Glycosphinogolipids (GSLs) are located in the outer leaf of the plasma membrane where they participate in a wide range of cell surface related events including regulation of neuronal growth and differentiation, and are receptors for a number of molecules, such as neurotransmitters, toxins, virus and interferon. Each cell type expresses a characteristic pattern of GSLs and displays a unique complement of ceramide molecular species. The nature of the ceramide molecular species has been shown to affect the antigenicity of GSLs and presumably also the activity of GSLs in their other functions. The long term goal of this project is to determine what factors regulate the synthesis of the unique complement of GSLs that are characteristic of a given cell type and their expression on the plasma membrane. It has been proposed that the variation in GSL molecular species observed in different tissues is the result of differences in the molecular species specificity of the glycosyltransferases which synthesize a particular class of GSL. To test this hypothesis, the molecular species specificity of both sialyltransferase and alpha-galactosyltransferase, the committed steps in the synthesis of gangliosides and globoside, will be studied in brain and liver, two tissues with different complements of ceramide molecular species. Both enzymes use the same lactosylceramide (LacCer) pool as a source of lipid substrate, but the molecular species profile of their respective products is different.
The specific aims of this proposal are to (1) study the molecular species specificity of sialyltransferase in a Golgi fraction of rat brain neurons. The results will be compared to those already obtained with the liver enzyme; (2) purify sialyltransferase from both liver and a neuronal fraction of rat brain; (3) reconstitute the purified enzyme into a defined lipid environment in order to study the specific mechanisms responsible for the changes in the molecular species specificity of the enzyme which occur when the lipid composition of the Golgi membrane is altered; and (4) determine the LacCer molecular species specificity of alpha- galactosyltransferase in both liver and neurons. These studies should contribute to understanding the regulation of GSL synthesis, a process important to normal development of the nervous system and to pathological processes such as Creutzfeld-Jakob disease and polyunsaturated fatty acid lipidosis.
