Analysis of the IgE-epitope of Der f 2, a major mite allergen, by in vitro mutagenesis

Der f2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f2 recognized by human IgE antibodies, artificial mutations were introduced to Der f2 protein. The IgE-binding activity of Der f2 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.