Identification of immunodominant IgE epitopes of the major house dust mite allergen Der f 24

Previously, a ubiquinol-cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HDM) species Dermatophagoides farinae (Der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein Der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in Der f and human UQCRB was performed. Four different recombinant Der f 24 and hybrid proteins formed by integrating Der f and human UQCRB sequences were expressed in Escherichia coli, purified using Ni-NTA resins, and IgE-binding activity was determined using IgE-western blotting and enzyme-linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE-dot blotting and IgE-ELISA with synthetic polypeptides and HDM-allergic sera. Three-dimensional (3D) structural modeling was used to analyze the position of the immuno-dominant IgE epitope. The amino acid sequence homology between Der f 24 and the human UQCRB protein was determined to be 39.34%. IgE-ELISA and western blot analysis showed that all of the Der f-human UQCRB hybrid proteins generated, except for the one lacking 59 residues of the N-terminal region of Der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N-terminal reacted with IgEs from HDM-allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32-aa N-terminal region of Der f 24 was localized to a structural protrusion, which may facilitate specific IgE-binding. These results indicate that the immunodominant IgE epitope of Der f 24 is located mainly in a 32-residue region of the N-terminus. These findings may inform the mechanisms of HDM allergy sensitization and allergy immunotherapy development.