Dokument: Mapping of coronary endothelial cell membrane proteome and comparative proteomic analysis of regulatory T cells in CD73 knockout mice
| Titel: | Mapping of coronary endothelial cell membrane proteome and comparative proteomic analysis of regulatory T cells in CD73 knockout mice | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=10180 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20090126-105804-9 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Master of Science Arjunan, Selvam [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. med. Schrader, Jürgen [Gutachter] Prof. Dr. Martin, William [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The surface located enzyme ecto-5’nucleotidase (CD73) is a GPI-linked membrane-bound glycoprotein which hydrolyzes extracellular AMP into adenosine. Recent studies have indicated that CD73 from endothelial cells (EC) and regulatory T cells plays an integral role in adenosine mediated suppression of inflammation. In the present study an attempt was made to study the molecular consequences of CD73 deletion at the level of the proteome using mass-spectrometry based differential proteomic approaches.
Initially the cationic silica bead method was applied to enrich the endothelial cell membrane from the mouse heart under in vivo condition. In summary, 31 membrane proteins and 40 intracellular proteins were identified. The results revealed that due to the ionic nature of the interaction of colloidal silica with the coronary endothelial membrane, cardiac homogenisation resulted in the partial loss of specificity. Furthermore the amount of protein recovered from one mouse heart is rather low and required pooling of ten hearts. These drawbacks precluded a more detailed analysis of the coronary EC membrane proteome under in vivo conditions. In a next experimental series, ECs were isolated from mouse aorta and mouse lung to be able to perform a differential proteomic analysis between WT and CD73-/- mutants under in vitro condition. Mouse aortic and lung endothelial cells were conveniently isolated by magnetic beads labeled with CD31 and CD102-dynal beads, respectively. To be within the sensitivity range of the mass spectrometer this approach required expansion of isolated aortic and lung EC cells by subcultivation. However, aortic ECs, showed substantial loss of purity upon subcultivation due to overgrowth with non endothelial cells and dedifferentiation. Similarly, expression of CD73 in lung endothelial cells was initially low and was almost fully lost upon subcultivation. These experiments again precluded a differential proteomic analysis. Immunohistochemistry of mouse spleen revealed that there is high expression of CD73 associated with cells of the red pulp which were most likely regulatory T cells (Treg). Regulatory T cells have recently been shown to possess an ecto-nucleotidase cascade, CD73 being a novel marker of Treg cells. Therefore, in a separate experimental series, CD4+CD25+FoxP3+ regulatory T cells were isolated from mouse spleen by magnetic cell separation (Miltenyi). For differential proteomic analysis of Treg cells from WT and CD73-/- mutants were isolated and individual peptides were derivatized with the dimethyl stable isotope method. This labeling strategy produced mass differences between isotopic pairs of 4 mass units. Identification was done by charge state and mass difference using the MSQuant algorithm. In summary, 355 proteins were identified. Interestingly 25 proteins showed significant changes. Among 25 proteins, 17 proteins were upregulated and 8 proteins were downregulated. Among the differentially expressed proteins the following deserve particular attention: Coactosin-like protein, Isoform 2 of 60 kDa heat shock protein , Proteasome subunit beta type-9 alpha type-3, which are 50%, 65%, 58% and 243% respectively upregulated, whereas T-cell specific GTPase was downregulated by 70% in CD73-/- mutants. These upregulated and downregulated proteins may participate in the modulation of the adenosine-mediated anti inflammatory response. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Medizinische Fakultät » Institute » Institut für Herz- und Kreislaufphysiologie | |||||||
| Dokument erstellt am: | 26.01.2009 | |||||||
| Dateien geändert am: | 23.01.2009 | |||||||
| Promotionsantrag am: | 08.12.2008 | |||||||
| Datum der Promotion: | 16.01.2009 |
