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Structural Proteomics of the Fungal Cell Wall
Fungi are surrounded by a thick layer of carbohydrates and proteins, which is essential for the cell’s viability – the fungal cell wall. Proteins are incorporated into this organelle in different ways: some are covalently linked to the carbohydrate moiety of the cell wall via Glycosylphosphatidylino...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2020
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| Zusammenfassung: | Fungi are surrounded by a thick layer of carbohydrates and proteins, which is essential for the cell’s viability – the fungal cell wall. Proteins are incorporated into this organelle in different ways: some are covalently linked to the carbohydrate moiety of the cell wall via Glycosylphosphatidylinositol (GPI)-anchors or alkali-sensitive linkages, others are indirectly attached to the cell wall via disulfide bonds. Cell wall proteins are involved in various cellular functions, such as cell wall biosynthesis, adhesion to external surfaces, or sensing.
The GPI-anchored cell wall proteome of the thermophilic model organism Chaetomium thermophilum was identified in the first part of this thesis. First, a prediction of GPI-proteins, anchored to the cell wall and the plasma membrane was done. The prediction was then complemented by mass-spectrometric identification of GPI-anchored cell wall proteins in isolated cell walls. The detected proteins were then analyzed concerning their functions and putative roles and interesting targets for pharmaceutical applications and fundamental research were established, including Gel1/2, Kre9/Knh1, and Ecm33. In addition, the ultrastructure of the C. thermophilum cell wall was analyzed via transmission electron microscopy, revealing short microfibrils in its outer layer and its similarity to the cell wall of S. cerevisiae.
The thesis then advances to the analysis of the A-domains of the Candida glabrata adhesins Awp1 and Awp3, which are members of adhesin cluster VI. Although the fungal pathogen lacks certain virulence factors – such as hyphae formation – C. glabrata infections are commonly observed; its large repertoire of adhesins is believed to be the reason therefore. Awp1A and Awp3A both consist of a β helix domain and an α crystallin domain. They are structurally similar to carbohydrate binding proteins, e. g. polysaccharide lyases, but carbohydrate binding could not be observed. A sequence similarity network (SSN) elucidates their high similarity to cluster V adhesins Awp2 and Awp4 and thereby reinforces previous classifications. The structures of Awp1 and Awp3 provide first insights into new types of adhesins in C. glabrata that include the adhesin clusters V and VI.
Furthermore, the G-protein coupled receptor Pth11 from C. thermophilum was analyzed. It contains an N-terminal CFEM domain – a domain exclusively found in fungal cell wall and plasma membrane proteins – that is predicted to be the ligand binding site. The CtPth11 CFEM domain consists of five α helices and reveals two potential binding sites, divided by F48. Distinct conformers of F48 allow formation of a tunnel through the domain. A model of the CtPth11 CFEM domain and transmembrane region – based on prediction of neighboring residues via sequence covariation analysis – shows that both potential binding sites are accessible. In a fragment screen, four fragments were bound in the same cavity; three of them could be fitted into their respective electron densities. These hydrophobic fragments are placed in the hydrophobic cavity, with only few additional interactions, which is in accordance with the proposal that Pth11 senses hydrophobic cues on the plant surface. |
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| Umfang: | 228 Seiten |
| DOI: | 10.17192/z2021.0105 |