Determination of the N- and C-terminal sequences required to bind human IgE of the major house dust mite allergen Der f 2 and epitope mapping for monoclonal antibodies.
B cell epitopes of the major house dust mite allergen Der f 2 from Dermatophagoides farinae were analysed using deletion mutants of Der f 2 expressed as fusion proteins in Escherichia coli. The reactivities of these partial Der f 2 molecules to human anti-mite IgE antibodies in atopic patients and to murine anti-Der f2 monoclonal antibodies (mAbs) were examined by immunoblotting. A C-terminal deletion mutant of Der f 2, 1-123, had almost the same reactivity to human IgE as the whole Der f 2 (1-129) and an N-terminal deletion mutant of Der f 2 (25-129) still had weak reactivity. On the other hand, in two deleted Der f 2 molecules, 1-120 and 30-129, reactivity was lost in spite of long overlapping sequences. These results suggest that the human IgE antibodies to Der f 2 in atopic patient sera recognize the conformational structures dependent on the tertiary structure of Der f 2, including disulfide bond formations, rather than the contiguous sequences of amino acids. The sequences 1-24, 25-29 and 121-123 were revealed as the minimum N- and C- terminal amino acid sequences required for IgE binding. Contrastingly, all three murine mAbs bound to the smaller deletion mutants, 1-90 and 67-129, suggesting that the cores of the epitopes for these mAbs exist in the 24 amino acid sequence of Der f 2, 67-90 overlapping the sequential human IgE epitope on Der p 2, the equivalent allergen from Dermatophagoides pteronyssinus. These findings are important for the understanding of the antigenic structure of Der f 2 and for the manipulation of the allergen for immunotherapy.
Takai
T
T
Bioscience Research and Development Laboratory, Biotechnology Section, Asahi Breweries Ltd, Ohta-ku, Tokyo, Japan.
Yuuki
T
T
Okumura
Y
Y
Mori
A
A
Okudaira
H
H
D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D039741:Antigens, Dermatophagoides; D018604:Epitope Mapping; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008925:Mites / Q000276:immunology; D011485:Protein Binding; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D007073:Immunoglobulin E