Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8 and Mal f 9

Malassezia furfur, formerly known as Pityrosporum orbiculare or P. ovale, i s a yeast that colonizes human skin. Normally, this yeast is nonpathogenic but under the influence of predisposing factors it may induce IgE reactivit y in patients with atopic dermatitis. Approximately 40-65% of atopic dermat itis patients have IgE antibodies and/or skin reactivity against M. furfur and a higher T-cell response against this yeast is found in atopic dermatit is patients than in healthy individuals. By making a cDNA library displayed on a phage surface, we previously cloned five different IgE-binding protei ns, Mal f 5, Mal f 6, MF 7, MF 8 and MF 9, from this yeast. The cDNAs encod ing these allergens were sequenced and expressed in Escherichia coli. The s equences of MF 7, MF 8 and MF 9 were not full length (missing their 5'-ends ) giving only partial gene products. To obtain complete cDNA sequences, we performed RACE-PCR to amplify the 5'-ends of each cDNA. These PCR products were sequenced and analyzed. The coding sequences of Mal f 7, Mal f 8 and M al f 9 encode proteins with ORFs of 141 (16.2 kDa), 179 (19.2 kDa) and 126 (14.0 kDa) amino-acid residues, respectively. None of the putative proteins showed significant sequence homology with other known proteins in the sear ched database. The proteins encoded by the complete cDNA sequences were exp ressed in E. coli as recombinant proteins. Immunoblotting and radioallergos orbant test data showed that all of the expressed recombinant proteins have the ability to bind serum IgE from atopic dermatitis patients and furtherm ore, the M. furfur extract could specifically inhibit this IgE binding.