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. T o determine the major epitopes on Der f2 recognized by human IgE antib odies, 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-terminu s. Site-directed mutagenesis with a single amino acid replacement by A la or Leu in both N- and C-terminal regions as well as a central porti on was performed to generate 42 single-site mutations. Amino acid repl acement around a disulfide bond of Cys8-Cys119 caused a marked decreas e in IgE-binding activity. Furthermore, a distinct decrease in IgE-bin ding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these res ults, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitop e structures recognized by human IgE antibodies.