The broad long-term goals of this project are to accurately define the glycosylation patterns of normal and heterologous extra-cellular glycoproteins secreted from the filamentous fungus Neurospora crassa. Neurospora has been recently used by the principal investigator as a host cell for production of useful heterologous mammalian proteins. Ongoing research in the laboratory includes the attempt to express human IgG antibodies in Neurospora. It is well established that the biologically """"""""correct"""""""" glycosylation of antibodies is an absolute requirement for their full function in the mammalian immune system. The glycoforms of different species, different cell types from the same species, and even the same cell type grown under various culture conditions have been shown to be highly variable. Any host cell which is proposed for use in a heterologous expression system must produce glycoforms consistent with the natural glycoform produced by the cell type generating the protein in nature. Glycoforms produced by Neurospora have never been systematically investigated. Homologous fungal proteins and heterologous mammalian proteins expressed in Neurospora cells and secreted to the cell surface or into the culture medium will be collected from the media or in pellets of cell wall-membrane preparations. Crude preparations and electrophoretically separated glycoproteins will be evaluated for glycoforms by the """"""""First Choice"""""""" glycosylation assay system developed by Boehringer Mannheim Corp. A systematic survey of these glycoforms will provide a first step in evaluating the usefulness of a Neurospora expression system for production of mammalian glycoproteins.
