title: Identification of critical amino acids in an immunodominant IgE epitope of Pen c 13, a major allergen from Penicillium citrinum.
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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Jui-Chieh
authors.initials: JC
authors.lastname: Chen
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Chiu
authors.forename: Kuang-Lun
authors.initials: KL
authors.lastname: Lee
authors.forename: Wei-Ning
authors.initials: WN
authors.lastname: Huang
authors.forename: Jiing-Guang
authors.initials: JG
authors.lastname: Chuang
authors.forename: Hsin-Kai
authors.initials: HK
authors.lastname: Liao
authors.forename: Lu-Ping
authors.initials: LP
authors.lastname: Chow
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000946:Antigens, Fungal / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D018604:Epitope Mapping / Q000379:methods; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology / Q000378:metabolism; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007073:Immunoglobulin E / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010407:Penicillium / Q000276:immunology* / Q000378:metabolism
chemical_list: D000485:Allergens; D000596:Amino Acids; D000946:Antigens, Fungal; D018985:Epitopes, B-Lymphocyte; D005656:Fungal Proteins; D016056:Immunodominant Epitopes; C405642:Pen n 13 allergen; D007073:Immunoglobulin E