Cross-Reactivity and 1.4-Å Crystal Structure ofMalassezia sympodialisThioredoxin (Mala s 13), a Member of a New Pan-Allergen Family

Abstract

We have identified thioredoxins (Trx) of Malassezia sympodialis, a yeast involved in the pathogenesis of atopic eczema, and of Aspergillus fumigatus, a fungus involved in pulmonary complications, as novel IgE-binding proteins. We show that these Trx, including the human enzyme, represent cross-reactive structures recognized by serum IgE from individuals sensitized to M. sympodialis Trx. Moreover, all three proteins were able to elicit immediate-type allergic skin reactions in sensitized individuals, indicating a humoral immune response based on molecular mimicry. To analyze structural elements involved in these reactions, the three-dimensional structure of M. sympodialis Trx (Mala s 13) has been determined at 1.4-Å resolution by x-ray diffraction analysis. The structure was solved by molecular replacement and refined to a crystallographic R factor of 14.0% and a free R factor of 16.8% and shows the typical Trx fold. Mala s 13 shares 45% sequence identity with human Trx and superposition of the solved Mala s 13 structure with those of human Trx reveals a high similarity with a root mean square deviation of 1.11 Å for all Cα atoms. In a detailed analysis of the molecular surface in combination with sequence alignment, we identified conserved solvent-exposed amino acids scattered over the surface in both structures which cluster to patches, thus forming putative conformational B cell epitopes potentially involved in IgE-mediated cross- and autoreactivity.

Overview of Aspergillus Allergens

“…Therefore it can be concluded that the Asp f 9 cDNA sequence is correct, and that the published Asp f 16 peptide sequence results from frameshift, other sequencing errors, or derives from a different A. fumigatus strain; (iii) Asp f 17, originally described as a cDNA encoding a 191 amino acid protein [24], is incomplete and the complete gene encodes a protein of 284 amino acids; and (iv) the postulated sequence of the Asp f 56 kDa allergen not included in the official allergen database, which was derived from a short peptide obtained from a biochemically purified protein [28] is not predicted to be encoded in any of the sequenced Aspergillus genomes. All other A. fumigatus allergens reported in the official allergen database as well as the sequences of the recently cloned thioredoxins (Asp f 28, Asp f 29) [29], cyclophilins (Asp f 11, Asp f 27) [30], and Phi A cell wall protein (Asp f 34, Glaser unpublished) sequences showed virtually a 100% identity with the genome sequence, confirming at genomic level the validity of cDNA cloning approaches for the elucidation of allergen repertoires. The whole repertoire of A. fumigatus allergens included in the official allergen list reported in Table 2 have been produced as highly pure recombinant allergens and evaluated for their IgE-binding capacity in vitro.…”

“…However, only three crystal structures derived from fungal allergens, ribotoxin (Asp f 1) [45, 46], MnSOD (Asp f 6) [65] from A. fumigatus and a Xylanase from Paecilomyces variotii [66] have been reported so far. Recently the crystal structures of a cyclophilin of A. fumigatus (Asp f 11) [61] and Malassezia sympodialis (M. sympodialis) (Mala s 6) [30], as well as the crystal structure of thioredoxin of M. sympodialis (Mala s 13) [29], and Mala s 1, a major allergen of M. sympodialis with unknown function [67] have been determined at high resolution.…”

“…Interestingly some of these proteins show cross-reactivity also with their homologous human proteins at both, humoral and cellular level. The best investigated structures at this level are human MnSOD [57,58], cyclophilin [30,61] and thioredoxin [29], which were shown to bind to serum IgE from A. fumigatus sensitised patients in vitro, to elicit specific skin test reactions in vivo, and to be potentially involved in the pathogenesis of ABPA and atopic eczema [18,63]. Although the role played by IgE-mediated autoreactivity to self-antigens remains controversial, the fact that the application of human MnSOD in patch test on healthy skin areas of atopic eczema patients sensitised to fungal MnSOD is sufficient to induce an eczematous reaction [57,63] strongly indicates that autoreactivity could contribute to the exacerbation of the symptoms in the absence of external exposure to environmental allergens.…”

“…The whole repertoire of A. fumigatus allergens included in the official allergen list reported in Table 2 have been produced as highly pure recombinant allergens and evaluated for their IgE-binding capacity in vitro. The majority of these proteins have also been tested for their ability to elicit positive skin tests in vivo [27,[29][30][31][32].…”

“…Basically cross-reactivity can be traced back to conserved proteins sharing common B and T cell epitopes as shown for many allergen structures [33]. Amongst these MnSOD (Asp f 6) [57,58], P 2 acidic ribosomal protein (Asp f 8) [59], cyclophilins (Asp f 11 and Asp f 27) [30,60,61] and thioredoxins (Asp f 28 and Asp f 29) [29] have been shown to belong to families of cross-reactive pan-allergens. Per definition pan-allergens share a high degree of sequence identity at primary structure level, thus belonging to protein families showing similar structural folding [33,62].…”