All living cells synthesize glycan chains. The process of glycosylation is catalyzed by highly specific glycosyltransferases that require specific sugar nucleotide donors. These donors are derived from monosaccharide precursors via a variety of cytosolic/nuclear biosynthetic pathways. The monosaccharide precursors arise from de novo or salvage pathways and can also come in from nutritional sources. Modifications of glycans such as sulfation and acetylation also involve specific nucleotide donors. The pathways for biosynthesis of all these donors are currently assumed to be the same for all cell types. It is also presumed that all molecules in these pathways are already discovered. The true concentrations of these nucleotide donors in the cytosol of living cells are largely unknown. These concentrations could affect glycosylation reactions, as can the concentrations of related nucleotide precursors and products. UCSD has 7 GM R01-funded investigators carrying out various projects that share the need to understand the regulation of synthesis and turnover of these nucleotide donors in intact cells. We have organized these investigators into an interactive group, facilitating knowledge transfer with in this field. We now propose to work together to set up several new and published methods for manipulating and analyzing the components of the nucleotide donor pathways in intact cells and cell lysates. Support of this """"""""Glue grant """""""" application will accelerate progress on some existing aims of the R01 grants, as well as generate new avenues to extend these aims. Examples of new questions to be explored include: do animal mutant cells with major blocs in glycan biosynthesis have altered levels of intracellular PAPS? Is fungal chitin synthase action in the intact cell affected by UDP-GlcNAc levels, and how does exogenous GlcNAc affect this level? How does nutritional mannose supply affect GDP-Man and GDP-Fuc pools in intact mice or in mice with PMI deficiency? What are the donors present in the primitive eukaryote Giardia lamblia, and how do they affect Giardia cell wall biosynthesis? What happens to the levels of sugar nucleotide donors during the process of mitosis, when glycan biosynthesis appears to cease? What is the fate of the nonhuman sialic acid Neu5Gc in human cells? Is there really a pool of CMP-Sia in the nucleus? What are the intracellular levels of UDP-GlcA, and how do they affect the efficiency of glucuronidation of drugs? Are there novel byproducts of sugar nucleotide pathways that are heretofore unknown? The approach we propose to these and other questions will insure that useful new knowledge will emerge, and that the sum of efforts will be greater than the parts.
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