title: 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 alpha-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 alpha2 and alpha3 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.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Siew Leong
authors.initials: SL
authors.lastname: Chan
authors.forename: Tan Ching
authors.initials: TC
authors.lastname: Ong
authors.forename: Yun Feng
authors.initials: YF
authors.lastname: Gao
authors.forename: Yuen Sung
authors.initials: YS
authors.lastname: Tiong
authors.forename: De Yun
authors.initials: de Y
authors.lastname: Wang
authors.forename: Fook Tim
authors.initials: FT
authors.lastname: Chew
authors.forename: Yu Keung
authors.initials: YK
authors.lastname: Mok
mesh_terms: D000485:Allergens / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D001666:Binding Sites, Antibody; D018360:Crystallography, X-Ray; D003888:Desensitization, Immunologic; D004391:Dust / Q000276:immunology; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D008925:Mites / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D004391:Dust; D010446:Peptide Fragments; C094701:allergen Blo t 5; D007073:Immunoglobulin E