The molecular basis of antigenic cross-reactivity between the group 2 miteallergens

Background: Mite group 2 allergens Der p 2, Der f 2, and fur m 2 are 14-kDa proteins of unknown function that share 83% to 85% amino acid sequence ide ntity. Isoforms of the allergens within each genus have been identified whi ch differ by 3 or 4 amino acids, but little is known of the influence of gr oup 2 polymorphisms on human IgE antibody binding. Objective: The purpose of this study was to investigate the importance of i nterspecies and isoform substitutions on murine mAb and IgE antibody bindin g and on the molecular structure of the group 2 allergens. Methods: Site-directed mutagenesis was used to incorporate the isoform amin o acid substitutions onto the Der p 2.0101 sequence. Recombinant allergens were expressed and purified from Escherichia coli and used to evaluate anti body binding by enzyme-linked immunosorbent assay (ELISA). Molecular modeli ng of the tertiary structure was used to analyze structural differences bet ween the various group 2 allergens. Results: The substitution of asparagine for aspartic acid at position 114 r estored mAb binding of rDer p 2.0101; the other Der p 2 isoforms and the 3 rDer f 2 isoforms also reacted in the 2-site ELISA. The correlation of IgE binding to the Der p 2 isoforms was excellent and tended to be higher in th e isoforms with the asparagine 114 substitution (r(2) = 0.87 vs r(2) = 0.95 ), rEur m 2.0101 bound to all mAb except 7A1: when compared with rDer p 2 f or IgE binding, rEur m 2.0101 gave a correlation coefficient of r(2) = 0.68 . Molecular modeling revealed that fur m 2 and the storage mite homologs Le p d 2 and Tyr p 2 retain the tertiary fold of Der p 2. fur m 2 has a conser ved surface, whereas Lep d 2 and Tyr p 2 present most of the amino acid sub stitutions on this surface. Lep d 2 and Tyr p 2 did not react with mAb or w ith sera from patients with IgE to Dermetophagoides species. Conclusion: The isoform substitutions of rDer p 2 can be distinguished by m Ab. The allergenic cross-reactivity between Der p 2, Der f 2, and fur m 2 i s a direct result of the conserved antigenic surface, whereas the lack of c ross-reactivity with Lep d 2 and Sr p 2 is a result of the multiple substit utions across this surface.