The long-term goal of this project is to contribute to the understanding of the roles of modifications in N-linked oligosaccharides in the synthesis, routing, secretion, and development accumulation of lysosomal enzymes. Dictyostelium discoideum is bing used as a model system for this study because of the availability of a large variety of mutant strains which are altered in each of these physiologically important processes. We will conduct a detailed analysis of the structure and steps in the biosynthesis of the highly modified (3H)Mannose-labeled, N-linked oligosaccharides found on a single, well characterized lysosomal enzyme, Alpha-mannosidase. Mutant strains which are defective in the synthesis, routing, packaging and secretion of single or multiple lysosomal enzymes appear to have altered oligosaccharides. Therefore, similar structural analyses will be performed on this enzyme in the mutant strains to determine whether changes in oligosaccharide structure lead to or are correlated with mislocalization of the enzymes. Since mammalian cells target amny, but not all, of their lysosomal enzymes as a result of oligosaccharide modifications, this study may provide significant insight into these processes. Developmentally specific post-translational modifications occur in Dictyostelium. Certain mutant strains fail to both develop properly and to accumulate normal amounts of lysosomal enzymes, apparently due to alterations in these modifications. The structure and biosynthesis of the oligosaccharides of Alpha-mannosidase in these mutants will also be analyzed. An understanding of how oligosaccharide structures are modified and their cnsequences for development may contribute to our understanding such diverse processes as embryogenesis, turmoigenesis, and age-related alterations in protein modification.
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