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. METHODS: Eighty-one sequences derived from an Aspergillus fumigatus cDNA library encoding putative allergens were examined for the presence of cell wall components. A new allergen (Asp f 34) was evaluated by Western blots, enzyme-linked immunosorbent assay (ELISA), peripheral blood mononuclear cell (PBMC) proliferation assays, and skin prick test (SPT). RESULTS: The cDNA encoding Asp f 34 contained an open reading frame predicting a protein of 185 amino acids with a molecular weight of 19.38 kDa, showing sequence homology to phiA, an essential protein for the formation of conidia in the genus Aspergillus. The recombinant Asp f 34 was binding IgE from sensitized individuals in Western blots. An ELISA survey showed that 94% of the ABPA and 46% of the A. fumigatus-sensitized individuals tested had Asp f 34-specific serum IgE. Asp f 34 induced allergen-specific proliferation exclusively of PBMCs from patients sensitized to the allergen. Eight patients with anti-Asp f 34 serum IgE tested reacted positively in SPT, whereas four A. fumigatus-sensitized individuals without Asp f 34-specific IgE and eight healthy controls scored negatively. CONCLUSIONS: A cell wall protein of the phialides of A. fumigatus was identified as a major allergen. Asp f 34 belongs to the Aspergillus-specific proteins of the phiA family and has relevant potential for a specific diagnosis of Aspergillus sensitization.

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. 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.