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C-Terminal Cysteine Residues Determine the IgE Binding of Aspergillus fumigatus Allergen Asp f 2¹

Banani Banerjee^2,*, Viswanath P. Kurup^*,, Paul A. Greenberger, Kevin J. Kelly^* and Jordan N. Fink^*

^* Allergy Immunology Division, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226; Zablocki Veterans Affairs Medical Center, Milwaukee, WI 53295; and Northwestern University Medical School, Chicago, IL 60611

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 C₂₀₄ and C₂₆₇ 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.

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				H. Ramachandran, B. Banerjee, P. A. Greenberger, K. J. Kelly, J. N. Fink, and V. P. Kurup Role of C-Terminal Cysteine Residues of Aspergillus fumigatus Allergen Asp f 4 in Immunoglobulin E Binding Clin. Vaccine Immunol., March 1, 2004; 11(2): 261 - 265. [Abstract] [Full Text] [PDF]