Secretome Analysis in Higher Basidiomycetes - Freely Secreted and Cell Wall Proteins from Coprinopsis cinerea

Abstract

Listen

Translate article

Basidiomycetes secrete numerous proteins not only into the extracellular space but as well into extracellular structures as the cell wall and the associated polysaccharide layer (hyphal sheath). A detailed overview of the secretome of Coprinopsis cinerea fractionated into freely secreted proteins, proteins of the hyphal sheath and cell wall proteins is given in this thesis. The cell wall proteins were further fractionated: into ionically bound proteins, into other covalently bound proteins and proteins associated by disulphide bridges and covalently bound proteins. The single extractable fractions were analyzed by proteomic methods and the proteins were identified by mass spectrometrie (LC-MS2). At the beginning of this work, an optimized protein preparation protocol applicable for various higher basidiomyctes was established. To analyze the fractionated secretome in the early exponential growth phase two proteomic techniques were applied: a 2-DE gel approach in combination with protein identification by LCMS2 and in parallel a one-dimensional (1-DE) shotgun approach for the identification of proteins by LC-MS2. Both methods showed a clear compartmentation between the cell wall proteome on the one hand and the freely secreted and the hyphal sheath proteome on the other hand. Only few of the identified proteins were overlapping between the free secretome and the hyphal sheath proteome on the one hand and the fractions of the cell wall proteome on the other hand. In total, 162 proteins in five different fractions (with overlappings) were identified in this experimental setup. The identified proteins from the free secretome and the hyphal sheath included mainly glycoside hydrolases, peptidases and oxidoreductases, all putative enzymes involved in nutrient supply. Within the cell wall, proteins with putative functions in the cell wall formation and restructuring (e.g. chitinases, mannosidases) were detected. Also enzymes known from intracellular processes were identified and thus could possibly be located in the cell wall of C. cinerea. Although the existence of such typically intracellular proteins in the cell wall of fungi was already shown previously, the extracellular function of these proteins is controversial. Further analysis of the fractionated secretome of C. cinerea over the time of cultivation revealed a dynamic secretome, possibly an adaption to the changing environmental conditions. The secretome showed a reduced complexity over the time. Contrary, single proteins such as peptidases and glycoside hydrolases changed significantly in their concentration over the time of cultivation, as visible in the 2-DE gels. In conclusion, these experimental setups revealed a dynamic and strictly compartmented secretome of C. cinerea. The analysis of the C. cinerea secretome by 2-DE showed that many of the extracellular proteins have extensive posttranslational modifications (PTM). The freely secreted proteins of C. cinerea were found to be highly glycosylated. In addition, experiments with radioactively labeled phosphate revealed evidence that specifically phosphorylated proteins are present in the secretome of C. cinerea. The analysis of the fractionated secretome from C. cinerea grown in liquid medium gave an overview concerning the nature and the dynamics of the secretome over the time and gave an overview of the secretome from C. cinerea in liquid cultures. However, for a deeper insight into substrate degradation and the cell wall formation of fungi, an analysis on natural substrate is crucial. In course of this work, it could be shown that the methods developed for C. cinerea in liquid culture are as well applicable to fungi growing on natural substrate, as demonstrated for Pleurotus ostreatus strain PC9 grown on wheat straw for a period of 31 days.