Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2

Abstract

Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ϳ100-fold reduced capacity to bind IgE, while preserving the native molecular fold, as proven by x-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and the CD4 ؉ T-cell phenotype in peripheral blood mononuclear cell cultures. Although T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T-cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.

Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant

“…Loss-of-function mutations in these two peptides resulted in a decrease in IgE affinity by 2-3 orders of magnitude. This implied the presence of a limited number of high affinity IgEbinding sites on this allergen and consequently the oligoclonal nature of anti-Rhi o 1 IgE antibody as also evident for other allergens (24,33). These two IgE-reactive peptides of Rhi o 1 were not conserved in other allergen sequences (data not shown) and therefore probably non-cross-reactive in nature.…”

Cockroach allergen exposure and risk of asthma

“…In addition, profiling of N-linked glycans from Bla g 2 using matrix-assisted laser desorption/ionization-mass spectrometry revealed a predominance of tri-antennary N-linked core di-fucose modified glycans with mannose-, galactose-, and/or N-acetyl glucosamine- (GlcNAc) terminated moiety ( Figure 1 ). Thus, we suspected that these glycan determinants are of insect (i.e., cockroach) due to the fact that di-fucosylation of the innermost GlcNAc moiety is commonly present in insects (56). These studies give a glimpse into the potential association between immunogenicity and particular structural features of glycans and their possible contributions to allergic diseases.…”

Indoor Allergens and Allergic Respiratory Disease

“…The antigenic surface of Bla g 2 has been analyzed by determining the structure of the allergen in complex with fragments (Fab or Fab′) of mAb that interfere with IgE antibody binding and by site-directed mutagenesis of residues involved in the epitopes [32–34, 35•]. These studies revealed IgE antibody binding sites and mechanisms of allergen-antibody interaction.…”

“…Allergens fused to viral domains or viral-like particles have shown immunomodulatory capacity [21]. Another strategy is to perform site-directed mutagenesis of known IgE epitopes, based on the X-ray crystal structures of the allergens alone or in complex with monoclonal antibodies that interfere with IgE antibody binding [9, 10••, 32, 34, 35•, 123, 124]. …”