Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor

To study the contribution of the 3 disulphide bonds in the major allergen Lep d 2 to the antigenic structure, site-directed mutagenesis was performed. Mutants with one or more cysteine residues altered were constructed with a histidine residue tag For purification purposes and expressed as recombinant proteins in E. coli. Seven mutants were analysed: 3 single mutants (Cys 8, Cys 21 and Cys 72), 3 double mutants (Cys 8-117, Cys 21-16 and Cys 72-77) and one mutant with all 6 cysteines altered (6 Cys). The evaluation of IgE reactivity in 10 allergic patients showed that the disulphide bond formed by cysteine 72 and 77 was the single most contributing bond to IgE binding. Mutants with disruption of the Cys 8-117 bond had a lesser reduction in IgE binding, even though this alteration seemed to influence the compact nature of Lep d 2. However, to abolish the IgE reactivity almost completely, all 6 cysteines had to be altered. A monoclonal antibody previously raised against Lepidoglyphus destructor showed a similar binding as human IgE with no reactivity to the Cys 72-77 or the 6 Cys mutant. Using skin prick test we found no reaction to the 6 Cys mutant at the concentrations tested (1-100 mu g/ml) in an Lepidoglyphus destructor allergic patient, while the T-cell reactivity was preserved. The 6 Cys mutant of Lep d 2 may, after further evaluation, be a candidate molecule for improved immunotherapy of Lepidoglyphus destructor allergy. (C) 1999 Elsevier Science Ltd. All rights reserved.