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

Abstract

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-γ. 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.

IL-33–Mediated Innate Response and Adaptive Immune Cells Contribute to Maximum Responses of Protease Allergen–Induced Allergic Airway Inflammation

“…The mature forms were then purified by anion exchange column and sizeexclusion column chromatography. Papain, rDer f 1, and rDer p 1 were treated with an irreversible inhibitor specific to cysteine proteases, E64 (Peptide Institute, Osaka, Japan), as described previously (10), with minor modifications. After the treatment, free E64 was removed by dialysis.…”

“…Protease activity of house dust mite (HDM) major group 1 allergens (Der f 1 and Der p 1), two of the most important allergens common worldwide, has been shown to cause barrier dysfunction, to induce the production of proinflammatory cytokines in epithelial cells and keratinocytes, to cleave various molecules, to modulate functions of various cell types, and to induce Th2 responses (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Papain, a papaya fruit-derived occupational protease allergen, stimulates epithelial cells (12,13) and mouse basophils in vitro (14,15) and induces Th2 responses (14)(15)(16)(17) and IL-33-dependent innate responses of lung eosinophilia in mice (18).…”

Modulation of Allergenicity of Major House Dust Mite Allergens Der f 1 and Der p 1 by Interaction with an Endogenous Ligand

“…At a high concentration of papain (100 g/ml), its cysteine protease activity stimulated murine bone marrow-derived basophils in vitro to produce Th2-inducing cytokines including IL-4 but not release histamine. Cysteine protease activity of our preparations of rDer p 1 and rDer f 1 also crucially commits to the sensitization process in mice (37). Although Phillips et al (60) reported that Der p 1 stimulated purified human basophils to release IL-4, whether its cysteine protease activity is responsible for the basophil activation is unknown because their Der p 1 preparation also contained serine proteases other than Der p 1 (28,61).…”

“…Der f 1-specific Abs were detected on plates coated with 5 g/ml rDer f 1 and blocked with ImmunoBlock (DS Pharma Biochemicals), and developed with HRP-conjugated Abs specific to the murine IgE and IgG subclasses as described previously (37,54,55) with some modifications as follows. Sera and detection Abs were diluted with solutions 1 and 2 of CanGetSignal (TOYOBO), respectively.…”

“…It has been proposed that the proteolytic activity of Der f 1 and Der p 1 is involved in the pathogenesis of allergies and sensitization to the production of IgE by reducing physical and biochemical tissue barriers, cleaving various molecules, and modulating the functions of various cells (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). We found that human sweat inhibited the proteolytic activities of recombinant Der f 1 and Der p 1 and used sweat as a skin-derived material for isolation of the inhibitor, which was subsequently determined to be cystatin A (31), also called stefin A (38,39).…”