Saposins are small proteins implicated in trafficking and loading of lipids onto Cluster of Differentiation 1 (CD1) receptor proteins that in turn present lipid antigens to T cells and a variety of T-cell receptors, thus playing a crucial role in innate and adaptive immune responses in humans. Despite their low sequence identity, the four types of human saposins share a similar folding pattern consisting of four helices linked by three conserved disulfide bridges. However, their lipid-binding abilities as well as their activities in extracting, transporting and loading onto CD1 molecules a variety of sphingo- and phospholipids in biological membranes display two striking characteristics: a strong pH-dependence and a structural change between a compact, closed conformation and an open conformation. In this work, we present a comparative computational study of structural, electrostatic, and dynamic features of human saposins based upon their available experimental structures. By means of structural alignments, surface analyses, calculation of pH-dependent protonation states, Poisson-Boltzmann electrostatic potentials, and molecular dynamics simulations at three pH values representative of biological media where saposins fulfill their function, our results shed light into their intrinsic features. The similarities and differences in this class of proteins depend on tiny variations of local structural details that allow saposins to be key players in triggering responses in the human immune system.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of the childhood caused by mutations in the gene encoding palmitoyl protein thioesterase 1 (PPT1). PPT1 localizes to late endosomes/lysosomes of non-neuronal cells and in neurons also to presynaptic areas. PPT1-deficiency causes massive death of cortical neurons and most tissues show an accumulation of saposins A and D. We have here studied endocytic pathways, saposin localization and processing in PPT1-deficient fibroblasts to elucidate the cellular defects resulting in accumulation of specific saposins. We show that PPT1-deficiency causes a defect in fluid-phase and receptor-mediated endocytosis, whereas marker uptake and recycling endocytosis remain intact. Furthermore, we show that saposins A and D are more abundant and relocalized in PPT-deficient fibroblasts and mouse primary neurons. Metabolic labeling and immunoprecipitation analyses revealed hypersecretion and abnormal processing of prosaposin, implying that the accumulation of saposins may result from endocytic defects. We show for the first time a connection between saposin storage and a defect in the endocytic pathway of INCL cells. These data provide new insights into the metabolism of PPT1-deficient cells and offer a basis for further studies on cellular processes causing neuronal death in INCL and other neurodegenerative diseases.

Prosaposin is a precursor of saposins A, B, C, and D. Saposins are indispensable for lysosomal hydrolysis of sphingolipids. The notion that prosaposin itself is likely involved in brain development led us to generate an anti-mouse prosaposin-specific antibody that do not cross-react with any of the processed saposins. We have used it to study expression of prosaposin in the brain of wild-type (WT) and saposin D knockout mice (Sap-D(-/-)). Immunoblot studies indicated that prosaposin, already abundant in the brain of WT, was dramatically increased in Sap-D(-/-). By immunohistochemistry, the brain of WT was rich in prosaposin in hippocampal CA3 pyramidal neurons, tufted cells and mitral cells in olfactory bulb, and cerebellar Purkinje cells. In Sap-D(-/-), immunoreactivity of prosaposin was increased in these neurons, most notably in the CA3 pyramidal neurons which contained prosaposin immuno-positive inclusion bodies in the endoplasmic reticulum. Further characterization of these prosaposin-rich neurons may provide new insights into the physiological functions of prosaposin in the nervous system.

Neurotrophic factor prosaposin (PS) is a precursor for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. Both saposins and PS are widely contained in various tissues. The brain, skeletal muscle, and heart cells predominantly contain unprocessed PS rather than saposins. PS and PS-derived peptides stimulate neuritogenesis and increase choline acetyltransferase activity in neuroblastoma cells and prevent programmed cell death in neurons. We previously detected increases in PS immunoactivity and its mRNA in the rat facial nucleus following facial nerve transection. PS mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. In the present study, we examined the changes in immunoreactivity of the PS receptors GPR37 and GPR37L1 in the rat facial nucleus following facial nerve transection. Following facial nerve transection, many small Iba1- and glial fibrillary acidic protein (GFAP)-positive cells with strong GPR37L1 immunoreactivity, including microglia and astrocytes, were observed predominately on the operated side. These results indicate that GPR37 mainly works in neurons, whereas GPR37L1 is predominant in microglia or astrocytes, and suggest that increased PS in damaged neurons stimulates microglia or astrocytes via PS receptor GPR37L1 to produce neurotrophic factors for neuronal recovery.

Saposins are lipid transfer proteins required for the degradation of sphingolipids in the lysosome. These small proteins bind lipids by transitioning from a closed, monomeric state to an open conformation exposing a hydrophobic surface that binds and shields hydrophobic lipid tails from the aqueous environment. Saposins form a range of multimeric assemblies to encompass these bound lipids and present them to hydrolases in the lysosome. This lipid-binding property of human saposin A has been exploited to form lipoprotein nanodiscs suitable for structural studies of membrane proteins. Here we present the crystal structure of a unique tetrameric assembly of murine saposin A produced serendipitously, following modifications of published protocols for making lipoprotein nanodiscs. The structure of this new saposin oligomer highlights the diversity of tertiary arrangement that can be adopted by these important lipid transfer proteins.

Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs.

Factors responsible for invasive and metastatic progression of prostate cancer (PCa) remain largely unknown. Previously, we reported cloning of prosaposin (PSAP) and its genomic amplification and/or overexpression in several androgen-independent metastatic PCa cell lines and lymph node metastases. PSAP is the lysosomal precursor of saposins, which serve as activators for lysosomal hydrolases involved in the degradation of ceramide (Cer) and other sphingolipids.

Glycosphingolipids are involved in intercellular signaling, adhe-sion, proliferation, and differentiation. Saposins A, B, C, and D are cofactors required for glycosphingolipid hydrolysis. Saposins A-D are present in series in a common precursor protein, prosaposin. Thus, glycosphingolipids amounts depend on prosaposin cellular levels. We previously reported that prosaposin and saposin B bind coenzyme Q10 in human cells. Coenzyme Q10 is an essential lipid of the mitochondrial electron transport system, and its reduced form is an important antioxidant. Coenzyme Q10 level decrease in aging and in various progressive diseases. Therefore, it is interesting to understand the cellular response to long-term coenzyme Q10 deficiency. We established a long-term coenzyme Q10 deficient cell model by using the coenzyme Q10 biosynthesis inhibitor, 4-nitrobenzoate. The levels of coenzyme Q10 were reduced by 4-nitrobenzoate in HepG2 cells. Administration of 4-nitrobenzoate also decreased prosaposin protein and mRNA levels. The cellular levels of coenzyme Q10 and prosaposin were recovered by treatment with 4-hydroxybenzoquinone, a substrate for coenzyme Q10 synthesis that counteracts the effect of 4-nitrobenzoate. Furthermore, the ganglioside levels were altered in 4-nitrobenzoate treated cells. These results imply that long-term coenzyme Q10 deficiency reduces cellular prosaposin levels and disturbs glycosphingolipid metabolism.

Because excessive glutamate release is believed to play a pivotal role in numerous neuropathological disorders, such as ischemia or seizure, we aimed to investigate whether intrinsic prosaposin (PS), a neuroprotective factor when supplied exogenously in vivo or in vitro, is up-regulated after the excitotoxicity induced by kainic acid (KA), a glutamate analog. In the present study, PS immunoreactivity and its mRNA expression in the hippocampal and cortical neurons showed significant increases on day 3 after KA injection, and high PS levels were maintained even after 3 weeks. The increase in PS, but not saposins, detected by immunoblot analysis suggests that the increase in PS-like immunoreactivity after KA injection was not due to an increase in saposins as lysosomal enzymes after neuronal damage, but rather to an increase in PS as a neurotrophic factor to improve neuronal survival. Furthermore, several neurons with slender nuclei inside/outside of the pyramidal layer showed more intense PS mRNA expression than other pyramidal neurons. Based on the results from double immunostaining using anti-PS and anti-GABA antibodies, these neurons were shown to be GABAergic interneurons in the extra- and intra-pyramidal layers. In the cerebral cortex, several large neurons in the V layer showed very intense PS mRNA expression 3 days after KA injection. The choroid plexus showed intense PS mRNA expression even in the normal rat, and the intensity increased significantly after KA injection. The present study indicates that inhibitory interneurons as well as stimulated hippocampal pyramidal and cortical neurons synthesize PS for neuronal survival, and the choroid plexus is highly activated to synthesize PS, which may prevent neurons from excitotoxic neuronal damage. To the best of our knowledge, this is the first study that demonstrates axonal transport and increased production of neurotrophic factor PS after KA injection.

Saposins A, B, C and D are derived from a common precursor, prosaposin (psap). The few patients with saposin C deficiency develop a Gaucher disease-like central nervous system (CNS) phenotype attributed to diminished glucosylceramide (GC) cleavage activity by acid beta-glucosidase (GCase). The in vivo effects of saposin C were examined by creating mice with selective absence of saposin C (C-/-) using a knock-in point mutation (cysteine-to-proline) in exon 11 of the psap gene. In C-/- mice, prosaposin and saposins A, B and D proteins were present at near wild-type levels, but the saposin C protein was absent. By 1 year, the C-/- mice exhibited weakness of the hind limbs and progressive ataxia. Decreased neuromotor activity and impaired hippocampal long-term potentiation were evident. Foamy storage cells were observed in dorsal root ganglion and there was progressive loss of cerebellar Purkinje cells and atrophy of cerebellar granule cells. Ultrastructural analyses revealed inclusions in axonal processes in the spinal cord, sciatic nerve and brain, but no excess of multivesicular bodies. Activated microglial cells and astrocytes were present in thalamus, brain stem, cerebellum and spinal cord, indicating regional pro-inflammatory responses. No storage cells were found in visceral organs of these mice. The absence of saposin C led to moderate increases in GC and lactosylceramide (LacCer) and their deacylated analogues. These results support the view that saposin C has multiple roles in glycosphingolipid (GSL) catabolism as well as a prominent function in CNS and axonal integrity independent of its role as an optimizer/stabilizer of GCase.

Saposins (SAPs) are small glycoproteins required for activation of sphingolipid hydrolysis by lysosomal enzymes. Four SAPs, SAP-A, -B, -C, and -D, are proteolytically cleaved from a single gene product termed prosaposin. The mature coding sequence of human SAP-A tagged with 6-histidine was expressed in Pichia pastoris and the recombinant protein was purified from the culture supernatant by simple purification steps with an immobilized metal ion affinity column, a Concanavalin A column, and reversed-phase HPLC. Secreted SAP-A contained both glycosylated and nonglycosylated forms. Both forms of SAP-A activated galactocerebroside and 4-methylumbelliferyl beta-d-glucoside hydrolysis by galactocerebrosidase and glucocerebrosidase. SAP-A expressed in P. pastoris should be useful for further structural and functional analysis of this protein.

Prosaposin (PSAP), a highly conserved glycoprotein, is a precursor of saposins A-D. Accumulating evidence suggests that PSAP is a neurotrophic factor that induces differentiation and prevents death in a variety of neuronal cells through the active region within the saposin C domain both in vivo and in vitro. Recently, GPR37 and GPR37L1 were recognized as PSAP receptors. In this study, we examined the alteration in expression of PSAP and its receptors in the cerebellum using rats injected with kainic acid (KA). The results show that PSAP was strongly expressed in the cytoplasm of Purkinje cells and interneurons in the molecular layer, and that PSAP expression in both types of neurons was markedly enhanced following KA treatment. Immunoblotting revealed that the expression of GPR37 was diminished significantly three days after KA injection compared with control rats; however, no changes were observed through immunostaining. No discernable changes were found in GPR37L1. These findings may help us to understand the role of PSAP and the GPR37 and GPR37L1 receptors in alleviating the neural damage caused by KA.

G protein-coupled receptor (GPR) 37 and GPR37L1 are known to modulate the dopaminergic neuron activity, and recently, they are identified as candidate prosaposin receptors. Intercellular prosaposin is proteolytically processed into four saposins, each of which acts as a sphingolipid hydrolase activator in the lysosome. In contrast, extracellular prosaposin exerts a trophic effect on neurons via GPR37 and GPR37L1. In this study, the expression patterns of GPR37 and GPR37L1 in the mouse digestive system were examined immunohistochemically. The islets of Langerhans of the pancreas showed intense immunoreactivity for GPR37 and GPR37L1. Weak immunoreactivity for GPR37 and GPR37L1 was found in the nerve plexuses of the esophagus and small and large intestines. Colocalization of GPR37 and tyrosine hydroxylase immunoreactivity was observed in the neuron of the nerve plexus of the large intestine. This study suggests the possibility that prosaposin affects the function of islet-secreting cells. Also, the expression of GPR37 and GPR37L1 in the nerve plexus suggests that prosaposin exerts a trophic effect not only in the central nervous system, but also in the enteric nervous system.

Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription-polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP, demonstrated by specific triglyceride transfer activity, is present in both mouse splenocytes and a CD1d-positive mouse NKT hybridoma. In a novel in vitro transfer assay, purified MTP directly transfers phospholipids, but not triglycerides, to recombinant CD1d. Chemical inhibition of MTP lipid transfer does not affect major histocompatibility complex class II presentation of ovalbumin, but considerably reduces CD1d-mediated presentation of alpha-galactosylceramide (alpha-galcer) and endogenous antigens in mouse splenic and bone marrow-derived dendritic cells (DCs), as well as in human APC lines and monocyte-derived DCs. Silencing MTP expression in the human monocyte line U937 affects CD1d function, as shown by diminished presentation of alpha-galcer. We propose that MTP acts upstream of the saposins and functions as an ER chaperone by loading endogenous lipids onto nascent CD1d. Furthermore, our studies suggest that a small molecule inhibitor could be used to modulate the activity of NKT cells.

Prosaposin (PSAP), a highly conserved glycoprotein, is a precursor of saposins A-D. Accumulating evidence suggests that PSAP is a neurotrophic factor, as well as a regulator of lysosomal enzymes. Recently, the orphan G-protein-coupled receptors GPR37 and GPR37L1 were recognized as PSAP receptors, but their functions have not yet been clarified. In this study, we examined the distribution of PSAP and its receptors in the dorsal root ganglion (DRG) during development using specific antibodies, and showed that PSAP accumulates primarily in lysosomes and is dispersed throughout the cytoplasm of satellite cells. Later, PSAP colocalized with two receptors in satellite cells, and formed a characteristic ring shape approximately 8 weeks after birth, during a period of rapid DRG development. This ring shape, which was only observed around larger neurons, is evidence that several satellite cells are synchronously activated. We found that sortilin, a transporter of a wide variety of intracellular proteins containing PSAP, is strongly localized to the inner side of satellite cells, which contact the neuronal surface. These findings suggest that PSAP and GPR37/GPR37L1 play a role in activating both satellite and nerve cells.

Carnocyclin A (CclA) is a potent antimicrobial peptide from Carnobacterium maltaromaticum UAL307 that displays a broad spectrum of activity against numerous Gram-positive organisms. An amide bond links the N and C termini of this bacteriocin, imparting stability and structural integrity to this 60-amino acid peptide. CclA interacts with lipid bilayers in a voltage-dependent manner and forms anion selective pores. Several other circular bacteriocins have been reported, yet only one (enterocin AS-48) has been structurally characterized. We have now determined the solution structure of CclA by NMR and further examined its anion binding and membrane channel properties. The results reveal that CclA preferentially binds halide anions and has a structure that is surprisingly similar to that of AS-48 despite low sequence identity, different oligomeric state, and disparate function. CclA folds into a compact globular bundle, comprised of four helices surrounding a hydrophobic core. NMR studies show two fluoride ion binding modes for CclA. Our findings suggest that although other circular bacteriocins are likely to have diverse mechanisms of action, many may have a common structural motif. This shared three-dimensional arrangement resembles the fold of mammalian saposins, peptides that either directly lyse membranes or serve as activators of lipid-degrading enzymes.

An infant presented with multifocal myoclonus and cyanotic hypoxemia immediately after birth, and severe feeding problems, a protein-losing enteropathy, massive ascites and grand-mal epilepsy marked his rapid downhill course, with death at 17 weeks. At 2 weeks, brain MRI revealed grey matter heterotopias in the parieto-occipital regions suggestive of a cortical morphogenetic disorder. In cultured skin fibroblasts, lipid storage and reduced activities of ceramidase, galactosylceramide beta-galactosidase and glucosylceramide beta-glucosidase were evident. Autopsy disclosed generalised lysosomal lipid storage with macrophages and adrenal cortex prominently affected. The pattern of stored lipids in cultured fibroblasts and in dewaxed spleen tissue blocks was compatible with a diagnosis of prosaposin (pSap) deficiency (pSap-d). Neuropathologically, there was a pronounced generalised neurolysosomal storage combined with a severe depletion of cortical neurons and extreme paucity of myelin and oligodendroglia. This pathology, in particular the massive neuronal loss, differed from that in other neurolipidoses and could be explained by the reduced hydrolysis of multiple sphingolipids and the loss of pSap's neurotrophic function. The absence of immunostainable saposins on tissue sections and the presence of a homozygous c.1 A > T mutation in the prosaposin gene confirmed the diagnosis. PSap-d may be an underdiagnosed condition in infants with severe neurological and dystrophic signs starting immediately after birth.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.

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.

The Niemann-Pick type C2 (NPC2) protein is a small, soluble, lysosomal protein important for cholesterol and sphingolipid transport in the lysosome. The immunological phenotype of NPC2-deficient mice was limited to an impaired thymic selection of Valpha14 natural killer T cells (NKT cells) and a subsequent reduction of NKT cells in the periphery. The remaining NKT cells failed to produce measurable quantities of interferon-gamma in vivo and in vitro after activation with alpha-galactosylceramide. In addition, thymocytes and splenocytes from NPC2-deficient mice were poor presenters of endogenous and exogenous lipids to CD1d-restricted Valpha14 hybridoma cells. Importantly, we determined that similar to saposins, recombinant NPC2 was able to unload lipids from and load lipids into CD1d. This transfer activity was associated with a dimeric form of NPC2, suggesting a unique mechanism of glycosphingolipid transfer by NPC2. Similar to saposin B, NPC2 dimers were able to load isoglobotrihexosylceramide (iGb3), the natural selecting ligand of NKT cells in the thymus, into CD1d. These observations strongly suggested that the phenotype observed in NPC2-deficient animals was directly linked to the efficiency of the loading of iGb3 into CD1d molecules expressed by thymocytes. This conclusion was supported by the rescue of endogenous and exogenous iGb3 presentation by recombinant NPC2. Thus, the loading of endogenous and exogenous lipids and glycolipids onto CD1d is dependent on various small, soluble lipid transfer proteins present in the lysosome.