Cloning, expression and characterization of two new IgE‐binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase

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Abstract
Malassezia sympodialis is an opportunistic yeast that colonizes human skin and may induce IgE and T cell reactivity in patients with atopic eczema/dermatitis syndrome (AEDS). Previously, we have cloned and expressed six recombinant allergens (rMala s 1 and rMala s 5 to rMala s 9) from this yeast. By combining high throughput screening and phage surface display techniques, 27 complete and partial IgE‐binding clones of M. sympodialis have been identified. Here we enlarged the panel of recombinant M. sympodialis allergens by RACE–PCR, cloning and nucleotide sequencing to obtain the coding sequences of two new IgE‐binding clones. The coding sequences of one of the clones showed similarity to the heat shock protein (HSP) family and the other to manganese superoxide dismutase (MnSOD), and both had a high degree of homology to human counterparts. The coding sequences were expressed in Escherichia coli as six‐histidine tagged recombinant proteins and generated products with molecular masses of 86.1 kDa for HSP and 22.4 kDa for MnSOD. Their IgE‐binding frequencies were shown to be 69% and 75%, respectively, to 28 sera from AEDS patients with serum IgE to M. sympodialis extract, indicating that HSP and MnSOD are major M. sympodialis allergens. In inhibition immunoblotting, M. sympodialis extract could inhibit the binding of serum IgE from AEDS patients to rHSP and rMnSOD in a concentration‐dependent manner. The high frequency of sera from AEDS patients, showing IgE binding to both HSP and MnSOD, indicates that these allergens, designated Mala s 10 and Mala s 11, could play a role in AEDS.