IgE epitope mapping of Pen ch 13 allergen

Abstract

Rationale We have identified an alkaline serine protease that is a major allergen of Penicillium chrysogenum (Pen ch 13). The objective of the present study is to identify the linear IgE-binding epitopes of Pen ch 13. Methods The sequential IgE epitopes on Pen ch 13 were investigated with phage-displayed overlapping peptides, synthetic peptides attached on membranes, recombinant wild type Pen ch 13 and mutants carrying substituted amino acid(s). Results IgE antibodies in 35 serum samples tested reacted with at least one of the 11 peptide fragments of Pen ch 13. Peptide f-2n (residues 31 to 61) showed a high intensity and the highest frequency (77%) of IgE-binding. SPOTs assay narrowed down the IgE-binding to residues 48-55 (GHADFGGR). Substitution of His49 and/or Phe52 on Pen ch 13 with methionine resulted in proteins with drastic loss of IgE binding in seven sera tested. A model was constructed based on the structure of Penicillium cyclopium subtilisin protease that has over 90% (256 out of 283 amino acids) sequence identity with Pen ch 13. The major epitope (GHADFGGR) on Pen ch 13 formed a loop-like structure and located at the surface of the allergen. Conclusions Several linear IgE-reactive epitopes and their critical core amino acid residues were identified for the Pen ch 13 allergen. Mapping of these results on a 3D model of the allergen provides valuable information to elucidate the molecular basis of allergenicity for Pen ch 13.