Effects of Double Mutation at Two Distant IgE-binding Sites in the Three-dimensional Structure of the Major House Dust Mite Allergen Der f 2 on IgE-binding and Histamine-releasing Activity

Abstract

Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.

• mite group 2 allergens
• IgE epitopes
• tertiary structure
• allergen engineering
• recombinant allergen