Recombinant Der p 1 and Der f 1 with in vitro Enzymatic Activity to Cleave Human CD23, CD25 and α₁-Antitrypsin, and in vivo IgE-Eliciting Activity in Mice

Abstract

Background: The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcΕRII), the α-subunit of the IL-2 receptor (CD25), and a protease inhibitor α₁-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice. Methods: Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human α₁-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA. Results: Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human α₁-antitrypsin, and elicited significant serum IgE levels in immunized mice. Conclusion: The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses.

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

“…Serum total IgE and allergen-specific Abs were measured by a sandwich ELISA, as described previously (10,(32)(33)(34)(35) with minor modifications. Plates were coated with papain (30 mg/ml for the experiment of Fig.…”

Major House Dust Mite Allergens Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1 Degrade and Inactivate Lung Surfactant Proteins A and D

“…Der f 1, one of the major allergens, is also a cysteine protease and shows 82% homology to Der p 1. Similar to Der p 1, a recombinant form of Der f 1 has been shown to cleave human CD23 and CD25 in vitro (32) and reduce barrier function of the skin through its proteolytic activity (33).…”

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

“…The cysteine protease activity of Der p 1 and Der f 1 has been suggested to be involved in the pathogenesis of allergies by facilitating the passage of their own and other allergens across the epithelium (21,22), cleaving and/or interacting with cell surface molecules and intrinsic protease inhibitors (23)(24)(25)(26)(27)(28)(29)(30), and modulating the function of various cells (23,27,(31)(32)(33)(34)(35)(36)(37)(38)(39). Herbert et al (21) speculated that cysteine protease activity of natural Der p 1 could be activated in vivo by glutathione in the airway on the basis of their in vitro observations.…”

“…(43)(44)(45)(46), have proteolytic activities (44 -46), show a similar substrate-specificity for cysteine protease activity as the natural types (16,28), and are free from mite-derived serine proteases (16). They exhibited enzymatic activity in vitro to cleave human CD23 and CD25 on the cell surface and human ␣1-antitrypsin, which had been reported to be cleaved by natural Der p 1 and IgE-eliciting activity in vivo in mice similar to natural Der p 1 and Der f 1 (28). The highly purified and fully active rDer p 1 and rDer f 1 will be the basis for allergen standardization and the design of safer and more effective allergen-vaccines and be useful for studies in vitro and in vivo to investigate the roles of their function and structure in allergic diseases.…”