C-Terminal Cysteine Residues Determine the IgE Binding of Aspergillus fumigatus Allergen 2

Banani Bonerjee*, V Kurup*. Paul Greenbergerf. K Kelly*, J Fink* *

Author address: 

College of Wisconsin and VAMC, Milwaukee, WI. tNorthwestem University Medical School, Chicago, IL Knowledge of the structure function


Knowledge of the structure function relationship of allergens is essential to understand the pathogenesis of allergic disease. Although Aspergillus fimigarus (Af) causes different allergic diseases such as allergic asthma, ABPA, and hypersensitivity pneumonitis. no information is currently available on the immune system interaction with Af allergens. Using recombinant DNA technology a number of Af allergens have been cloned and characterized. The knowledge of the primary amino acid sequence of these allergens is of value in understanding the humoral and cell mediated immune responses in patients. In order to define the involvement of cysteines in antibody allergen interaction. various deletion mutants of Asp f 2, a major Af allergen, were constructed. The recombinant constructs encompassing different cysteine residues were cloned and overexpressed using 2 different prokaryotic expression systems. The specificity of antibody epitope interaction was further defined by designing synthetic peptides including cysteine residues C z5, and Cz,, at the C-terminal region. All recombinant constructs and synthetic peptides were analyzed for circular dichroism (CD) for structural differences. The synthetic peptides were studied for their IgE binding by ELISA using ABPA patient sera. Recombinant construct Asp f 2B (68-203) with 4 cysteine residues Cs,, Css, C,, and C,,, failed to show IgE binding with sera from Af sensitized patients, whereas, Asp f 2A (I-203) with an additional Cl4 at the N-terminal region and Asp f 2C (68-268) with 3 more cysteines at Cz,, C,,, and C,,, near the C-terminal region exhibited IgE antibody binding with ABPA sera. This finding indicates that specific conformation and/or tertiary structure of the allergen is essential for the binding of IgE. In order to demonstrate the specificity of cysteine residues and their positions in IgE binding ability, we devised additional constructs by incorporating the remaining cysteines C,,, C,,, and C,,, one at a time to Asp f 2B. Only construct Asp f 2B4 (68-267) with all the 3 cysteines at the Cterminal region showed IgE binding. The involvement of C-terminal cysteine C,,, in IgE binding was confirmed by no reactivity of the fragment Asp f 2B3 (68-266) missing only the CZ6, residue. On evaluation, the synthetic linear peptide (254-268) PSNCHTHEGGQLHCT and cyclic peptide (256-268) NCHTHEGGQLHCT with a bond between Cz and C,, exhibited similar IgE binding as the complete Asp f 2 molecule. Understanding the IgE cysteine interaction may yield useful information on the conformational structure of allergens and may provide strategies for immune intervention for Af mediated allergic diseases.

abstract No: 


Full conference title: 

2000 American Academy of Allergy, Asthma, and Immunology Annual Meeting
    • AAAAI 2000 (56th)