Reduction in IgE binding to allergen variants generated by site-directed mutagenesis: contribution of disulfide bonds to the antigenic structure of the major house dust mite allergen Der p 2.
Site-directed mutagenesis was used to investigate the contribution of disulfide bonds to the antigenic structure of Der p 2. Single amino acid variants were generated at cysteine residues, preventing the formation of disulfide bonds at positions 21-27, 73-78, and 8-119. The variants were tested for binding to murine monoclonal antibodies (mAb) and human IgE antibodies (Ab) in an inhibition enzyme immunoassay. Removal of the disulfide linking the amino-carboxy termini (C8-C119) had no effect on mAb binding, however, IgE Ab binding was reduced by up to 10-fold. The other two disulfides form small loops and disruption of these bonds gave different binding patterns. The variant lacking the C21-C27 bond showed up to a 40-fold reduction in antibody binding, while the variant lacking the C73-C78 bond showed more than a 100-fold reduction in IgE Ab binding and failed to bind 3 of 4 mAb. Intradermal skin testing with the C73-C78 variant supported the in vitro findings; the variant was 10 to 100-fold less reactive than rDer p 2. These two bonds thus make markedly different contributions to stabilizing the antigenic determinants of Der p 2. The results suggest that the C73-C78 bond plays a critical role in stabilizing the antigenic structure of this major mite allergen.
Smith
A M
AM
Department of Medicine, University of Virginia, Charlottesville 22908, USA.
Chapman
M D
MD
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D039741:Antigens, Dermatophagoides; D001483:Base Sequence; D004220:Disulfides; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D004220:Disulfides; D006023:Glycoproteins; D007073:Immunoglobulin E