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...

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.

Sphingosine-1-phospate (S1P) is a bioactive lysophospholipid signaling molecule that serves important roles in normal development and physiological processes, including modulating the immune, cardiovascular, and central nervous systems ( 1-4 ). S1P is a key player in the sphingolipid signaling cascade and is produced from ceramide (CER) and sphingosine (SPH) through the action of sphingosine kinase (SPHK). While CER and SPH are intracellular promoters of apoptosis, S1P has opposite action and, in general, protects cells from apoptotic stimuli. Several experimental fi ndings from independent research groups implicate S1P as a key mediator of multiple survival and growth-promoting pathways ( 5 ). The extracellular functions of S1P are initiated by the binding of the bioactive lipid to a set of fi ve G protein-coupled receptors (GPCRs) belonging to the S1P receptor family ( 6 ). The balance between CER/SPH levels versus S1P provides a rheostat that determines whether a cell is sent into the death Abstract Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and infl ammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affi nity and specifi city for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can outcompete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte traffi cking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, fi rst-in-class therapeutic acting as a "molecular sponge" to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be benefi cial.

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