Alternaria alternata mannitol dehydrogenase, a fungal allergen

Abstract

Rationale Mannitol dehydrogenase (MtDH) has previously been shown to be the major allergen of Cladosporium herbarum. Since cross-reactivity has been demonstrated for several fungal allergens we have cloned the homolog protein of Alternaria alternata and tested it in respect to its IgE-reactivity. Methods A. alternata MtDH was cloned by screening of an in vivo excised A. alternata cDNA library (pBluescript SK) using the C. herbarum homolog as a hybridization probe. In order to obtain a recombinant non fusion and a 6xHIS fusion protein, the coding sequence was subcloned into the pMW172 and the pHIS-parallel2 vector, respectively. Expression of the recombinant proteins was performed in the E. coli strain Bl21(DE3). The recombinant non fusion protein was purified by ammonium sulphate precipitation, hydrophobic interaction chromatography, anion exchange chromatography and Cibacron Blue F3G-A affinity chromatography. IgE immunoblots were performed utilizing the 6xHIS fusion protein purified by Ni 2+ complexed Chelating Sepharose affinity chromatography. Results A. alternata MtDH has been cloned. The coding sequence of the full-length cDNA clone comprises 798 bp and 266 amino acids, respectively. Protein sequence analysis revealed an identity of 74% and a homology of 80,5% between the MtDHs of A. alternata and C. herbarum. The purified recombinant protein was enzymatically active as shown by spectrophotometrical analysis. In IgE-immunoblots 9 out of 28 (34%) A. alternata patients reacted with the 6xHIS-MtDH. Conclusion Mannitol dehydrogenase was shown to be a new important allergen of A. alternata.