Effects of proline mutations in the major house dust mite allergen Der f 2on IgE-binding and histamine-releasing activity

Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino-acid residues. Wild-type and six prol ine mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed i n Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The appar ent molecular masses analyzed by gel-filtration were 14-15 kDa. The IgE-bin ding capacity in the sera of seven mite-allergic patients, inhibitory activ ity for IgE-binding to immobilized wild-type Der f 2, and activity to stimu late peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild-type Der f 2 in their CD spectrum, showed reduced IgE-binding, reduced inhibitory acti vity, and less histamine-releasing activity than the wild-type. P34A also s howed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are cl osely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE-binding site common to al l tested patients. P66A showed reduced IgE-binding in two sera out of seven . P26A and P79A showed no reduced allergenicity. However, in immunoblot ana lysis after SDS/PAGE under reduced conditions, P79A showed no or markedly r educed IgE-binding while the other mutants showed IgE-binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy.