Age- and sex-associated changes in prosaposin and its receptors in the lacrimal glands of rats.

Prosaposin, a saposin precursor, is a potent neurotrophic factor found in several tissues and various biological fluids. Saposin-deficient patients have different ophthalmic disorders, indicating a relationship between ocular health and prosaposin. However, there is little information about prosaposin on the ocular surface. Because ocular functions are diverse and depend on age and sex, we examined whether prosaposin and its receptors, G protein-coupled receptor 37 (GPR37) and GPR37L1, are expressed in the major ocular glands, the extra orbital lacrimal gland (ELG), and harderian gland (HG) of rats and whether sex and aging affect their expression. Immunohistochemical analyses revealed that prosaposin and its receptors were expressed in the ELGs and HGs of rats, although their expression varied based on the type of gland, age, and sex. Prosaposin, GPR37, and GPR37L1 were expressed in the basolateral membranes and cytoplasm of acinar cells of the ELGs, and their immunoreactivities were higher in female rats of menopausal age than age-matched male rats. However, such age- and sex-related differences in the immunoreactivities of prosaposin, GPR37, and GPR37L1 were not observed in the HGs. Triple immunofluorescence labelling revealed that prosaposin, GPR37, and GPR37L1 were co-localised in the acinar and ductal cells in the ELGs, although the degrees of colocalization varied according to the age and sex of the rats. Together, the present results showed that prosaposin and its receptors were expressed in the major ocular glands of rats, and their immunoreactivities to the ELGs differed considerably with age and sex.

Comparative structure-function characterization of the saposin-like domains from potato, barley, cardoon and Arabidopsis aspartic proteases.

The present study characterized the aspartic protease saposin-like domains of four plant species, Solanum tuberosum (potato), Hordeum vulgare L. (barley), Cynara cardunculus L. (cardoon; artichoke thistle) and Arabidopsis thaliana, in terms of bilayer disruption and fusion, and structure pH-dependence. Comparison of the recombinant saposin-like domains revealed that each induced leakage of bilayer vesicles composed of a simple phospholipid mixture with relative rates Arabidopsis>barley>cardoon>potato. When compared for leakage of bilayer composed of a vacuole-like phospholipid mixture, leakage was approximately five times higher for potato saposin-like domain compared to the others. In terms of fusogenic activity, distinctions between particle size profiles were noted among the four proteins, particularly for potato saposin-like domain. Bilayer fusion assays in reducing conditions resulted in altered fusion profiles except in the case of cardoon saposin-like domain which was virtually unchanged. Secondary structure profiles were similar across all four proteins under different pH conditions, although cardoon saposin-like domain appeared to have higher overall helix structure. Furthermore, increases in Trp emission upon protein-bilayer interactions suggested that protein structure rearrangements equilibrated with half-times ranging from 52 to 120s, with cardoon saposin-like domain significantly slower than the other three species. Overall, the present findings serve as a foundation for future studies seeking to delineate protein structural features and motifs in protein-bilayer interactions based upon variability in plant aspartic protease saposin-like domain structures.

Saposins utilize two strategies for lipid transfer and CD1 antigen presentation.

Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.

Chronological changes in prosaposin in the developing rat brain.

Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronal cell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized in the lysosomes of the neurons. We also examined the chronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRNA forms, especially the one without a nine-base insertion, increased significantly from embryonic day 15 to postnatal day 7, then decreased gradually until postnatal day 28. Abundant prosaposin expression in the perinatal stages indicates a potential role of prosaposin in the early development of the rat brain.

Peroxisome proliferator-activated receptor γ-regulated cathepsin D is required for lipid antigen presentation by dendritic cells.

It is well established that dendritic cells (DCs) take up, process, and present lipid Ags in complex with CD1d molecules to invariant NKT cells. The lipid-activated transcription factor, peroxisome proliferator-activated receptor γ (PPARγ), has previously been shown to regulate CD1d expression in human monocyte-derived DCs, providing a link between lipid metabolism and lipid Ag presentation. We report that PPARγ regulates the expression of a lysosomal protease, cathepsin D (CatD), in human monocyte-derived DCs. Inhibition of CatD specifically reduced the expansion of invariant NKT cells and furthermore resulted in decreased maturation of saposins, a group of lipid transfer proteins required for lysosomal lipid Ag processing and loading. These results reveal a novel mechanism of lipid Ag presentation and identify CatD as a key component of this machinery and firmly place PPARγ as the transcriptional regulator linking lipid metabolism and lipid Ag processing.

Biochemistry and neurobiology of prosaposin: a potential therapeutic neuro-effector.

Prosaposin, a 66 kDa glycoprotein, was identified initially as the precursor of the sphingolipid activator proteins, saposins A-D, which are required for the enzymatic hydrolysis of certain sphingolipids by lysosomal hydrolases. While mature saposins are distributed to lysosomes, prosaposin exists in secretory body fluids and plasma membranes. In addition to its role as the precursor, prosaposin shows a variety of neurotrophic and myelinotrophic activities through a receptor-mediated mechanism. In studies in vivo, prosaposin was demonstrated to exert a variety of neuro-efficacies capable of preventing neuro-degeneration following neuro-injury and promoting the amelioration of allodynia and hyperalgesia in pain models. Collective findings indicate that prosaposin is not a simple house-keeping precursor protein; instead, it is a protein essentially required for the development and maintenance of the central and peripheral nervous systems. Accumulating evidence over the last decade has attracted interests in exploring and developing new therapeutic approaches using prosaposin for human disorders associated with neuro-degeneration. In this review we detail the structure characteristics, cell biological feature, in vivo efficacy, and neuro-therapeutic potential of prosaposin, thereby providing future prospective in clinical application of this multifunctional protein.

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.

Molecular mechanism for neuro-protective effect of prosaposin against oxidative stress: its regulation of dimeric transcription factor formation.

Prosaposin triggers G-protein-coupled receptor (GPCR)-mediated protein kinase B (Akt)/extracellular signal-regulated kinase (ERK) phosphorylation cascades to exert its neurotrophic and myelinotrophic activity capable of preventing neural cell death and promoting neural proliferation and glial differentiation. In the present study, we investigated the down-stream neurotrophic signaling mechanism of prosaposin by which rat pheochromocytoma (PC-12) cells are protected from cell death induced by oxidative stress. When PC-12 cells were exposed to H2O2, the cells underwent abrupt shrinkage followed by apoptosis. Prosaposin treatment at as low as 1 nM protected PC-12 cells from cell death by the oxidative stress with the activation of an ERK phosphorylation cascade. Simultaneously, prosaposin blocked the oxidative stress induced-Akt phosphorylation that acts on the down-stream of caspase-3 activation. A MEK inhibitor, PD98059, or a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, abolished the survival effect of prosaposin on the oxidative stress-induced cell death. Furthermore, prosaposin blocked the oxidative stress-induced phosphorylations of c-Jun N-terminal kinase (JNK) and p38 stress-activated protein kinase. We further investigated the effect of prosaposin treatment on the phosphorylation of activating protein-1 (AP-1) complex components, c-Jun and activating transcription factor (ATF)-3. Western blot analysis demonstrated that prosaposin treatment at 100 ng/ml decreased the levels of c-Jun and ATF-3 induced by H2O2 stimulation. Our results suggest that prosaposin aids survival of PC-12 cells from oxidative stress not only by reducing the phosphorylation levels of JNK and p38, but also by regulating the c-Jun/AP-1 pathway.

Neurological deficits and glycosphingolipid accumulation in saposin B deficient mice.

Saposin B derives from the multi-functional precursor, prosaposin, and functions as an activity enhancer for several glycosphingolipid (GSL) hydrolases. Mutations in saposin B present in humans with phenotypes resembling metachromatic leukodystrophy. To gain insight into saposin B's physiological functions, a specific deficiency was created in mice by a knock-in mutation of an essential cysteine in exon 7 of the prosaposin locus. No saposin B protein was detected in the homozygotes (B-/-) mice, whereas prosaposin, and saposins A, C and D were at normal levels. B-/- mice exhibited slowly progressive neuromotor deterioration and minor head tremor by 15 months. Excess hydroxy and non-hydroxy fatty acid sulfatide levels were present in brain and kidney. Alcian blue positive (sulfatide) storage cells were found in the brain, spinal cord and kidney. Ultrastructural analyses showed lamellar inclusion material in the kidney, sciatic nerve, brain and spinal cord tissues. Lactosylceramide (LacCer) and globotriaosylceramide (TriCer) were increased in various tissues of B-/- mice supporting the in vivo role of saposin B in the degradation of these lipids. CD68 positive microglial cells and activated GFAP positive astrocytes showed a proinflammatory response in the brains of B-/- mice. These findings delineate the roles of saposin B for the in vivo degradation of several GSLs and its primary function in maintenance of CNS function. B-/- provide a useful model for understanding the contributions of this saposin to GSL metabolism and homeostasis.

Saposin B mobilizes lipids from cholesterol-poor and bis(monoacylglycero)phosphate-rich membranes at acidic pH. Unglycosylated patient variant saposin B lacks lipid-extraction capacity.

Sphingolipid activator proteins (SAPs), GM2 activator protein (GM2AP) and saposins (Saps) A-D are small, enzymatically inactive glycoproteins of the lysosome. Despite of their sequence homology, these lipid-binding and -transfer proteins show different specificities and varying modes of action. Water-soluble SAPs facilitate the degradation of membrane-bound glycosphingolipids with short oligosaccharide chains by exohydrolases at the membrane-water interface. There is strong evidence that degradation of endocytosed components of the cell membrane takes place at intraendosomal and intralysosomal membranes. The inner membranes of the lysosome differ from the limiting membrane of the organelle in some typical ways: the inner vesicular membranes lack a protecting glycocalix, and they are almost free of cholesterol, but rich in bis(monoacylglycero)phosphate (BMP), the anionic marker lipid of lysosomes. In this study, we prepared glycosylated Sap-B free of other Saps by taking advantage of the Pichia pastoris expression system. We used immobilized liposomes as a model for intralysosomal vesicular membranes to probe their interaction with recombinantly expressed Sap-B. We monitored this interaction using SPR spectroscopy and an independent method based on the release of radioactively labelled lipids from liposomal membranes. We show that, after initial binding, Sap-B disturbs the membrane structure and mobilizes the lipids from it. Lipid mobilization is dependent on an acidic pH and the presence of anionic lipids, whereas cholesterol is able to stabilize the liposomes. We also show for the first time that glycosylation of Sap-B is essential to achieve its full lipid-extraction activity. Removal of the carbohydrate moiety of Sap-B reduces its membrane-destabilizing quality. An unglycosylated Sap-B variant, Asn215His, which causes a fatal sphingolipid storage disease, lost the ability to extract membrane lipids at acidic pH in the presence of BMP.

Saposin B is the dominant saposin that facilitates lipid binding to human CD1d molecules.

CD1d molecules bind lipid antigens in the endocytic pathway, and access to the pathway is important for the development of CD1d-restricted natural killer T (NKT) cells. Saposins, derived from a common precursor, prosaposin, are small, heat-stable lysosomal glycoproteins required for lysosomal degradation of sphingolipids. Expression of prosaposin is required for efficient lipid binding and recognition of human CD1d molecules by NKT cells. Despite high sequence homology among the four saposins, they have different specificities for lipid substrates and different mechanisms of action. To determine the saposins involved in promoting lipid binding to CD1d, we expressed prosaposin deletion mutants lacking individual saposins in prosaposin-negative, CD1d-positive cells. No individual saposin proved to be absolutely essential, but the absence of saposin B resulted in the lowest recognition of alpha-galactosylceramide by NKT cells. When recombinant exogenous saposins were added to the prosaposin-negative cells, saposin B was the most efficient in restoring CD1d recognition. Saposin B was also the most efficient in mediating alpha-galactosylceramide binding to recombinant plate-bound CD1d and facilitating NKT cell activation. Saposin B could also mediate lipid binding to soluble CD1d molecules in a T cell-independent assay. The optimal pH for saposin B-mediated lipid binding to CD1d, pH 6, is higher than that of lysosomes, suggesting that saposin B may facilitate lipid binding to CD1d molecules throughout the endocytic pathway.

Neurolysosomal pathology in human prosaposin deficiency suggests essential neurotrophic function of prosaposin.

A neuropathologic study of three cases of prosaposin (pSap) deficiency (ages at death 27, 89 and 119 days), carried out in the standard autopsy tissues, revealed a neurolysosomal pathology different from that in the non-neuronal cells. Non-neuronal storage is represented by massive lysosomal accumulation of glycosphingolipids (glucosyl-, galactosyl-, lactosyl-, globotriaosylceramides, sulphatide, and ceramide). The lysosomes in the central and peripheral neurons were distended by pleomorphic non-lipid aggregates lacking specific staining and autofluorescence. Lipid storage was borderline in case 1, and at a low level in the other cases. Neurolysosomal storage was associated with massive ubiquitination, which was absent in the non-neuronal cells and which did not display any immunohistochemical aggresomal properties. Confocal microscopy and cross-correlation function analyses revealed a positive correlation between the ubiquitin signal and the late endosomal/lysosomal markers. We suppose that the neuropathology most probably reflects excessive influx of non-lipid material (either in bulk or as individual molecules) into the neurolysosomes. The cortical neurons appeared to be uniquely vulnerable to pSap deficiency. Whereas in case 1 they populated the cortex, in cases 2 and 3 they had been replaced by dense populations of both phagocytic microglia and astrocytes. We suggest that this massive neuronal loss reflects a cortical neuronal survival crisis precipitated by the lack of pSap. The results of our study may extend the knowledge of the neurotrophic function of pSap, which should be considered essential for the survival and maintenance of human cortical neurons.

Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin.

After a yeast two-hybrid screen identified prosaposin as a potential interacting protein with the nicotinic acetylcholine receptor (nAChR) subunit alpha10, studies were performed to characterize prosaposin in the normal rodent inner ear. Prosaposin demonstrates diffuse organ of Corti expression at birth, with gradual localization to the inner hair cells (IHCs) and its supporting cells, inner pillar cells, and synaptic region of the outer hair cells (OHCs) and Deiters' cells (DCs) by postnatal day 21 (P21). Microdissected OHC and DC quantitative reverse transcriptase-PCR and immunohistology localizes prosaposin mRNA to DCs and OHCs, and protein predominantly to the apex of the DCs. Subsequent studies in a prosaposin knock-out (KO) (-/-) mouse showed intact but slightly reduced hearing through P19, but deafness by P25 and reduced distortion product otoacoustic emissions from P15 onward. Beginning at P12, the prosaposin KO mice showed histologic organ of Corti changes including cellular hypertrophy in the region of the IHC and greater epithelial ridge, a loss of OHCs from cochlear apex, and vacuolization of OHCs. Immunofluorescence revealed exuberant overgrowth of auditory afferent neurites in the region of the IHCs and proliferation of auditory efferent neurites in the region of the tunnel of Corti. IHC recordings from these KO mice showed normal I-V curves and responses to applied acetylcholine. Together, these results suggest that prosaposin helps maintain normal innervation patterns to the organ of Corti. Furthermore, prosaposin's overlapping developmental expression pattern and binding capacity toward the nAChR alpha10 suggest that alpha10 may also play a role in this function.

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.

Saposin C promotes survival and prevents apoptosis via PI3K/Akt-dependent pathway in prostate cancer cells.

BACKGROUND: In addition to androgens, growth factors are also implicated in the development and neoplastic growth of the prostate gland. Prosaposin is a potent neurotrophic molecule. Homozygous inactivation of prosaposin in mice has led to the development of a number of abnormalities in the male reproductive system, including atrophy of the prostate gland and inactivation of mitogen-activated protein kinase (MAPK) and Akt in prostate epithelial cells. We have recently reported that prosaposin is expressed at a higher level by androgen-independent (AI) prostate cancer cells as compared to androgen-sensitive prostate cancer cells or normal prostate epithelial and stromal cells. In addition, we have demonstrated that a synthetic peptide (prosaptide TX14A), derived from the trophic sequence of the saposin C domain of prosaposin, stimulated cell proliferation, migration and invasion and activated the MAPK signaling pathway in prostate cancer cells. The biological significances of saposin C and prosaposin in prostate cancer are not known. RESULTS: Here, we report that saposin C, in a cell type-specific and dose-dependent manner, acts as a survival factor, activates the Akt-signaling pathway, down-modulates caspase-3, -7, and -9 expression and/or activity, and decreases the cleaved nuclear substrate of caspase-3 in prostate cancer cells under serum-starvation stress. In addition, prosaptide TX14A, saposin C, or prosaposin decreased the growth-inhibitory effect, caspase-3/7 activity, and apoptotic cell death induced by etoposide. We also discovered that saposin C activates the p42/44 MAP kinase pathway in a pertussis toxin-sensitive and phosphatidylinositol 3-kinase (PI3K) /Akt-dependent manner in prostate cancer cells. Our data also show that the anti-apoptotic activity of saposin C is at least partially mediated via PI3K/Akt signaling pathway. CONCLUSION: We postulate that as a mitogenic, survival, and anti-apoptotic factor for prostate cancer cells, saposin C or prosaposin may contribute to prostate carcinogenesis at its early androgen-dependent or metastatic AI state.

Regulation of aging and innate immunity in C. elegans.

The free-living soil nematode Caenorhabditis elegans is a versatile model for the study of the genetic regulation of aging and of host-pathogen interactions. Many genes affecting multiple processes, such as neuroendocrine signalling, nutritional sensing and mitochondrial functions, have been shown to play important roles in determining the lifespan of C. elegans. The DAF-2-mediated insulin signalling pathway is the major pathway that regulates aging in this nematode and this role appears universal; neuroendrocrine signalling also affects aging in Drosophila and mice. Recent studies have shown that the innate immune function in C. elegans is modulated by signalling from the TGF-beta-like, the p38 MAPK and the DAF-2 insulin pathways. The requirement for the DAF-2 pathway in modulating aging and immunity suggests that these processes may be linked at the molecular level. It is well known that as humans age, immunosenescence occurs in which there is a general degradation of immune efficiency. However, the molecular mechanisms involved in this process remain unclear. In this review, we discuss the molecular mechanisms that modulate aging and immune response and attempt to suggest molecular links between these two processes.

Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation.

Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.

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.