The long term goal of this work is to study the function of oligosaccharides in the soil amoeba Dictyostelium discoideum and then to extrapolate these results to higher organisms. The focus of this work is on the structure, biosynthesis and function of two novel types of glycosylation found in different populations of vesicle associated proteins in this amoeba. One of these is the specific methylation of mannose-6-phosphate (Man6P) residues by a phosphate methyl transferase. In mammalian cells, Man6P is used to target newly made lysosomal enzymes to the lysosome, but the role of methylation for lysosomal enzyme targeting or other functions in Dictyostelium is unknown. Partial purification and biochemical characterization of this transferase will be done. This will be followed by creation mutants that specifically disrupt the methyltransferase gene to assess its function. The other type of glycosylation is called serine phosphoglycosylation, and it is initiated by the transfer of N-acetylglucosamine-l-phosphate (GlcNAc-alpha-l-P) to serine residues. The modification occurs on some lysosomal proteins in vegetative cells and later in development, in spore cell-specific organelles called prespore vesicles. This modification may also be involved in targeting. Other sugars may be linked to GIcNAc-l-P, and this study will examine the structural diversity of the these chains, how they are made and what structural features of the acceptor proteins induce or permit phosphoglycosylation. A novel mutagenesis procedure will be used to generate mutants that cannot add these chains because the GlcNAc-alpha-1-P transferase gene is disrupted. The consequences of its loss will be examined for intracellular protein movement, targeting and development. The availability of an antibody against GlcNAc-alpha-1-P, and a specific enzyme assay will be used to search for phosphoglycosylation in higher organisms. Preliminary studies that show its presence in the cell adhesion molecule T-cadherin will be expanded to include further work on its biosynthesis and structure. If either of these modifications is used for lysosomal enzyme targeting in Dictyostelium, this may have implications for other novel ways of targeting proteins in mammalian cells.
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