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- Materials and methods
Background: Thioredoxins are cross-reactive allergens involved in the pathogenesis of atopic eczema and asthma. Cross-reactivity to human thioredoxin can contribute to the exacerbation of severe atopic diseases.
Methods: Human thioredoxin, Asp f28 and Asp f29, two thioredoxins of Aspergillus fumigatus, and thioredoxin of Malassezia sympodialis were cloned and produced as recombinant proteins. Allergenicity and cross-reactivity to thioredoxins in allergic bronchopulmonary aspergillosis patients were assessed by enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, immunoblot analysis, proliferation assays and skin tests. Molecular homology modelling was used to identify conserved, surface-exposed amino acids potentially involved in immunoglobulin E (IgE)-binding.
Results: All thioredoxins, including the human enzyme, bind IgE from patients with allergic bronchopulmonary aspergillosis and induce allergen-specific proliferation in peripheral blood mononuclear cells and positive skin reactions in thioredoxin-sensitized patients. Inhibition experiments showed that the thioredoxins are cross-reactive indicating humoral immune responses based on molecular mimicry. To identify structural surface elements involved in cross-reactivity, the three-dimensional structures were modelled based on solved thioredoxin structures. Analysis of the molecular surfaces combined with sequence alignments allowed identification of conserved solvent exposed amino acids distantly located in the linear sequences which cluster to patches of continuous surface areas. The size of the surface areas conserved between human and fungal thioredoxins correlates well with the inhibitory potential of the molecules in inhibition ELISA indicating that the shared amino acids are involved in IgE-binding.
Conclusions: Conserved, solvent exposed residues shared between different thioredoxins cluster to continuous surface regions potentially forming cross-reactive conformational B-cell epitopes responsible for IgE-mediated cross-reactivity and autoreactivity.
Skin test surveys suggest that 3–10% of the worldwide population is affected by fungal allergy (1, 2), and sensitization to Aspergillus species is quite common in patients with asthma or cystic fibrosis (CF) (3, 4). Among the moulds involved in pulmonary complications Aspergillus fumigatus plays a predominant role (5).
The different A. fumigatus-related pulmonary diseases can be broadly classified according to their clinical conditions into systemic mycoses, saprophytic colonization and allergic diseases (6). Aspergillus fumigatus-related allergy include rhinitis, sinusitis, allergic asthma and allergic bronchopulmonary aspergillosis (ABPA) (7). ABPA is the most severe complication related to A. fumigatus sensitization, affects almost exclusively patients with severe asthma or CF, and is difficult to diagnose (8). Although ABPA as a syndrome can only be diagnosed based on clinical signs, serologic tests are used to confirm a suspected ABPA and recombinant A. fumigatus allergens have been proven useful to discriminate between A. fumigatus sensitization and ABPA in both, asthmatic and CF patients (4, 9, 10). Aspergillus fumigatus allergic patients mount immunoglobulin E (IgE)-immune responses to secreted fungal allergens present as aeroallergens like Asp f1 (11), or secreted proteases (12). ABPA patients, however, have or had the fungus growing in the lung (13) and, as a result of fungal damage due cellular defence mechanisms, become exposed also to nonsecreted proteins. Classical examples of intracellular allergens are manganese-dependant superoxide dismutase (MnSOD, rAsp f6) (14, 15) differentially expressed only during germination (16) and ribosomal P2 protein (rAsp f8) (17). IgE-responses to these allergens are detected almost exclusively in sera of patients with ABPA. In concordance with the presence of rAsp f6 or rAsp f8-specific serum IgE, sensitized ABPA patients strongly react in skin test challenges to these allergens (14, 17) as well as to homologous proteins (18, 19). Interestingly, phylogenetically highly conserved proteins like MnSOD (18), thioredoxin (Trx) (20, 21) and cyclophilin (22, 23) are members of large pan-allergen families exhibiting a high degree of cross-reactivity also with their homologous human self-antigens (14, 15, 17, 20–24). Here we show that two cross-reactive thioredoxins are recognized by sera of patients with ABPA. As already shown for patients suffering from atopic eczema (20) and for baker’s asthma (21), also ABPA patients sensitized to fungal thioredoxin recognize human thioredoxin as an IgE-binding self-antigen further corroborating the involvement of IgE-mediated autoreactivity in the pathogenesis of chronic atopic disorders.
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- Materials and methods
IgE-binding proteins sharing homology belong to groups of phylogenetically conserved protein families called pan-allergens. The most interesting cross-reactive proteins are those showing sequence homology between environmental allergens and human self-antigens, which can lead to humoral and cell-mediated autoreactivity (14, 17, 19, 20, 23). Patients suffering from severe atopic diseases often show elevated IgE-levels against self-antigens indicating that the pathogenesis of allergic diseases can be influenced by cross-reactive structures. The most convincing evidence that self-reactivity is involved in the pathogenesis of atopic diseases derives from the demonstration that simple application of human MnSOD to healthy skin areas of patients with atopic eczema induces eczematous reactions (15). Here we performed a study on cross- and self-reactivity to thioredoxins in patients with ABPA. Asp f28, Asp f29, Mala s13, and human Trx were all recognized by serum IgE from the majority of the 40 ABPA patients tested (Fig. 2). The allergens induced proliferation exclusively in PBMC’s from ABPA patients showing detectable amounts of Trx-specific serum IgE, whereas PBMC’s of healthy controls and of A. fumigatus-sensitized individuals without Trx-specific serum IgE consistently failed to proliferate (Fig. 4). However, the demonstration that proteins act as allergen is their ability to elicit immediate skin reactions. Among 15 individuals tested, positive SPT to all Trxs were observed only in patients with detectable serum IgE antibodies to Trx. SPT reactions depend on rapid degranulation of mast cells and mediator release, particularly histamine, triggered through cross-linking of high-affinity IgE-receptors (30). The skin test results show that all Trxs elicit IgE cross-linking on mast cells in vivo, and reactivity to human Trx suggests humoral autoimmune responses.
Extended, although incomplete, cross-reactivity between all thioredoxins was demonstrated by inhibition ELISA (Fig. 3). This indicates that primary sensitization to fungal Trxs induces polyclonal responses eliciting additional IgE-responses to surface areas not conserved between the homologous structures.
A partial explanation for the incomplete cross-reactivity can be obtained by an extended structural analysis. Antibody–antigen interfaces bury a surface area of 560–900 Å2 involving 10–22 amino acids of the antigen which interact with the antibody (31, 32). Detailed structural study of Fv-lysozyme complexes revealed that the productive binding is mediated by only five to six energetically important residues constituting an ‘energetic’ epitope with a surface area of approximately 250–300 Å2 (29). We compared the modelled structures of Asp f28 and Asp f29 to the solved crystal structure of human Trx (33) by structural alignments. The identical solvent exposed residues, scattered over the linear sequence (Fig. 5), cluster to four independent patches on the surface of human Trx (Fig. 6). Patch 1 is well conserved in all Trxs and fulfils the criteria for a conformational B-cell epitope (20). Patch 2 covers a surface of 414 Å2 and lies in a region with sequential and structural deviation in Asp f29 and Mala s13 compared to human Trx and Asp f28 (Figs. 5 and 6). This potential B-cell epitope is only shared between human Trx and Asp f28. Patch 3 shows the largest conserved surface between human Trx and Asp f29 (527 Å2), whereas two and four amino acids mutations in Mala s13 and Asp f28, respectively, contribute to a reduction of the patch size to 433 and 344 Å2, still fulfilling the criteria required for energetic B-cell epitopes. Patch 4 is only conserved between human Trx and Mala s13 but considering the small surface (239 Å2) is unlikely to contribute to IgE-binding. Notably the gradual reduction of the total solvent-accessible surface areas shared between human Trx, Asp f28 (1433 Å2), Asp f29 (1301 Å2) and Mala s13 (1300 Å2), coincides with the decreasing inhibitory capacity of the proteins in competition ELISAs (Fig. 3B). These results suggest that the conserved patches are indeed involved in IgE-binding and cross-reactivity. This structural information could be exploited to engineer a Trx molecule with reduced IgE-binding capacity. Such molecules could be useful for the treatment of ABPA patients with an autoimmune background due to thioredoxin sensitization. However, because A. fumigatus produces a complex mixture of IgE-binding molecules (7) it is unlikely that a single molecule would be useful as a general vaccine against A. fumigatus-related allergic complications.
In summary, the data presented provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human Trx in ABPA clearly traceable back to conserved surface-exposed amino acids. Molecular mimicry is the most plausible explanation for the reactions observed (18, 23).