C-terminal cysteine residues determine the IgE binding of Aspergillus fumigatus allergen Asp f 2.

The knowledge of the structure function relationship of the allergen is essential to design allergenic variants with reduced IgE binding capacity but intact T cell reactivity. Asp f 2 is a major allergen from the fungus Aspergillus fumigatus and >90% of A. fumigatus-sensitized individuals displayed IgE binding to Asp f 2. In the present study, we evaluated the involvement of C-terminal cysteine residues in IgE binding conformation of Asp f 2. The deletion mutants were constructed by adding three C-terminal cysteines of the native Asp f 2 one at a time to the non-IgE binding Asp f 2 (68-203). The point mutants of Asp f 2 (68-268) with C204A and C257A substitutions were constructed to study the role of C-terminal cysteines in IgE binding. Immunological evaluation of reduced and alkylated Asp f 2 and its mutants were conducted to determine the contribution of free sulfhydryl groups as well as the disulfide bonds in allergen Ab interaction. Four-fold increase in IgE Ab binding of Asp f 2 (68-267) compared with Asp f 2 (68-266) and complete loss in IgE binding of C204A mutant of Asp f 2 (68-268) indicate the involvement of C(204) and C(267) in IgE binding conformation of Asp f 2. A significant reduction in IgE binding of wild and mutated Asp f 2 after reduction and alkylation emphasizes the importance of cysteine disulfide bonds in epitope Ab interaction. The hypoallergenic variants may be explored further to develop safe immunotherapeutic strategy for allergic disorders.