Recombinant Der p 1 and Der f 1 with in vitro enzymatic activity to cleave human CD23, CD25 and alpha1-antitrypsin, and in vivo IgE-eliciting activity in mice.
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/FcepsilonRII), the alpha-subunit of the IL-2 receptor (CD25), and a protease inhibitor alpha1-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 alpha1-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 alpha1-antitrypsin, and elicited significant serum IgE levels in immunized mice.


CONCLUSIONS
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.
Takai
Toshiro
T
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Hongo, Tokyo, Japan. t-takai@med.juntendo.ac.jp
Kato
Takeshi
T
Ota
Mikiko
M
Yasueda
Hiroshi
H
Kuhara
Takatoshi
T
Okumura
Ko
K
Ogawa
Hideoki
H
D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology* / Q000378:metabolism*; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood*; D051379:Mice; D017455:Receptors, IgE / Q000097:blood / Q000378:metabolism; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D017455:Receptors, IgE; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases; C451159:Dermatophagoides farinae antigen f 1; C451162:Dermatophagoides pteronyssinus antigen p 1