Identification of prosaposin as a neurotrophic factor.

Abstract

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...

Protective Effect of a Prosaposin-Derived, 18-Mer Peptide on Slowly Progressive Neuronal Degeneration after Brief Ischemia

“…The molecular mechanisms underlying this neuroprotective effect of 18MP is not fully understood. However, it has been suggested that the high-affinity prosaposin receptor is present on the surface of neuroblastoma cells (O'Brien et al, 1994). Prosaposin at extracellular concentrations greater than 1 ng/mL induces a mitogen-activated protein kinase phosphorylation in PC12 cells (Campana et al, 1996).…”

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity

“…However, this disease phenotype appears also, when functional sap-B is absent. 1 A neurotrophic function of the precursor prosaposin protein itself has been elucidated, 7,8 and a role in ganglioside binding and transport has been hypothesized. 9 Recently, the structure of a saposin-like protein, porcine NK-lysin, representative of a family of sequence related proteins sharing a possible common fold, has been determined, comprising five amphipathic α-helices, one at the centre, the others at the two opposite sides, folded into a single globular domain with three disulphide bonds.…”

Novel CDNF/MANF family of neurotrophic factors

“…Neurotrophic activities of SAPLIPs, e.g., prosaposin, saposin C, and a peptide derived from them have been reported in vitro (O'Brien et al, 1994(O'Brien et al, , 1995 and in vivo (Kotani et al, 1996;Liu et al, 2001), suggesting that the neurotrophic activity may reside in the N-terminal domain of CDNF and MANF (Parkash et al, 2009). It is also possible that the neurotrophic activities and cytoprotection against ER stress are mediated by the same structural motif.…”