Galactomannan-based targeting of glycoproteins for secretion and cell wall incorporation

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This project addresses the question of how fungal cells are able to correctly assemble their cell walls and secrete enzymes into their environment. Fungi negatively impact society by causing food spoilage, damaging materials and buildings, and being agents of plant and animal diseases. They are also used by industry for producing citrate and a number of important protein products. An understanding of the mechanisms involved in fungal cell wall formation will facilitate efforts to control the growth of fungi in many practical situations. A better understanding of the fungal secretion process could also facilitate increased production of industrial proteins. An important aspect of this research project is to provide training opportunities for postdoctoral researchers and undergraduate students pursuing careers as research scientists. Additionally, there is a program to involve high school science teachers in the Buffalo area in the research.The project will define how the model filamentous fungus, Neurospora crassa, correctly targets cell wall proteins to the cell wall and secreted proteins to the environment. Both groups of glycoproteins are secreted by the fungi into the cell wall space and must be targeted either for incorporation into the wall or for release to the environment. Previous work has demonstrated the importance of an N-linked galactomannan structure for the incorporation of cell wall proteins into the cell wall. The project tests the hypothesis that modifications to the N-linked oligosaccharides are used to direct cell wall proteins into the wall and to allow secreted glycoproteins to be released into the environment. The N-linked modifications present on cell wall and secreted proteins will be examined in a number of mutant isolates using techniques from glycobiology to identify and characterize galactomannan structures. The DFG-5 alpha-1,6-mannanase plays a critical role in generating the galactomannan structure needed for incorporation of glycoproteins into the wall, and the research will characterize the enzymatic activity of DFG-5 and the interactions between DFG-5 and cell wall glycoproteins. These experiments will utilize a HIS6-tagged version of DFG-5 to purify the enzyme. DFG-5 enzymatic activity will be assessed using substrate galactomannans, and protein/protein interactions will be characterized in co-purification and co-immunoprecipitation experiments using antibodies directed against cell wall and secreted glycoproteins. Previous research from the investigator’s groups demonstrated that lichenin (a glucan with a repeating beta-1,4-glucose-beta-1,4-glucose-beta-1,3-glucose trisaccharide structure) is used to attach cell wall proteins into the cell wall and the project will identify and characterize lichenin synthases. This will be done using molecular genetics to identify putative lichenin synthases and anti-bodies directed against lichenin to demonstrate the synthesis of lichenin. In conjunction with the published literature, the research will provide a comprehensive picture of fungal cell wall biogenesis and will answer the question of how fungal cells distinguish between secreted and cell wall glycoproteins and target them to their correct extracellular locations.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.