Abstract

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.

Author Keywords: IgE; antigenic structure; allergy

Abbreviations: mAb, monoclonal antibody (ies); Ab, antibody (ies); Ag, antigen; GST, glutathione-S-transferase; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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