Analysis of the cross-reactivity and of the 1.5 Å crystal structure of the Malassezia sympodialis Mala s 6 allergen, a member of the cyclophilin pan-allergen family

Abstract

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.

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].…”