A Stretch of 17 Amino Acids in the Prosaposin C Terminus Is Critical for Its Binding to Sortilin and Targeting to Lysosomes

Prosaposin, the precursor of four lysosomal cofactors required for the hydrolysis of sphingolipids, is transported to the lysosomes via the alternative receptor, sortilin. In this study, we identified a specific domain of 17 amino acids within the C terminus of prosaposin involved in binding to this sorting receptor. We generated six prosaposin deletion constructs and examined the effect of truncation by coimmunoprecipitation and confocal microscopy. The experiments revealed that the first half of the prosaposin C terminus (aa 524–540), containing a saposin-like motif, was required and necessary to bind sortilin and to transport it to the lysosomes. Based on this result, we introduced twelve site-directed point mutations within the first half of the C terminus. Although the interaction of prosaposin with sortilin was pH dependent, the mutation of hydrophilic amino acids that usually modulate pH-dependent protein interactions did not affect the binding of prosaposin to sortilin. Conversely, a tryptophan (W530) and two cysteines (C528 and C536) were essential for its interaction with sortilin and for its transport to the lysosomes. In conclusion, our investigation demonstrates that a saposin-like motif within the first half of the prosaposin C terminus contains the sortilin recognition site. (J Histochem Cytochem 58:287–300, 2010)     

The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM₂AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.     

Identification of a novel sequence involved in lysosomal sorting of the sphingolipid activator protein prosaposin

Prosaposin is synthesized as a 53-kDa protein, post-translationally modified to a 65-kDa form and further glycosylated to a 70-kDa secretory product. The 65-kDa protein is associated to Golgi membranes and is targeted to lysosomes, where four smaller nonenzymatic saposins implicated in the hydrolysis of sphingolipids are generated by its partial proteolysis. The targeting of the 65-kDa protein to lysosomes is not mediated by the mannose 6-phosphate receptor. The Golgi apparatus appears to accomplish the molecular sorting of the 65-kDa prosaposin by decoding a signal from its amino acid backbone. This investigation deals with the characterization of the sequence involved in this process by deleting the saposin functional domains A, B, C, and D and the highly conserved N and C termini of prosaposin. The truncated cDNAs were subcloned into expression vectors and transfected to COS-7 cells. The destination of the mutated proteins was assessed by immunocytochemistry. Deletion of the C terminus did not interfere with the secretion of prosaposin but abolished its transport to lysosomes. Deletion of saposins and the N-terminal domain did not affect the lysosomal or secretory routing of prosaposin. A chimeric construct of albumin and the C terminus of prosaposin was not directed to lysosomes. However, albumin connected to the C terminus and one or more functional domains of prosaposin reached lysosomes, indicating that the C terminus and at least one saposin domain are required for this process. In summary, we are reporting a novel sequence involved in the targeting of prosaposin to lysosomes.     

Study of the mouse sortilin gene: Effects of its transient silencing by RNA interference in TM4 Sertoli cells

Using databases of the mouse genome in combination with a sequence deduced from a mouse sortilin cDNA originated in our laboratory, we found the sortilin gene to map to a region of chromosome 3. The mouse sortilin gene contains 19 short exons separated by introns of various sizes. The study elucidated the exon-intron boundaries. Some introns extend over more than 24 kb. In the cytoplasmic domain of the translation product, we found a dileucine motif and three other motifs known to constitute the active sorting signal of the mannose 6-phosphate receptor (M6P-R). We also tested the hypothesis that sortilin is involved in the sorting of prosaposin (SGP-1) to the lysosomes. Prosaposin was initially identified in Sertoli cells, found in large amounts in the lysosomal compartment and implicated in the degradation of residual bodies released by the spermatids during spermiation. Interestingly, the targeting of prosaposin to the lysosomes is independent of the M6P-R. This investigation demonstrated that sortilin was required for the trafficking of prosaposin to the lysosomes in TM4 cells. The requirement of sortilin was shown using a siRNA probe to block the translation of sortilin mRNA. Sortilin-deficient cells were not able to route prosaposin to the lysosomal compartment but continue to transport cathepsin B, since this hydrolase uses the M6P-R to be routed to the lysosomes. These results indicate that sortilin appears to be involved in the lysosomal trafficking of prosaposin.     

The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins

Prosaposin (SGP-1) and GM2 activator protein (GM2AP) are soluble sphingolipid activator proteins (SAPs) that are targeted to the lysosomal compartment of Sertoli cells to aid hydrolases in the breakdown of glycosphingolipids. To reach the lysosome, most soluble proteins must interact with the mannose 6-phosphate receptor (MPR). To be sorted from the Golgi, the MPR must bind to the Golgi associated, gamma-adaptin homologous, ARF binding proteins (GGAs), a group of monomeric adaptor proteins responsible for the recruitment of clathrin. It is well established, however, that the lysosomes of I-cell disease (ICD) patients have near normal levels of several lysosomal proteins, including prosaposin and GM2AP. ICD results from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases. Thus, prosaposin and GM2AP can traffic to lysosomes in a MPR independent manner. Previous work has demonstrated that an interaction with sphingomyelin in the Golgi membrane is necessary for the targeting of prosaposin by an unknown receptor. Using a TM4 Sertoli cell line, we tested the hypothesis that prosaposin and GM2AP are targeted to the lysosomal compartment via the sortilin receptor, which has been recently shown to have a GGA binding motif. Interestingly, dominant-negative GGAs, unable to bind clathrin to shuttle from the Golgi, prevented the trafficking of prosaposin and GM2AP to lysosomes. A dominant negative construct of sortilin lacking the GGA binding domain retained prosaposin and GM2AP in the Golgi. In conclusion, our results showed that the trafficking of prosaposin and GM2AP to the lysosome is dependent on sortilin.     

Sortilin and prosaposin localize to detergent-resistant membrane microdomains

Most soluble lysosomal hydrolases are sorted in the trans-Golgi network (TGN) and delivered to the lysosomes by the mannose 6-phosphate receptor (M6PR). However, the non-enzymic sphingolipid activator protein (SAP), prosaposin, as well as certain soluble lysosomal hydrolases, is sorted and trafficked to the lysosomes by sortilin. Based on previous results demonstrating that prosaposin requires sphingomyelin to be targeted to the lysosomes, we hypothesized that sortilin and its ligands are found in detergent-resistant membranes (DRMs). To test this hypothesis we have analyzed DRM fractions and demonstrated the presence of sortilin and its ligand, prosaposin. Our results showed that both the M6PR and its cargo, cathepsin B, were also present in DRMs. Cathepsin H has previously been demonstrated to interact with sortilin, while cathepsin D interacts with both sortilin and the M6PR. Both of these soluble lysosomal proteins were also found in DRM fractions. Using sortilin shRNA we have showed that prosaposin is localized to DRM fractions only in the presence of sortilin. These observations suggest that in addition to interacting with the same adaptor proteins, such as GGAs, AP-1 and retromer, both sortilin and the M6PR localize to similar membrane platforms, and that prosaposin must interact with sortilin to be recruited to DRMs.     

The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin

Most soluble lysosomal proteins bind the mannose 6-phosphate receptor (M6P-R) to be sorted to the lysosomes. However, the lysosomes of I-cell disease (ICD) patients, a condition resulting from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases, have near normal levels of several lysosomal proteins, including the sphingolipid activator proteins (SAPs), GM2AP and prosaposin. We tested the hypothesis that SAPs are targeted to the lysosomal compartment via the sortilin receptor. To test this hypothesis, a dominant-negative construct of sortilin and a sortilin small interfering RNA (siRNA) were introduced into COS-7 cells. Our results showed that both the truncated sortilin and the sortilin siRNA block the traffic of GM2AP and prosaposin to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated that GM2AP and prosaposin are interactive partners of sortilin. Furthermore, a dominant-negative mutant GGA prevented the trafficking of prosaposin and GM2AP to lysosomes. In conclusion, our results show that the trafficking of SAPs is dependent on sortilin, demonstrating a novel lysosomal trafficking.     

Neurotrophic signalling pathway triggered by prosaposin in PC12 cells occurs through lipid rafts

Prosaposin is a neurotrophic factor that has been demonstrated to mediate trophic signalling events in different cell types; it distributes to surface membranes of neural cells and also exists as a secreted protein in different body fluids. Prosaposin was demonstrated to form tightly bound complexes with a variety of gangliosides, and a functional role has been suggested for ganglioside-prosaposin complexes. In this work, we provide evidence that exogenous prosaposin triggers a signal cascade after binding to its target molecules on lipid rafts of pheochromocytoma PC12 cell plasma membranes, as revealed by scanning confocal microscopy and linear sucrose gradient analysis. In these cells, prosaposin is able to induce extracellular signal-regulated kinase phosphorylation, sphingosine kinase activation, and consequent cell death prevention, acting through lipid rafts. These findings point to the role of lipid rafts in the prosaposin-triggered signalling pathway, thus supporting a role for this factor as a new component of the multimolecular signalling complex involved in the neurotrophic response.     

Saposins: structure, function, distribution, and molecular genetics.

Saposins A, B, C, and D are small heat-stable glycoproteins derived from a common precursor protein, prosaposin. These mature saposins, as well as prosaposin, activate several lysosomal hydrolases involved in the metabolism of various sphingolipids. All four saposins are structurally similar to one another including placement of six cysteines, a glycosylation site, and conserved prolines in identical positions. In spite of the structural similarities, the specificity and mode of activation of sphingolipid hydrolases differs among individual saposins. Saposins appear to be lysosomal proteins, exerting their action upon lysosomal hydrolases. Prosaposin is a 70 kDa glycoprotein containing four domains, one for each saposin, placed in tandem. Prosaposin is proteolytically processed to saposins A, B, C and D, apparently within lysosomes. However, prosaposin also exists as an integral membrane protein not destined for lysosomal entry and exists uncleaved in many biological fluids such as seminal plasma, human milk, and cerebrospinal fluid, where it appears to have a different function. The physiological significance of saposins is underlined by their accumulation in tissues of lysosomal storage disease patients and the occurrence of sphingolipidosis due to mutations in the prosaposin gene. This review presents an overview of the occurrence, structure and function of these saposin proteins.     

Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase.

We report that prosaposin treatment induced extracellular signal-regulated kinases (ERKs) and sphingosine kinase activity, increased DNA synthesis, and prevented cell apoptosis. Prosaposin treatment induced pheochromocytoma cells (PC12) to enter the S phase of the cell cycle; this effect was inhibited by the MEK inhibitor PD98059, indicating that prosaposin-induced ERK phosphorylation is required for stimulation of DNA synthesis. The prosaposin effect was also inhibited by pertussis toxin, indicating that the prosaposin receptor is a G-protein-coupled receptor. Prosaposin rescued PC12 cells from apoptosis induced by staurosporine or ceramide. Sphingosine kinase activity was increased by prosaposin treatment. We propose that this effect is a mechanism underlying the proliferative and anti-apoptotic functions of prosaposin. Prosaposin appears to be a key regulatory factor in the ceramide-S-1-P rheostat, which regulates cell fate.     

Secretion of sphingolipid hydrolase activator precursor, prosaposin

Sphingolipid hydrolases are activated by activator proteins or saposins. The precursor protein has been expected from the studies on the cDNA for saposins. Here we demonstrate that prosaposin occurs in various kinds of human secretory fluids such as cerebrospinal fluid, semen, milk, pancreatic juice, and bile. However, mature type saposins were not detected in these fluids. In human milk the amount of prosaposin changed during the lactating period; it became high in concentration within a few days after delivery, decreased during the transitional milk lactating stage, and then increased again toward the mature milk lactating stage. Prosaposin was released from human platelets in response to stimulation by thrombin, but mature saposins were not. From the time course of the release of prosaposin induced by thrombin and from the fact that weak platelet agonists, ADP, epinephrine, and collagen, did not cause the release of prosaposin, prosaposin secretion from platelets seemed to be from lysosome like granules. We postulate that some prosaposin works as a precursor for saposins in the lysosomes and the other serves as an extracellular protein with other specific roles.     

Temporal Changes in Prosaposin Expression in the Rat Dentate Gyrus after Birth

Neurogenesis in the hippocampal dentate gyrus occurs constitutively throughout postnatal life. Adult neurogenesis includes a multistep process that ends with the formation of a postmitotic and functionally integrated new neuron. During adult neurogenesis, various markers are expressed, including GFAP, nestin, Pax6, polysialic acid-neural cell adhesion molecule (PSA-NCAM), neuronal nuclei (NeuN), doublecortin, TUC-4, Tuj-1, and calretinin. Prosaposin is the precursor of saposins A–D; it is found in various organs and can be excreted. Strong prosaposin expression has been demonstrated in the developing brain including the hippocampus, and its neurotrophic activity has been proposed. This study investigated changes in prosaposin in the dentate gyrus of young and adult rats using double immunohistochemistry with antibodies to prosaposin, PSA-NCAM, and NeuN. Prosaposin immunoreactivity was intense in the dentate gyrus at postnatal day 3 (P3) and P7, but decreased gradually after P14. In the dentate gyrus at P28, immature PSA-NCAM-positive neurons localized exclusively in the subgranular zone were prosaposin-negative, whereas mature Neu-N-positive neurons were positive for prosaposin. Furthermore, these prosaposin-negative immature neurons were saposin B-positive, suggesting that the neurons take up and degrade prosaposin. In situ hybridization assays showed that prosaposin in the adult dentate gyrus is dominantly the Pro+9 type, a secreted type of prosaposin. These results imply that prosaposin secreted from mature neurons stimulates proliferation and maturation of immature neurons in the dentate gyrus.     

Role of sulfated glycoprotein-1 (SGP-1) in the disposal of residual bodies by sertoli cells of the rat

Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10–15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley-Liss, Inc.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Regulation of cell proliferation and apoptosis through fibrocystin-prosaposin interaction

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). It is widely accepted that cystogenesis is owing to aberrant cell proliferation and apoptosis, increased fluid secretion, and extracellular matrix abnormality. Fibrocystin/polyductin (FPC), the encoded protein product by PKHD1, is a single transmembrane protein and believed to be a novel receptor-like molecule. FPC has been located mainly on the plasma membrane and cilium/basal body. However, its biological functions remain poorly understood. To investigate the roles of FPC in the pathogenesis of ARPKD, we searched for FPC-interacting proteins by yeast two-hybrid assay, and found a novel partner, prosaposin. Prosaposin is a glycoprotein with multiple functions. With GST pull-down assay and co-immunoprecipitation, we confirmed the interaction between FPC and prosaposin. In order to study the effects of FPC-prosaposin interaction on cell proliferation and apoptosis, we have made stable cell lines in which FPC was overexpressed or knocked down alone or in combination with prosaposin overexpression. By MTT assay, we found that FPC knockdown and prosaposin overexpression increased cell proliferation, respectively, while overexpression of FPC C-tail did the opposite. With apoptosis assay, we found that overexpression of FPC C-tail promoted cell apoptosis. However, overexpression of prosaposin significantly enhanced cell survival in FPC knockdown cells. All these findings indicated that FPC and prosaposin may play significant roles in regulation of cell proliferation and apoptosis. Taken together, we have disclosed a novel signaling pathway of FPC, which may be important for the pathogenesis of ARPKD.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

Role of sphingolipids in the transport of prosaposin to the lysosomes.

Prosaposin is the precursor of four lysosomal saposins that promote the degradation of glycosphingolipids (GSLs) by acidic hydrolases. GSLs contain a hydrophobic ceramide moiety, which acts as a membrane anchor, and a hydrophilic oligosaccharide chain that faces the lumen of the Golgi apparatus and extracellular spaces. By using fumonisin B1, PDMP and D609, we tested the hypothesis that sphingolipids mediate the transport of prosaposin to the lysosomes. Fumonisin B1 interferes with the synthesis of ceramide, PDMP blocks the formation of glucosylceramide and D609 blocks the formation of sphingomyelin. Fumonisin B1 produced a 59;-85% decrease in the density of gold particles in the lysosomes of CHO and NRK cells immunolabeled with anti-prosaposin antibody, and a 55% reduction in the lysosomes of CHO cells stably transfected with an expression vector containing a human prosaposin cDNA. To examine whether the mannose 6-phosphate receptor pathway was affected by this treatment, NRK and CHO cells treated or not with fumonisin B1 were labeled with anti-cathepsin A antibody. The results showed no significant differences in labeling of the lysosomes, suggesting that the effect of fumonisin B1 was specific. When fumonisin B1 and D609 were added to the media of transfected CHO cells, a decrease in immunofluorescence with anti-prosaposin antibody was observed by confocal microscopy. PDMP did not cause any reduction in immunoreactivity, indicating that sphingolmyelin appears to be involved in this process. In conclusion, our data support the hypothesis that sphingolipids, possibly sphingomyelin, are involved in the transport of prosaposin to the lysosomes.     

The lysosomal transport of prosaposin requires the conditional interaction of its highly conserved d domain with sphingomyelin

Lysosomal prosaposin (65 kDa) is a nonenzymic protein that is transported to the lysosomes in a mannose 6-phosphate-independent manner. Selective deletion of the functional domains of prosaposin indicates that the D domain and the carboxyl-terminal region are necessary for its transport to the lysosomes. Inhibitors of sphingolipid biosynthesis, such as fumonisin B(1) (FB(1)) and tricyclodecan-9-yl xanthate potassium salt (D609), also interfere with the trafficking of prosaposin to lysosomes. In this study, we examine sphingomyelin as a direct candidate for the trafficking of prosaposin. Chinese hamster ovary and COS-7 cells overexpressing prosaposin or an albumin/prosaposin construct were incubated with these inhibitors, treated with sphingolipids, and then immunostained. Sphingomyelin restored the immunostaining in lysosomes in both FB(1)- and D609-treated cells and ceramide reestablished the immunostaining in FB(1)-treated cells only. D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits glycosphingolipids, had no effect on the immunostaining pattern. To determine whether sphingomyelin has the same effect on the transport of endogenous prosaposin, testicular explants were treated with FB(1) and D609. Sphingomyelin restored prosaposin immunogold labeling in the lysosomes of FB(1)- and D609-treated Sertoli cells, whereas ceramide restored the label in FB(1) treatment only. Albumin linked to the D and COOH-terminal domains of prosaposin was used as a dominant negative competitor. The construct blocked the targeting of prosaposin and induced accumulation of membrane in the lysosomes, demonstrating that the construct uses the same transport pathway as endogenous prosaposin. In conclusion, our results showed that sphingomyelin, the D domain, and its adjacent COOH-terminal region play a crucial role in the transport of prosaposin to lysosomes. Although the precise nature of this lipid-protein interaction is not well established, it is proposed that sphingomyelin microdomains (lipid rafts) are part of a mechanism ensuring correct intercellular trafficking of prosaposin.     

The exon 8-containing prosaposin gene splice variant is dispensable for mouse development, lysosomal function, and secretion

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids.