Sphingolipids, which are ubiquitous components of animal membranes, are especially abundant in brain, and are important constituents of the outer leaflet of plasma membranes. In this application, studies on the mechanism involved in the stimulation of lysosomal sphingolipidhydrolases by saposins (sphingolipid activator proteins) and on the function of saposins in brain development are proposed. Saposins are small, heat-stable glycoproteins that are required by lysosomal hydrolases and are also implicated as carriers of sphingolipids in other functions such as membrane formation and modification and brain development. Four saposins (A, B, C, and D) are produced by partial proteolysis of a precursor, prosaposin. Their physiological significance is suggested by a wide tissue distribution, subcellular localization which includes nuclei, conservation during evolution, and of particular relevance to neurology the finding that they are present in highest concentration in the developing brain. Genetic deficiencies of saposins lead to profound neurological deficits. Recent advances in the molecular biology of sphingolipid metabolism, in part achieved in this laboratory, for the first time provide the opportunity to define the physiological role of saposins by examination of structure-function relationships.
Three specific aims are proposed.
Aim 1 concerns the mechanism of the stimulative effects of saposins in lysosomal sphingolipid hydrolysis. The interactions of saposins A and C with pure glucosylceramide beta- glucosidase, saposin B with pure GM1 ganglioside beta-galactosidase, and saposin D with pure sphingomyelinase will be investigated in detail with and without added respective substrates and acidic lipids, such as GM1 ganglioside. Gel filtration and affinity chromatography will be used for this purpose. In addition, saposins will be partially modified by proteolysis, deglycosylation, or derivitization of specific amino acid moieties, such as cysteine, aspartic acid, glutamic acid, and lysine. These fragments or derivatives will be tested for stimulation of specific enzyme activity to obtain further insight into the structure-function relationship of saposins.
In Aim 2, the significance and function of saposins and prosaposin during development of nervous system will be investigated. Changes in amount, distribution, and biogenesis of these proteins in various regions of rat brain during development will be investigated. Saposins, prosaposin, and their homologs will be determined by measuring stimulative activities and by crossed immunoelectrophoresis. Amounts of mRNA in these tissues will be estimated by Northern blots. Based on the report of occurrence of saposins in nuclei, the specific binding of saposins with protein/nucleic acid of rat brain nuclei will be investigated by using radioactive saposins and immobilized saposins.
In Aim 3, to determine the degree of conservation of the structure of saposins and to identify portions of the sequence important to saposin function, changes in structure of saposins will be studied by determining structures of cDNA encoding saposins in three animals of key importance from various stages of evolution.
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