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The opportunistic yeast Malassezia sympodialis belongs to the normal cutaneous flora but can also cause IgE-mediated sensitization in patients suffering from atopic eczema (AE). We investigated 706 individuals by ImmunoCAPm70 and skin-prick tests with a crude M. sympodialis extract. In AE patients, we further performed skin prick tests, atopy patch tests, ELISA, and peripheral blood mononuclear cells proliferation assays with recombinant M. sympodialis allergens (rMala s 1 and 5-9). In 52/97 patients with AE-specific IgE against M. sympodialis was detectable. Almost no reactivity to M. sympodialis was seen in patients suffering from other allergic diseases (4/571) and no reactivity at all was seen in healthy controls (0/38). Skin tests showed variable recognition patterns against the different molecular structures with a predominant sensitization to rMala s 1, 5, 6, and 9, confirmed also by specific serum IgE to these allergens. Interestingly, IgE- and T-cell-mediated reactivity against M. sympodialis was also found in patients with the intrinsic form of AE. Thus, sensitization to M. sympodialis is specific for AE patients and occurs in both the extrinsic and intrinsic variant of eczema. Recombinant yeast allergens represent a useful tool to study molecular structures and differential sensitization patterns in the pathogenesis of AE.

Cyclophilins constitute a family of proteins involved in many essential cellular functions. They have also been identified as a panallergen family able to elicit IgE-mediated hypersensitivity reactions. Moreover, it has been shown that human cyclophilins are recognized by serum IgE from patients sensitized to environmental cyclophilins. IgE-mediated autoreactivity to self-antigens that have similarity to environmental allergens is often observed in atopic disorders. Therefore comparison of the crystal structure of human proteins with similarity to allergens should allow the identification of structural similarities to rationally explain autoreactivity. A new cyclophilin from Aspergillus fumigatus (Asp f 27) has been cloned, expressed and showed to exhibit cross-reactivity in vitro and in vivo. The three-dimensional structure of cyclophilin from the yeast Malassezia sympodialis (Mala s 6) has been determined at 1.5 A (1 A=0.1 nm) by X-ray diffraction. Crystals belong to space group P4(1)2(1)2 with unit cell dimensions of a=b=71.99 A and c=106.18 A. The structure was solved by molecular replacement using the structure of human cyclophilin A as the search model. The refined structure includes all 162 amino acids of Mala s 6, an active-site-bound Ala-Pro dipeptide and 173 water molecules, with a crystallographic R- and free R-factor of 14.3% and 14.9% respectively. The overall structure consists of an eight-stranded antiparallel beta-barrel and two alpha-helices covering the top and bottom of the barrel, typical for cyclophilins. We identified conserved solvent-exposed residues in the fungal and human structures that are potentially involved in the IgE-mediated cross-reactivity.

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Airborne fungal spores have been implicated as causative factors in respiratory allergy, particularly asthma. However, the prevalence of fungal sensitization is not known mainly due to the lack of standardized fungal extracts and to the overwhelming number of fungal species able to elicit IgE-mediated reactions. Recent work based on high-throughput cloning of fungal allergens revealed that fungi are able to produce extremely complex repertoires of species-specific and cross-reactive allergens. There is evidence that fungal sensitization also contributes to auto-reactivity against self-antigens due to shared epitopes with homologous fungal allergens. Detailed studies at structural and immunological level indicate molecular mimicry as a basic mechanism involved in perpetuation of severe chronic allergic diseases. The real challenge at present is not related to cloning or production of a large number of different fungal allergens but rather to the assessment of the clinical relevance of each single structure. To date, substitution of complex extracts presently used in the diagnosis of fungal allergy by single, perfectly standardized components seems feasible in contrast to specific immunotherapy which is still not developed. Recombinant fungal allergens might create new perspectives in diagnosis and therapy of fungal allergy.

Type I allergic reactions occur by immediate release of anaphylactogenic mediators due to cross‐linking of IgE bound to the high‐affinity FcϵRI on the surface of effector cells ofsensitized individuals after allergen exposure. IgE‐mediated hypersensitivity against normally innocuous environmental antigens is of clinical importance because of an increasing incidence of asthma and severe atopic diseases causing raising health care burdens to the society. A vast variety of different molecular structures has been shown to be able to induce hypersensitivity reactions. However, the high structural homology between phylogenetically conserved allergenic proteins present in different, apparently unrelated sources of exposure seems to play an important role in IgE‐mediated poly‐sensitization. These allergen families, formally termed pan‐allergens, represent proteins sharing a high degree of sequence homology. Here we report cloning, production and serological investigations of a new pan‐allergen family, the cyclophilins, found to be cross‐reactive across species including humans. IgE‐mediated cross‐reactivity against autoantigens may contribute to perpetuation of severe atopic disorders even in the absence of exogenous allergen exposure. The molecular definition of pan‐allergen families may substantially contribute to reduce the number of structures needed for diagnosis and therapy of allergic diseases based on highly pure, standardized recombinant allergens.

Manganese superoxide dismutase (MnSOD) of Aspergillus fumigatus, a fungus involved in many pulmonary complications, has been identified as IgE-binding protein. It has been shown also that MnSODs from other organisms, including human, are recognized by IgE Abs from individuals sensitized to A. fumigatus MnSOD. Comparison of the fungal and the human crystal structure should allow the identification of structural similarities responsible for IgE-mediated cross-reactivity. The three-dimensional structure of A. fumigatus MnSOD has been determined at 2-Å resolution by x-ray diffraction analysis. Crystals belonged to space group P212121 with unit cell dimensions of a = 65.88 Å, b = 98.7 Å, and c = 139.28 Å. The structure was solved by molecular replacement using the structure of the human MnSOD as a search model. The final refined model included four chains of 199–200 amino acids, four manganese ions, and 745 water molecules, with a crystallographic R-factor of 19.4% and a free R-factor of 23.3%. Like MnSODs of other eukaryotic organisms, A. fumigatus MnSOD forms a homotetramer with the manganese ions coordinated by three histidines, one aspartic acid, and one water molecule. The fungal and the human MnSOD share high similarity on the level of both primary and tertiary structure. We identified conserved amino acids that are solvent exposed in the fungal and the human crystal structure and are therefore potentially involved in IgE-mediated cross-reactivity.

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 concentrationdependent 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.