C-Terminal Cysteine Residues Determine the IgE Binding ofAspergillus fumigatusAllergen Asp f 2

Abstract

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.

Role of C-Terminal Cysteine Residues ofAspergillus fumigatusAllergen Asp f 4 in Immunoglobulin E Binding

“…Interestingly, the Western blot analysis of the IgE antibody binding of denatured Asp f 4 reported that in Asp f 2, there was a fourfold increase in IgE antibody binding when cysteine was added at the 267th position. However, a loss of binding was noted when C204 was mutated to alanine (2). The results of the present study also support the significance of some of the cysteine residues in their specific binding to IgE.…”

“…In Birch pollen allergen Bet v 1 and house dust mite allergens Der p 1 and Der p 2, discontinuous IgE binding epitopes have been demonstrated and the IgE antibody binding was destroyed by fragmentation or mutagenesis of cysteine residues responsible for the formation of disulfide bonds (14,20,22). Our studies with another A. fumigatus allergen, Asp f 2, showed that alteration of cysteine residues at positions 204 and 257 induced marked differences in the IgE binding (2). Since no information on the structure function of Asp f 4 is available, we undertook the present study to investigate the role of cysteine residues present in Asp f 4 in the immune responses in ABPA.…”

“…Although sera from some ABPA patients reacted with all three fragments, there were differences in their immune responses, particularly in the realm of IgE antibody binding. It has been reported that the disulfide bonds formed between the different cysteine residues play an important role in the conformational structure of the allergens and this has contributed to IgE antibody binding (2,14,15,(20)(21)(22). Earlier studies by Olsson et al have demonstrated that in Lep d 2, the disulfide bond formed between cysteines 72 and 77 (Cys 72-77) was the single most important contributing factor in IgE binding (16).…”

The complex role of mast cells in fungal infections

“…Most studies on the role of MCs in chronic lung disease due to A. fumigatus focus on IgE‐mediated allergic bronchopulmonary aspergillosis (ABPA) . ABPA is associated with early allergic and late‐phase inflammatory responses of the lungs to A. fumigatus antigens that occur in a minority (less than 1%) of patients with asthma and, more often (7%‐10%), in patients with cystic fibrosis .…”

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum

“…[17, 18] and Dermatophytes [19]. Proteolytic activities identified from these fungi feature the families/subfamilies of elastase-like, chymotrypsin-like, subtilisin-like, and trypsin-like serine proteases, aspartic proteases, metalloproteases and cysteine proteases [16, 20, 21]. Amongst the proteases, elastase activity has emerged as the main indication for virulence and has been linked to germination and penetration into mice lungs [22], deterioration of respiratory function [14], and lung injury [23].…”