Nuclear Magnetic Resonance Structure and IgE Epitopes of Blo t 5, a Major Dust Mite Allergen

Abstract

A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel α-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices α2 and α3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76–91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.

NMR Structure and IgE Epitopes of Blo t 21, a Major Dust Mite Allergen from Blomia tropicalis

“…Among these five residues, the corresponding residues of Asp-79, Glu-84, and Glu-89 in Blo t 5 were also identified as three of the four essential IgE-binding epitopes in Blo t 5 ( Fig. 3A) (8). These data suggest that the main IgE-binding epitope of Blo t 21 is located in similar region to that observed in Blo t 5 and that these two allergens could share at least some common IgE epitopes.…”

“…The three-dimensional NMR structure of Blo t 21 shows that it consists of three anti-parallel ␣-helices assembled in a helical bundle resembling that of Blo t 5 (8,30) (4).…”

“…In Blo t 5, alanine substitution of the residue corresponding to Asp-79 demonstrated the most drastic reduction in IgE binding (8), whereas in Blo t 21, residue Glu-89 seemed to play a more important role in IgE binding. We found that the four conserved residues, i.e., Glu-74, Asp-79, Glu-84, and Glu-89, in Blo t 21, and the corresponding residues in Blo t 5 were oriented differently (Fig.…”

“…A linear epitope comprising four charged residues surrounding the turn region connecting ␣2 and ␣3 has been identified to be involved in IgE binding (8). Subsequently, the crystal structure of the homologous group 5 allergen, Der p 5, was determined (10).…”

“…The NMR structure of Blo t 5 was determined as a helical bundle consisting of three anti-parallel ␣-helices (8,9). A linear epitope comprising four charged residues surrounding the turn region connecting ␣2 and ␣3 has been identified to be involved in IgE binding (8).…”