Background: The Per a 3 is a species-specific allergen of the American cockroach (Periplaneta americana) related to insect hemolymph proteins and includes four known isoallergens. This study aimed to identify Per a 3 linear IgE-binding epitopes.
Methods: Per a 3 recombinant fragments were generated from the recombinant Per a 3.01 allergen (685 amino acid residues) by using existing restriction sites or by using polymerase chain reaction products, and expressed in Escherichia coli. Antigenicities were assessed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and binding inhibition with human IgE.
Results: Human IgE recognized recombinant fragments 340–425, 466–579, 502–595, and 595–636 as revealed by immunoblotting and ELISA. On the other hand, the N-terminal fragment 1–399, recombinants 410–443, 472–551, 502–579, 606–636, and the C-terminal fragment 636–685 were unable to bind human IgE. Amino acid sequences 400–409, 466–471, 580–595, and 595–605 were shown to be required for IgE binding to the Per a 3.01 allergen, suggesting that the C-terminus contains most of the IgE-binding sites. Four peptides corresponding to these IgE-binding amino acid sequences were synthesized. These peptides reacted with most sera (62.5–87.5%) tested as revealed by ELISA, demonstrating a heterogeneous IgE-binding response. Moreover, preincubation of IgE-positive recombinant proteins and synthetic peptides with atopic IgE resulted in marked inhibition of the IgE binding to Per a 3.01 allergen. Amino acid sequences 400TVLRDPVFYQ409, 466NNVDQI471, 580VDKGHNYCGYPENLLI595, and 595IPKGKKGGQAY605 of the major recombinant American cockroach Per a 3.01 allergen were involved in IgE binding.
Conclusion: These findings will advance our understanding of the antigenic structures responsible for allergenicity to the American cockroach, thereby providing strategies for the development of immunotherapies.