Saposins are sphingolipid activator proteins, four of which are derived from a single precursor, prosaposin, by proteolytic processing. These small heat-stable glycoproteins (12-14 kDa) are required for the lysosomal hydrolysis of a variety of sphingolipids. Characterization of these four activator proteins, two of which were recently discovered, and their importance in human health and disease are reviewed in this article.

Prosaposin was identified as a neurotrophic factor stimulating neurite outgrowth in murine neuroblastoma (NS20Y) cells and choline acetyltranferase (ChAT) activity in human neuroblastoma (SK-N-MC) cells. The four naturally occurring saposs, which are derived by proteolytic processing of prosaposin, were tested for activity. Saposin C was found to be active, whereas saposns A, B, and D were inactive as neurotrophic factors. Dose-response curves demonstrated that nanomolar concentrations of prosaposin and saposin C stimulated neurite outgrowth and increased ChAT activity. Prosaposin and saposin C exerted activity by a mechanlsm independent of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3. Binding assays uing saposin C as a ilgand gave two saturable binding constants, a hig-affnity (Kd = 19 pM) and a low-affinity (Kd = 1 uM) constant, with 2000 and 15,000 sites per NS20Y cell, respectively. Phosphorylation stimulation experiments demonstrated that brief treatment with prosaposin or saposin C enhanced phospholation of a variety of proteins, some of which contained phosphorylated tyrosine(s). Since both cell lines were also stimulated by ciary neurotrophic factor (CNTF) as well as prosaposin, inhibition was tested by utilizing an anti-gpl30 monoclonal antibody, which s lly inhibited CNTF stimulation; thls antibody did not inhibit prosaposin or saposi C smulation. These results indicate that prosaposin and saposin C are neurotrophic factors which initiate signal transduction by binding to a high-afinity receptor that induces protein phosphorylation.Prosaposin is the precursor of the lysosomal saposin proteins, which are required for hydrolysis ofglycosphingolipids by lysosomal hydrolases (1, 2). In addition to its role as a lysosomal precursor, prosaposin is presumed to have additional functions, since it exists as a secretory protein in human milk, cerebrospinal fluid, and seminal plasma (3-5); it is present in unprocessed form in high concentrations in human (6) and rat (7) brain; its mRNA is abundant in brain and dorsal root ganglia during embryonic development (8); it is present predominantly in neurons after immunostaining (9); and it occurs as an integral membrane component (10).Recently we demonstrated that prosaposin binds gangliosides with high affinity and facilitates their transfer from micelles to membranes (11). Since gangliosides have been shown to promote neurite outgrowth in cultured neuronal cells (12-14), we investigated whether prosaposin was also active. In this report we identify prosaposin as a potent neurotrophic factor and locate the active region to the saposin C domain. (PBS), and fixed in Bouins solution (30 min). After the fixative had been removed, neurite outgrowth was scored under a phase-contrast microscope. Cells bearing neurites longer than 1 cell diameter were scored as positive, and 100 cells were counted in triplicate from different portions of each dish. Each assay was carried out in duplicate dishes. The average error of duplicates (40 assay...

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Saposin A, a heat-stable 16-kDa glycoprotein, was isolated from Gaucher disease spleen and purified to homogeneity. Chemical sequencing from its amino terminus and of peptides obtained by digestion with protease from Staphylococcus aureus strain V-8 demonstrated that saposin A is derived from proteolytic processing of domain 1 of its precursor protein, prosaposin. Processing of prosaposin (70 kDa) also generates three other previously reported saposin proteins, B, C, and D, from its second, third, and fourth domains. Similar to saposin C, saposin A stimulates the hydrolysis of 4-methylumbelliferyl ,B-glucoside and glucocerebroside by ,B-glucosylceramidase and of galactocerebroside by ,B-galactosylceramidase, mainly by increasing the maximal velocity of both reactions. Saposin A is as active as saposin C in these reactions. Saposin A has no significant effect on other sphingolipid and 4-methylumbelliferyl glycoside hydrolases tested. Saposin A has two potential glycosylation sites that appear to be glycosylated. After deglycosylation, saposin A had a subunit molecular mass of 10 kDa and was as active as native saposin A. However, reduction and alkylation abolished the activation. A three-dimensional model comparing saposins A and C reveals significant sequence homology between them, especially preservation of conserved acidic and basic residues in their middle regions. Each appears to possess a conformationally rigid hydrophobic pocket stabilized by three internal disulffide bridges, with amphipathic helical regions interrupted by helix breakers. (16). Unlike saposin B, saposin C appears to interact directly with both enzymes to stimulate activity (17). The primary structure of saposin C was determined by chemical sequencing of its amino acids (18,19) and by deducing its sequence from nucleotide sequencing of a cDNA encoding prosaposin, its precursor (20). Saposin C has been reported to be deficient in a single patient with a variant form of Gaucher disease (21).Recently, the complete nucleotide sequence of a cDNA encoding prosaposin, the precursor of saposins B and C, was elucidated (20). Prosaposin is a 511-amino acid glycoprotein, and examination of its amino acid sequence reveals four saposin-like domains, two of which are saposins B and C; these are flanked by two additional domains, which we call saposin A and saposin D. Each domain is approximately 80-amino acid residues long; has nearly identical placement of cysteine residues, glycosylation sites, and helical regions; and is flanked by proteolytic cleavage sites. Molecular models indicate that the proteins derived from each domain can fold to give rise to a conformationally rigid hydrophobic pocket held together by three disulfide bridges. Proteolytic cleavage of prosaposin at each domain boundary was predicted to give rise to four saposin proteins (20). We recently have isolated saposin D, the protein arising from domain four of prosaposin and demonstrated it to be a specific sphingomyelin phosphodiesterase (EC 3.1.4.12) activator (22). In this...

Prosaposin, recently identified as a neurotrophic factor (1), is the precursor of saposins A, B, C, and D. The neurotrophic activity of prosaposin resides in the saposin C domain. We have pinpointed the active sequence to a linear 12-mer located in the NH2-terminal sequence of saposin C (LIDNNKTEKEIL). Nanomolar concentrations of a 22-mer peptide encompassing this region stimulated neurite outgrowth and choline acetyltransferase activity, and prevented cell death in neuroblastoma cells. In primary cerebellar granule cells, the 22-mer also stimulated neurite outgroth. Studies of the neuroblastoma line NS20Y using a radiolabeled 18-mer from the neurotrophic region identified a high-affinity (Kd = 70 pM) binding site indicative of receptor-ligand interaction. The 22-mer stimulated protein phosphorylation of several proteins, some of which were tyrosine-phosphorylated after brief exposure similar to saposin C. Circular dichroism studies demonstrated that the 22-mer was converted from a random to a helical structure by addition of ganglioside GM1. The results are consistent with receptor-ligand binding by the peptide initiating a signal transduction cascade and resulting in neuronal differentiation.