The long term objectives of this project are to contribute to the understanding of the factors which regulate the synthesis of glycosphingolipids (GSLs) and their expression on specific cellular membranes. Different cell types synthesize unique GSL classes with characteristic ceramide compositions. Unlike most tissues, brain contains GSL with complex ceramide molecular species, and it has been shown that there are changes in both class and ceramide molecular species composition of the GSL with development and cellular differentiation. Such information suggests that these glycolipids participate in the regulation of neuronal growth and development. GSLs are synthesized by sequential addition of monosaccharide residues, but the factors responsible for the unique variations of ceramide moieties are not yet understood. It is possible that the variations seen are due to the specificity of the enzymes which synthesize the various classes of GSL, by the exchange of fatty acids (deacylation-reacylation cycle) after the synthesis of the carbohydrate chain, or the compartmentalization of specific precursor molecular species into multienzyme complexes which synthesize specific end-products. Recent methodological developments (separation of individual molecular species and purification of GSL transfer protein) have made it possible to examine these alternatives. Mouse neuroblastoma 2A cells, which contain different ceramide long chain bases in different GSL classes will be used to test the above hypotheses by studying the partitioning of lactosylceramide molecular species between galactosyltransferase and sialosyltransferase, the committed steps in the synthesis of the globo and ganglio series. The possible occurrence of deacylation-reacylation will be tested with doubly-labeled molecular species of GSL. Retinoic acid treated neuroblastoma cells, which differentiate and show a shift in their GSL pattern will be examined in an effort to determine what factors are responsible for the GSL changes. These studies should contribute to understanding the regulation of GSL synthesis, processes important to normal development of the nervous system and pathological processes such as occur in Creutzfeld-Jakob disease.
