Crystal structure and some properties of a major house dust mite allergen, Derf 2

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Abstract

Pyroglyphid house dust mites are a major source of allergens in house dust. Mite allergens sensitize and induce asthma, rhinitis, and eczema in a large portion of patients with allergic diseases. Here, the crystal structure of a major mite allergen, Derf 2, derived from Dermatophagoides farinae was solved by single isomorphous replacement method with anomalous scattering (SIRAS) at 2.1 Å resolution. The present study also demonstrated that the conformation of the allergen was critical in the determination of Th1/Th2 shift based on physicochemical and immunological analyses. This indicates that rigidly folded and singly dispersed structure is essentially required for the generation of Th2 type cells by the allergen, while conformational variant protein leads to Th1 skewing, irrespective of the same amino acid sequence. This structure/function relationship may allow us to develop a novel strategy for hyposensitization therapy in patients with allergic diseases triggered by house dust mite allergens.

Section snippets

Materials and methods

Preparation of Derf 2 and its conformational variant. Recombinant Derf 2 protein was prepared as described previously [19]. In brief, Escherichia coli Rosseta(DE3)pLys (Merck KgaA, Darmstadt, Germany) was transfected with an expression plasmid harboring cDNA for Derf 2 and induced for protein expression with 1 mM IPTG. The resultant inclusion bodies were dissolved in buffer containing 8 M urea and 1 M guanidine, and refolded in arginine-based buffer. After dialysis against 10 mM Tris–HCl buffer, pH

Crystallization and structure determination

In search of hypoallergenic variants of Derf 2 for the use in hyposensitization therapy for allergic asthma, we previously demonstrated that the structure of allergen is intrinsically involved in allergenicity. Since only preliminary X-ray data were available [30], we first attempted to prepare crystals of Derf 2 and solve the X-ray structure in the present study. As shown in Fig. 1A, Derf 2 was crystallized in a mixture consisting of 20 mg/ml Derf 2, 23% PEG4000, 0.06 M ammonium sulfate, 0.1 M

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology, Japanese Government, the Inamori Foundation, Sumitomo Pharmaceutical Co., Ltd., and the Foundation for Biomedical Research and Innovation. An expression plasmid encoding cDNA for Derf 2 was kindly provided by Dr. Toyokazu Yokota, Asahi Breweries, Tokyo, Japan.

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