Analysis of the IgE-epitope of Der f 2, a major mite allergen, by in vitro mutagenesis.
Der f 2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f 2 recognized by human IgE antibodies, artificial mutations were introduced to Der f 2 protein. The IgE-binding activity of Der f 2 was significantly decreased by deletion of 10 amino acids at the N-terminus or nine amino acids at the C-terminus. Site-directed mutagenesis with a single amino acid replacement by Ala or Leu in both N- and C-terminal regions as well as a central portion was performed to generate 42 single-site mutations. Amino acid replacement around a disulfide bond of Cys8-Cys119 caused a marked decrease in IgE-binding activity. Furthermore, a distinct decrease in IgE-binding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these results, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitope structures recognized by human IgE antibodies.
Central Research Laboratories, Asahi Breweries Ltd, Tokyo, Japan.
C
C
Nishiyama
M
M
Fukada
Y
Y
Usui
N
N
Iwamoto
T
T
Yuuki
Y
Y
Okumura
H
H
Okudaira
D000485:Allergens / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D039741:Antigens, Dermatophagoides; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D007073:Immunoglobulin E / Q000032:analysis* / Q000737:chemistry; D008925:Mites / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000276:immunology*
D000485:Allergens; D000596:Amino Acids; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D016056:Immunodominant Epitopes; D007073:Immunoglobulin E