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Crucial Commitment of Proteolytic Activity of a Purified Recombinant Major House Dust Mite Allergen Der p1 to Sensitization toward IgE and IgG Responses¹

Yuko Kikuchi^*,, Toshiro Takai^2,*, Takatoshi Kuhara^*, Mikiko Ota^*,, Takeshi Kato^*, Hideki Hatanaka^¶, Saori Ichikawa^*,||, Tomoko Tokura^*, Hisaya Akiba, Kouichi Mitsuishi, Shigaku Ikeda, Ko Okumura^*, and Hideoki Ogawa^*,

^* Atopy (Allergy) Research Center, Department of Dermatology, and Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; Department of Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan; ^¶ Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan; and ^|| Department of Materials and Biological Sciences, Faculty of Science, Japan Women’s University, Tokyo, Japan

The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.