Open AccessTwo‐dimensional electrophoresis of Malassezia allergens for atopic dermatitis and isolation of Mal f 4 homologs with mitochondrial malate dehydrogenase
Author(s) -
Onishi Yoshimi,
Kuroda Masanobu,
Yasueda Hiroshi,
Saito Akemi,
SonoKoyama Eiko,
Tunasawa Susumu,
HashidaOkado Takashi,
Yagihara Tomoko,
Uchida Katsuhisa,
Yamaguchi Hideyo,
Akiyama Kazuo,
Kato Ikunoshin,
Takesako Kazutoh
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00247.x
Subject(s) - atopic dermatitis , malate dehydrogenase , allergen , complementary dna , immunoglobulin e , allergy , biology , amino acid , peptide sequence , malassezia , microbiology and biotechnology , molecular mass , immunology , chemistry , gene , biochemistry , enzyme , antibody
The yeast Malassezia furfur is a natural inhabitant of the human skin microflora that induces an allergic reaction in atopic dermatitis. To identify allergens of M. furfur , we separated a crude preparation of M. furfur antigens as discrete spots by 2‐D PAGE and detected IgE‐binding proteins using sera of atopic dermatitis patients. We identified the known allergens, Mal f 2 and Mal f 3, and determined N‐terminal amino acid sequences of six new IgE‐binding proteins including Mal f 4. The cDNA and genomic DNA encoding Mal f 4 were cloned and sequenced. The gene was mitochondrial malate dehydrogenase and encoded Mal f 4 composed of 315 amino acids and a signal sequence of 27 amino acids. We purified Mal f 4, which had a molecular mass of 35 kDa from a membrane fraction of a lysate of cultured cells. Thirty of 36 M. furfur ‐allergic atopic dermatitis patients (83.3%) had elevated serum levels of IgE to purified Mal f 4, indicating that Mal f 4 is a major allergen. There was a significant correlation of the Phadebas RAST unit values of Mal f 4 and the crude antigen, but not between Mal f 4 and the known allergen Mal f 2.