Effects of proline mutations in the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity.
Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino-acid residues. Wild-type and six proline mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed in Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The apparent molecular masses analyzed by gel-filtration were 14-15 kDa. The IgE-binding capacity in the sera of seven mite-allergic patients, inhibitory activity for IgE-binding to immobilized wild-type Der f 2, and activity to stimulate peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild-type Der f 2 in their CD spectrum, showed reduced IgE-binding, reduced inhibitory activity, and less histamine-releasing activity than the wild-type. P34A also showed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are closely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE-binding site common to all tested patients. P66A showed reduced IgE-binding in two sera out of seven. P26A and P79A showed no reduced allergenicity. However, in immunoblot analysis after SDS/PAGE under reduced conditions, P79A showed no or markedly reduced IgE-binding while the other mutants showed IgE-binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy.
Takai
T
T
Bioscience Research and Development Laboratory, Asahi Breweries, Ltd, Ibaraki, Japan. toshiro.takai@asahibeer.co.jp
Ichikawa
S
S
Hatanaka
H
H
Inagaki
F
F
Okumura
Y
Y
D000485:Allergens / Q000276:immunology* / Q000494:pharmacology*; D019943:Amino Acid Substitution; D000818:Animals; D039741:Antigens, Dermatophagoides; D001491:Basophils / Q000187:drug effects / Q000502:physiology; D001665:Binding Sites; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology*; D006636:Histamine Release / Q000187:drug effects*; D006801:Humans; D007124:Immunoenzyme Techniques; D007073:Immunoglobulin E / Q000378:metabolism*; D008925:Mites; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology
D000485:Allergens; D039741:Antigens, Dermatophagoides; D004220:Disulfides; D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D011392:Proline