Effects of double mutation at two distant IgE-binding sites in the three-dimensional structure of the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity.
Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.
Takai
T
T
Bioscience Research and Development Laboratory, Asahi Breweries, Ltd., Kitasoma-gun, Ibaraki, Japan.
Hatanaka
H
H
Ichikawa
S
S
Yokota
T
T
Inagaki
F
F
Okumura
Y
Y
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D039741:Antigens, Dermatophagoides; D001491:Basophils / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D066298:In Vitro Techniques; D008925:Mites / Q000235:genetics* / Q000276:immunology*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000485:Allergens; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E