Direct visualization of saposin remodelling of lipid bilayers

Saposins A, B, C and D are soluble, non-enzymatic proteins that interact with lysosomal membranes to activate the breakdown and transfer of glycosphingolipids. The mechanisms of hydrolase activation and lipid transfer by saposins remain unknown. We have used in situ atomic force microscopy (AFM) with simultaneous confocal fluorescence microscopy to investigate the interactions of saposins with lipid membranes. AFM images of the effect of saposins A, B and C on supported lipid bilayers showed a time and concentration-dependent nucleated spread of membrane transformation. Saposin B produced deep gaps that ultimately filled with granular material, while saposins A and C lead to localized areas of membrane that were reduced in height by approximately 1.5 nm. Fluorescence-labeled saposin C co-localized with the transformed areas of the bilayer, indicating stable binding to the membrane. Fluorescence resonance energy transfer confirmed a direct interaction between saposin C and lipid. Under certain conditions of membrane lipid composition and saposin concentration, extensive bilayer lipid removal was observed. We propose a multi-step mechanism that integrates the structural features and amphipathic properties of the saposin proteins.     

Conformational and amino acid residue requirements for the saposin C neuritogenic effect.

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50-60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.     

Structural requirements for lysosomal targeting of the prosaposin precursor protein

Although the Man-6-P-independent lysosomal sorting of prosaposin, a precursor of four saposins (A, B, C, and D) is not understood, a protein/lipid interaction is considered. Immunocytochemical analysis revealed that each single saposin linked to the C-terminus of prosaposin and to secretory albumin, drives the chimeric protein to lysosomes in COS-7 cells. Quantitative image analysis demonstrated that saposins are targeted with different efficiency (P<0.05) and in a less smooth manner than the precursor. Despite a very close homology, the charge distribution at the surface of 3D comparative models between saposins appeared different. Western blotting monitored prosaposin in cells also as a di- or trimeric form, whereas the chimeric saposins as monomeric. This implies that each amphipathic saposin-like motif may be a part of the overall structural requirements for binding of the precursor to the membrane lipids of transport vesicle. The crystal structure of saposin B demonstrating two dimeric units for lipid binding supports current findings.     

Interaction of saposins, acidic lipids, and glucosylceramidase

Activity of lysosomal glucosylceramidase is stimulated by two small glycoproteins, saposin A and C, which are, together with two other similar glycoproteins, derived from a single precursor protein. This enzyme is also stimulated by naturally occurring acidic lipids, such as phosphatidylserine and gangliosides. Using highly purified glucosylceramidase, saposins, and acidic lipids, the mechanism of enzyme stimulation was studied by investigating complex formation between the three components and by examining effects on activity caused by changing amounts of saposins and acidic lipids, individually or in combination. The results indicated that acidic lipids form a water-soluble complex with glucosylceramidase but not with saposins and that saposins and acidic lipids each bind to the enzyme at two different sites for the activation. Based on these observations, the previously proposed three-binding sites model of glucosylceramidase, activator, and substrate was modified to one composed of four binding sites: one for carbohydrate of the substrate, one for aglycon, one for acidic lipids, and one for saposins.     

Saposin C-LBPA interaction in late-endosomes/lysosomes

Acidic phospholipids and saposins associations are involved in the degradation process of glycosphingolipids/sphingolipids in late endosomes/lysosomes. In this report, we showed the colocalization of saposin C and lysobisphosphatidic acid (LBPA) in human fibroblasts by using cytoimmunofluorescence analysis. This colocalization pattern was not seen with other saposins. Large numbers of saposins A, B, and D illustrated the staining patterns that differ from LBPA. In addition, ingested anti-LBPA antibody altered the location of saposin C in human wild-type fibroblasts. In vitro assays demonstrated that saposin C at nM concentrations induced membrane fusion of LBPA containing phospholipid vesicles. Under the same condition, other saposins had no fusion induction on these vesicles. These results suggested a specific interaction between saposin C and LBPA. Total saposin-deficient fibroblasts showed a massive accumulation of multivesicular bodies (MVBs) by electron microscopic analysis. No significant increase of MVBs was found in saposins A and B deficient cells. Interestingly, the accumulated MVBs were significantly reduced by loading saposin C alone into the total saposin-deficient cells. Therefore, we propose that saposin C-LBPA interaction plays a role in the regulation of MVB formation in cells.     

Structural and membrane-binding properties of saposin D.

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5-Cys78, Cys8-Cys72 and Cys36-Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule is mainly modified by high-mannose-type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid-binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid-containing membranes.     

Differential membrane interactions of saposins A and C: implications for the functional specificity

Saposins are small, heat-stable glycoprotein activators of lysosomal glycosphingolipid hydrolases that derive from a single precursor, prosaposin, by proteolytic cleavage. Three of these saposins (B, C, and D) share common structural features including a lack of tryptophan, a single glycosylation sequence, the presence of three conserved disulfide bonds, and a common multiamphipathic helical bundle motif. Saposin A contains an additional glycosylation site and a single tryptophan. The oligosaccharides on saposins are not required for in vitro activation functions. Saposins A and C were produced in Escherichia coli to contain single tryptophans at various locations to serve as intrinsic fluorescence reporters, i.e. as topological probes, for interaction with phospholipid membranes. Maximum emission shifts, aqueous and solid quenching, and resonance energy transfer were quantified by fluorescence spectroscopy. Amphipathic helices at the amino- and carboxyl termini of saposins A and C were shown to insert into the lipid bilayer to about five carbon bond lengths. In comparison, the middle region of saposins A or C were either embedded in the bilayer or solvent-exposed, respectively. Conformational changes of saposin C induced by phosphatidylserine interaction suggested the reorientation of functional helical domains. Differential interaction models are proposed for the membrane-bound saposins A and C. By site-directed mutagenesis of saposin A and C, their membrane topological structures were correlated with their activation effects on acid beta-glucosidase. These findings show that proper orientation of the middle segment of saposin C to the outside of the membrane surface is critical for its specific and multivalent interaction with acid beta-glucosidase. Such membrane interactions and orientations of the saposins determine the proximity of their activation and/or binding sites to lysosomal hydrolases or lipoid substrates.     

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.     

Mesotrypsin and Caspase-14 Participate in Prosaposin Processing: POTENTIAL RELEVANCE TO EPIDERMAL PERMEABILITY BARRIER FORMATION

A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A–D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A–D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo.     

Sphingolipid hydrolase activator proteins and their precursors

Activator proteins for sphingolipid hydrolases (saposins) are small acidic, heat-stable glycoproteins that stimulate the hydrolysis of sphingolipids by lysosomal enzymes. The molecular mass of each stimulator is about 10 kDa, but glycosylated forms of higher mass exist too. The distribution and developmental changes in two saposins and their precursor proteins were studied with the aid of monospecific antibodies against saposin-B and saposin-C. They show a wide distribution in rat organs and forms intermediate between saposin and prosaposin (the precursor protein containing four different saposin units) could be seen. The amount of saposin and the degree of processing from prosaposin are quite different in different tissues. The saposins are the dominant forms in spleen, lung, liver, and kidney, while skeletal muscle, heart, and brain contain mainly precursor forms. In human blood, leukocytes contain mainly saposin, while plasma contains mainly precursor forms and platelets show many forms. Their subcellular distribution was studied using rat liver. The saposins of approximately 20 kDa are dominant in the light mitochondrial, mitochondrial, and microsomal fractions, following the distribution of the activity of a lysosomal marker enzyme. The nuclear fraction exhibits bands corresponding to non-glycosylated saposin. The soluble fraction contained much precursor forms. A developmental study of rat brain showed that the concentration of saposin precursors increased with age.     

Immunolocalization of the saposin-like insert of plant aspartic proteinases exhibiting saposin C activity. Expression in young flower tissues and in barley seeds

The plant-specific insert (PSI) of cypro11 gene-encoding cyprosin, an aspartic proteinase from Cynara cardunculus , has been cloned by polymerase chain reaction (PCR) into a bacterial expression vector. A rearranged form of this PSI in which the N- and C-terminal sequences were permutated to make it more similar to the structural arrangement observed in saposins was also cloned and expressed in the same system. The biological activities of the two purified recombinant proteins were compared to those of human saposins B and C. The proteins showed similar activity to saposin C, i.e. capacity to activate human glucosylceramidase. At a concentration of 5 µ M , wild-type PSI, saposin C, and rearranged PSI activated human glucosylceramidase two-, three-, and five-fold, respectively. The K_m for 4-methylumbelliferyl β-glucopyranoside was around 7 µ M in the presence of any of the three activators (5 µ M ). The neurotropic activity using NS20Y cells and lipid-binding properties of the plant recombinant proteins were tested. The two plant proteins showed lipid-binding properties similar to those of saposins but did not have any effect on neurite outgrowth. Immunolocalization of PSI showed its expression in protective tissues in flower meristem – protodermis, in C. cardunculus and embryonic root cap and coleorhiza in mature barley grains – as well as husk, pericarp, and the aleurone layer. Possible biological functions suggested for the plant homologue to saposins besides the general activation of enzymes involved in lipid metabolism would be involvement in plant defence.     

Structure and evolution of the human prosaposin chromosomal gene.

The gene for prosaposin was characterized by sequence analysis of chromosomal DNA to gain insight into the evolution of this locus that encodes four highly conserved sphingolipid activator proteins or saposins. The 13 exons ranged in size from 57 to 1200 bp, while the introns were from 91 to 3812 bp in length. The regions encoding saposins A, B, and D each had three exons, while that for saposin C had only two. This sequence included the regions that encode the carboxy terminus of the signal peptide, the four mature prosaposin proteins, and the 3' untranslated region. Primer extension studies indicated that over 99% of the coding sequence was contained in these 19,985 bp. Use of PCR and reverse PCR techniques indicated that the most 5' coding approximately 140 bp contained large introns and at least two small exons. Analyses of the intronic positions in the saposin regions indicated that this gene evolved from an ancestral gene by two duplication events and at least one gene rearrangement involving a double crossover after introns had been inserted into the gene.     

Saposins (Sphingolipid Activator Proteins) in the Twitcher Mutant Mouse

The twitcher mutant mouse, the animal model of Krabbe disease (human globoid cell leukodystrophy), is characterized by apparent deficiency of galactosylceramide beta-galactosidase activity. Saposin A and C, the heat-stable small sphingolipid activator glycoproteins, stimulate the activity of galactosylceramide beta-galactosidase as well as glucosylceramide beta-glucoside. The role of these saposins in the twitcher mutation was investigated. Boiled supernatant fractions, which contained saposins, were prepared from homogenates of twitcher brain, liver, kidney, and spleen. These preparations showed an almost identical effect on the activity of purified glucosylceramide beta-glucosidase (measured by hydrolysis of 4-methylumbelliferyl-beta-glucoside) with similar preparations from control tissues. The effect on the activity of galactosylceramide beta-galactosidase as well as 4-methylumbelliferyl-beta-glucoside beta-glucosidase in the twitcher brain and liver homogenates by authentic saposin A and C was similar to that in control tissues. These results suggest that the twitcher mutation does not affect the concentrations of saposin A or C or their interaction with galactosylceramide beta-galactosidase.     

Determination of saposin proteins (sphingolipid activator proteins) in human tissues

Saposins are small glycoproteins which are required for sphingolipid hydrolysis by lysosomal hydrolases. Each saposin (A, B, C, and D) stimulates a different enzymatic activity. A new simple HPLC method to determine the levels of saposins A, C, and D in tissue was developed. Tissues were homogenized in 20 vol of water, boiled, and centrifuged. The supernatant was lyophilized and redissolved in 5 ml of water. A 1.5-ml sample of the solution was applied to a reverse-phase HPLC column (C4 column) and eluted with an acetonitrile gradient. Most contaminants eluted from the column prior to the saposins, which were eluted later as a cluster of peaks. This cluster was collected and then analyzed by another HPLC system equipped with an AX-300 anion-exchange column using a NaCl gradient. Saposins D, A, and C eluted from the AX-300 column separately and in that order. Quantitation of the saposins was made by measuring the sizes of each peak. Standard curves made from pure saposins showed that quantification was linear over a range from 1 to 5 micrograms. Saposin B was measured by its stimulation activity on pure human liver GM1 ganglioside beta-galactosidase. Stimulation was linear up to 80 micrograms of saposin B. Application of this method to analysis of human tissues for their saposin content is presented.     

Sphingolipid activator proteins in a human hereditary renal disease with deposition of disialogangliosides

Summary Congenital nephrotic syndrome of the Finnish type is a recessively inherited renal disease with glomerular deposits of the disialogangliosideO-acetyl-GD3. Sphingolipid activator proteins (saposins) stimulate the degradation of glycosphingolipids by lysosomal enzymes, and defects in saposins cause accumulation of substrate lipids in the affected tissues in lysosomal storage diseases. Here we report a study of the role of saposins in the accumulation ofO-acetyl-GD3 in kidneys of congenital nephrotic syndrome patients. At the mRNA level, the expression of saposin precursor in diseased kidneys appeared normal, and the nucleotide sequence analysis of cDNA clones did not reveal abnormalities in the prosaposin gene. Immunohistologically, saposins were localized mainly to the epithelial cells of the distal renal tubules or to the parietal epithelial cells of glomeruli. In the nephrotic syndrome kidneys, the staining pattern was highly granular and appeared mostly in the apical part of the epithelial lining, unlike the control kidneys. These results show that a major site of ganglioside metabolism is located in the distal nephron. Furthermore, these results suggest that saposins are not directly involved in the metabolism of the terminal sialic acids of disialogangliosides in the nephrotic syndrome kidneys.     

The primary structure of mouse saposin.

The primary structure of mouse sphingolipid activator protein (saposin) was determined by cDNA sequencing. The amino acid sequence predicted by the cDNA sequence revealed that mouse saposin was highly homologous to human saposin and also to rat sertoli cell glycoprotein. Mouse saposin also has four functional domains, which are structurally similar to each other, and each domain has cysteines, prolines, and a potential glycosylation site at an almost identical position. An amino acid comparison between human and mouse saposins revealed that the similarity was approximately 70%, and human saposin lacks thirty-one amino acids between domains C and D. Heterogeneities of mRNA were found in both the coding and noncoding regions.     

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.     

Saposin D solubilizes anionic phospholipid-containing membranes.

Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles. We have presently found that, upon lipid binding, Sap D solubilizes the membranes, as shown by the clearance of the vesicles turbidity. The results of gel filtration, density gradient centrifugation, and negative staining electron microscopy demonstrate that this effect is due to the transformation of large vesicles to smaller particles. The solubilizing effect of Sap D is highly dependent on pH, the lipid/saposin ratio, and the presence of anionic phospholipids; small variations in each of these conditions markedly influences the activity of Sap D. The present study documents the interaction of Sap D with membranes as a complex process. Anionic phospholipids attract Sap D from the medium; when the concentration of the saposin on the lipid surface reaches a critical value, the membrane breaks down into recombinant small particles enriched in anionic phospholipids. Our results suggest that the role played by Sap D is more general than promoting sphingolipid degradation, e.g. the saposin might also be a key mediator of the solubilization of intralysosomal/late endosomal anionic phospholipid-containing membranes.     

Seminolipid and its precursor/degradative product,galactosylalkylacylglycerol, in the testis of saposin A- and prosaposin-deficient mice

Sphingolipid activator proteins (saposins A, B, C, and D) are derived from a common precursor protein (prosaposin) and specifically activate in vivo degradation of glycolipids with short carbohydrate chains. A mouse model of prosaposin deficiency (prosaposin-/-) closely mimics the human disease with an elevation of multiple glycolipids. The recently developed saposin A-/- mice showed a chronic form of globoid cell leukodystrophy, establishing the essential in vivo role of saposin A as an activator for galactosylceramidase to degrade galactosylceramide. Seminolipid, the principal glycolipid in spermatozoa, and its precursor/degradative product, galactosylalkylacylglycerol (GalEAG), were analyzed in the testis of the two mouse mutants by electrospray ionization mass spectrometry. Saposin A-/- mice showed the normal seminolipid level, while that of prosaposin-/- mice was approximately 150% of the normal level at the terminal stage. In contrast, GalEAG increased up to 10 times in saposin A-/- mice, whereas it decreased with age in the wild-type as well as in prosaposin-/- mice. These analytical findings on the two saposin mutants may shed some light on the physiological function of seminolipid and GalEAG.