Andreas G. Glaser

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

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.

Cross-reactivity among fungal allergens: a clinically relevant phenomenon?

Atopic patients suffering from allergic asthma, allergic rhinitis, or atopic eczema often have detectable levels of serum IgE antibodies to fungi. Although the association between fungal sensitisation and different forms of allergic diseases, including allergic asthma and life‐threatening allergic bronchopulmonary aspergillosis, is well established, the clinical relevance of cross‐reactivity among different fungal species remains largely unknown. Recent progress in molecular cloning of fungal allergens and the availability of more than 40 completely sequenced fungal genomes facilitates characterisation, cloning, and production of highly pure recombinant allergens, identification of homologous and orthologous allergens widespread among the fungal kingdom, in silico prediction, and experimental in vitro and in vivo verification of cross‐reactivity between homologous pan‐allergens. These studies indicate that cross‐reactivity is an important component of fungal sensitisation.

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

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.

Fungal Allergies: A Yet Unsolved Problem

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.

Immunoglobulin-E-Mediated Reactivity to Self Antigens: A Controversial Issue

Immunoglobulin E (IgE) reactivity to self antigens is well established in vitro by ELISA, inhibition ELISA, Western blot analyses and T cell proliferation experiments. In vivo, IgE-binding self antigens are able to elicit strong type I reactions in sensitized individuals and, in the case of human manganese superoxide dismutase, to elicit eczematous reactions on healthy skin areas of patients suffering from atopic eczema. The reactions against self antigens sharing structural homology with environmental allergens can be plausibly explained by molecular mimicry between common B cell epitopes. For the second class of IgE-binding self antigens without sequence homology to known allergens, it is still unclear if the structures are able to induce a B cell switch to IgE production, or if the reactivity is due to sequence similarity shared with not yet detected environmental allergens. However, in all cases, cross-reactivity is never complete, indicating either a lower affinity of IgE antibodies to self allergens than to the homologous environmental allergens or the presence of additional B cell epitopes on the surface of the environmental allergens, or both. Increasing evidence shows that self allergens could play a decisive role in the exacerbation of long-lasting atopic diseases. However, the only observation supporting a clinical role of IgE-mediated autoreactivity is confined to the fact that IgE levels against self antigens correlate with disease severity.

Molecular and immunological characterization of Asp f 34, a novel major cell wall allergen of Aspergillus fumigatus

Background:  Although fungal spores have been recognized as triggers of respiratory allergy and asthma, only two allergenic fungal cell wall components have so far been described.

Auto- and cross-reactivity to thioredoxin allergens in allergic bronchopulmonary aspergillosis

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.

Mutational analysis of amino acid residues involved in IgE-binding to the Malassezia sympodialis allergen Mala s 11.

The yeast Malassezia sympodialis, which is an integral part of the normal cutaneous flora, has been shown to elicit specific IgE- and T-cell reactivity in atopic eczema (AE) patients. The M. sympodialis allergen Mala s 11 has a high degree of amino acid sequence homology to manganese superoxide dismutase (MnSOD) from Homo sapiens (50%) and Aspergillus fumigatus (56%). Humoral and cell-mediated cross-reactivity between MnSOD from H. sapiens and A. fumigatus has been demonstrated. Taken together with the recent finding that human MnSOD (hMnSOD) can act as an autoallergen in AE patients sensitised to M. sympodialis, we hypothesized that cross-reactivity could also occur between hMnSOD and Mala s 11, endogenous hMnSOD thus being capable of stimulating an immune response through molecular mimicry. Herein we demonstrate that recombinant Mala s 11 (rMala s 11) is able to inhibit IgE-binding to recombinant hMnSOD and vice versa, indicating that these two homologues share common IgE-binding epitopes and providing an explanation at a molecular level for the autoreactivity to hMnSOD observed in AE patients sensitised to Mala s 11. Using molecular modelling and mapping of identical amino acids exposed on the surface of both Mala s 11 and hMnSOD we identified four regions each composed of 4-5 residues which are potentially involved in IgE-mediated cross-reactivity. Mutated rMala s 11 molecules were produced in which these residues were altered. Native-like folding was verified by enzymatic activity tests and circular dichroism. The rMala s 11 mutants displayed lower IgE-binding in comparison to wild-type rMala s 11 using plasma from AE patients. In particular, mutation of the residues E29, P30, E122 and K125 lowered the IgE-binding to Mala s 11. The results of this study provide new insights in the molecular basis underlying the cross-reactivity between Mala s 11 and hMnSOD.

Structural aspects and clinical relevance of Aspergillus fumigatus antigens/allergens

Robotics-based high throughput screening of Aspergillus fumigatus cDNA libraries displayed on phage surfaces revealed at last 81 different structures able to bind IgE from serum of patients sensitized to this fungus. Among these, species-specific as well as phylogenetically highly conserved structures and such with unknown function have been detected. A subset of cDNAs have been used to produce and characterize the corresponding recombinant allergens which have proven to be useful diagnostic reagents allowing specific detection of A. fumigatus sensitization and differential diagnosis of allergic bronchopulmonary aspergillosis. Phylogenetically highly conserved structures like manganese-dependent superoxide dismutase, P2 acidic ribosomal protein, cyclophilins and thioredoxins induce, beyond sensitization, IgE antibodies able to cross-react with the corresponding homologous self antigens. These reactions, likely to contribute to the exacerbation and perpetuation of allergic bronchopulmonary aspergillosis, can be traced back to shared conformational B-cell epitopes build up from conserved amino acid residues scattered over the surface of the molecules as shown by detailed analyses of the crystal structures.

Overview of Aspergillus Allergens

Fungi in general and, Aspergillus fumigatus (A. fumigatus) in particular, are able to produce complex patterns of IgE-binding molecules. Robotics-based high throughput screening of A. fumigatus cDNA libraries displayed on phage surfaces revealed at last 81 different sequences encoding structures potentially able to bind to serum IgE of sensitised individuals suffering from A. fumigatus-related complications. Although not all of these allergens have been characterised in detail, A. fumigatus still represents the best investigated allergenic source. A total of 23 A. fumigatus allergens are recorded by the official allergen list of the International Union of Immunological Societies (http://www.allergen.org) and this is by far the longest allergen list reported for a single allergenic source. The IgE-binding molecules include species-specific as well as phylogenetically highly conserved cross-reactive structures and such with unknown function. A subset of cDNAs have been used to produce and characterise the corresponding recombinant allergens which have proven to be useful diagnostic reagents allowing specific detection of A. fumigatus sensitisation and differential diagnosis of allergic bronchopulmonary aspergillosis. Structures highly conserved through different species like manganese-dependent superoxide dismutase, P2 acidic ribosomal protein, cyclophilins and thioredoxins induce, beyond sensitisation, IgE antibodies able to cross-react with the corresponding homologous self-antigens. The frequently observed cross-reactivity is traceable back to shared discontinuous B-cell epitopes as shown by detailed analyses of the crystal structures.