Part I: Integration of proteomics and bioinformatics tools to analyze the differential lung proteome in a mouse model of Pen c 13 allergen-induced allergic airway inflammation;Part II: Hypoallergenic fusion protein derived from B cell epitopes of the Pen
| Autor: | Jui-Chieh Chen, 陳瑞傑 |
|---|---|
| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 Part I Fungal allergens are associated with the development of asthma, and some have been characterized as proteases. Here, we established an animal model of allergic airway inflammation in response to continuous exposure to proteolytically active Pen c 13, a major allergen secreted by Penicillium citrinum. In functional analyses, Pen c 13 exposure led to increased airway hyperresponsiveness, significant inflammatory cell infiltration, mucus overproduction, and collagen deposition in the lung, dramatically elevated serum levels of total IgE and Pen c 13-specific IgE and IgG1, and increased production of the Th2 cytokines IL-4, IL-5, and IL-13 by splenocytes stimulated in vitro with Pen c 13. To examine the mechanisms, we performed two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis combined with nano-LC-MS-MS, followed by Ingenuity Pathways Analysis (IPA) to map significant functional networks. The highest-scoring network that associated with acute allergic pulmonary eosinophilia and cell movement in the Functions and Diseases analysis was selected for further dissection. Additionally, canonical pathways, including actin cytoskeleton, leukocyte extravasation, integrin, NRF2-mediated oxidative stress response, FAK, tight junction, and acute phase response, were also highlighted. Using IPA tools to identify potential targets, galectin-3 and laminin might be involved in novel pathogenic mechanisms. Finally, we focused on junctional proteins, because, in addition to opening of the epithelial barrier by environmental proteases possibly being the initial step in the development of asthma, these proteins are also associates with actin rearrangement. Taken together, Pen c 13 exposure causes junctional structure alterations and actin cytoskeletal rearrangements, resulting in increased permeability and airway structural changes. These effects probably change the lung microenvironment and foster the allergic sensitization. Part II Specific immunotherapy (SIT) that is in use at present involves the administration of allergen extracts to patients leading to the clinical tolerance of the allergens and cure for allergic symptoms. However, the risk of therapy-induced side effects limits its broad application. Recent studies have revealed that the epitope complexity of allergen extracts can be recreated using recombinant allergens, and hypoallergenic derivatives of these can be engineered to increase treatment safety. In present study, we developed the nonanaphylactic peptides derived from B cell epitopes of Pen c 13, an immunodominant human allergen secreted by Penicillium citrinum identified as an alkaline serine protease, to be a generally applicable strategy for the therapy of allergy. To find linear epitopes on Pen c 13, mapping of allergenic epitopes was performed by cleaved peptides which cover most of the protein sequence. The results showed that at least ten different linear IgE-binding epitopes located throughout the Pen c 13. Of these, peptide S16 (A148-E166) and S22 (A243-K274) were recognized by sera from 90% and 100% of the patients tested. In addition, the specificity of IgE binding was confirmed by ELISA inhibition assays. The peptide S22 was selected for dissection of its IgE-binding ability, and therefore we exerted molecular modeling and B-cell epitope predicted server to predict six most possible residues involved in IgE binding. Furthermore, the peptide S22 was split into two parts which comprise N-terminal (A243-A260) or C-terminal (T261-K274) part fused to GST. The result of the serum screening showed that the majority of IgE-binding ability resides indeed in its C-terminal fragment. Final, six most possible residues within C-terminus of the peptide S22 were substituted for alanine individual by point mutations; one of the mutants of Pen c 13 (T261-K274), K274A, had dramatically reduced IgE reactivity and may be designed hypoallergenic forms of the allergen, which develop a safe and efficient therapeutic strategy for treating human allergic diseases in the future. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|