

Abstract

Background: Manganese superoxide dismutase (MnSOD) from Aspergillus fumigatus has been demonstrated to be an allergen showing a high degree of homology with phylogenetically distant MnSODs. We describe cloning, production and characterization of MnSODs from different species. Methods: MnSODs were cloned by PCR, expressed as inclusion body protein in Escherichia coli, purified by Ni²⁺-chelate affinity chromatography and characterized. Results: The MnSODs from A. fumigatus, man, yeast, Drosophila melanogaster and E. coli show about 50% identity and 70% similarity on the primary structure. All proteins were produced at a high level in E. coli and refolded to achieve enzymatic activity. The proteins were able to bind IgE from sera of individuals sensitized to the A. fumigatus MnSOD. Conclusions: MnSOD represents a novel pan-allergen restricted to individuals sensitized to A. fumigatus.

Comparison of the Crystal Structures of the Human Manganese Superoxide Dismutase and the Homologous Aspergillus fumigatus Allergen at 2-Å Resolution

“…In the case of recombinant MnSOD, a native-like conformation could be confirmed by measurement of the enzymatic activity of the protein. Moreover, inhibition experiments using different MnSODs and serum of individuals sensitized to A. fumigatus MnSOD showed that the enzymes share common IgE-binding epitopes (14). Sequential site-directed mutagenesis of the conserved, surface-displayed amino acid residues described above will allow experimental determination of the contribution of each single residue to the clinically observed crossreactivity of A. fumigatus-allergic individuals toward MnSODs from different species.…”

“…The protein was reconstituted by dialysis against water containing 0.1 mM MnCl 2 . Molecular size and purity were analyzed by denaturing, reducing SDS-PAGE and native-like folding was confirmed by determination of the enzymatic activity (14). For crystallization experiments the recombinant protein was concentrated to 15 mg/ml by ultrafiltration (Centrex UF-2; Schleicher & Schuell, Dassel, Germany).…”

“…The MnSOD of Aspergillus fumigatus, a fungus involved in many pulmonary complications ranging from benign colonization of the lung to life-threatening diseases (12), has been isolated as an IgE-binding protein from a cDNA library displayed on the surface of filamentous phage (13). Furthermore, it has been shown that the homologous proteins from Saccharomyces cerevisiae, Drosophila melanogaster, and E. coli are able to bind IgE from individuals sensitized to A. fumigatus MnSOD (14). These data suggest that MnSODs from phylogenetically distant species represent a family of cross-reactive structures involved in allergic reactions.…”

“…An additional 24 amino acids are identical or similar in the two sequences and at least 30% solvent exposed in both structures (shown in green boxes). and E. coli are able to bind IgE from A. fumigatus-sensitized individuals (14), suggesting IgE cross-reactivity between these organisms. Sequence alignment of the MnSODs from these organisms together with the MnSODs from A. fumigatus and human results in an identity of 19% and a similarity of 34% of the alignment length (48 identical plus 38 similar amino acids).…”

Generation of Th1 T cell responses directed to a HLA Class II restricted epitope from theAspergillusf16 allergen

“…allergen.org). Most of the identified allergens elicit humoral immune responses [26][27][28][29], others are toxic to PBMC or are strongly homologous to human proteins, while some appear to prime primarily a Th2 T cell response [30][31][32][33]. Two allergens have been shown to stimulate both T and B cell responses from patients with ABPA, Asp f2 and Asp f16 [34,35].…”