Identification of Critical Amino Acids in an immunodominant IgE epitope of pen c 13, a major allergen from penicillium citrinum
Journal
PLoS ONE
Journal Volume
7
Journal Issue
4
Date Issued
2012
Author(s)
Abstract
Background: Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. Methodology/Principal Findings: Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A 148-E 166) and S22 (A 243-K 274) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T 261-K 274), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity. Conclusions/Significance: Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases. ? 2012 Chen et al.
SDGs
Other Subjects
allergen; fungus antigen; immunoglobulin E; Pen c 13; serine proteinase; unclassified drug; allergen; amino acid; epitope; fungal protein; fungus antigen; immunoglobulin E; Pen n 13 allergen; adult; aged; allergy; amino acid analysis; antibody combining site; antigen antibody reaction; antigen binding; article; B lymphocyte; carboxy terminal sequence; child; client server application; computer model; computer prediction; controlled study; epitope mapping; experimental study; female; fungus allergy; human; major clinical study; male; mass spectrometry; molecular model; nonhuman; Penicillium citrinum; peptide analysis; school child; screening test; site directed mutagenesis; skin test; theoretical study; chemistry; hypersensitivity; immunology; metabolism; methodology; Penicillium; Penicillium citrinum; Allergens; Amino Acids; Antigens, Fungal; Binding Sites, Antibody; Epitope Mapping; Epitopes, B-Lymphocyte; Fungal Proteins; Humans; Hypersensitivity; Immunodominant Epitopes; Immunoglobulin E; Mutagenesis, Site-Directed; Penicillium
Type
journal article