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Nuclear Magnetic Resonance Structure and IgE Epitopes of Blo t 5, a Major Dust Mite Allergen¹

Siew Leong Chan^2,*, Tan Ching Ong^*, Yun Feng Gao^*, Yuen Sung Tiong^*, De Yun Wang, Fook Tim Chew^* and Yu Keung Mok^3,*

^* Department of Biological Sciences and Department of Otolaryngology, National University of Singapore, Singapore, Singapore

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 -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 2 and 3 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Academic Research Fund, National University of Singapore to Y.K.M. (R-154-000-349-112) and by the Biomedical Research Council grant from the Agency for Science Technology and Research (A*STAR), Singapore to F.T.C. (04/1/21/19/315). F.T.C. and Y.K.M. are members of the Structural Biology and Proteomics Research group of the Life Science Institute, National University of Singapore.

² Current address: Burnham Institute for Medical Research, La Jolla, CA 92037.

³ Address correspondence and reprint requests to Dr. Yu Keung Mok, Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore, Singapore 117543. E-mail address: dbsmokh{at}nus.edu.sg

⁴ Abbreviations used in the paper: NMR, nuclear magnetic resonance; CD, circular dichroism; NOESY, Nuclear Overhauser Effect Spectroscopy.

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