[Show abstract][Hide abstract] ABSTRACT: Gaucher disease (GD) is characterized by accumulation of glucosylceramide (GC) in the cells of monocyte/macrophage system. The degradation of GC is controlled by glucosylceramidase (GCase) and saposin (Sap) C, a member of a family of four small glycoproteins (Saps A, B, C and D), all derived by proteolytic processing of a common precursor, prosaposin (PSAP). Saps contain six cysteine residues, forming three disulfide bridges, that affect their structure and function. Sap C is an essential activator of GCase and its deficit impairs the GCase activity causing GD. In the present study the biological properties of cells from four recently described GD patients carrying mutations in the Sap C domain of the PSAP gene have been characterized. Two patients had mutations involving a cysteine residue, whereas the other two had a L349P mutation. It was found that: (i) in the four Sap C-deficient cells PSAP was normally processed and sorted, the lack of Sap C being mainly due to the Sap C instability in late endosomal/lysosomal environment; (ii) the decrease/absence of Sap C affected the GCase intracellular localization; (iii) the lowest level of Sap C and enhanced autophagy were observed in the cells, which carried a Sap C mutation involving a cysteine residue; (iv) the four Sap C-deficient fibroblasts stored GC, ceramide and cholesterol, the last two lipids being clearly localized in lysosomes; (v) a correlation was observed between the type of Sap C mutation and the Gaucher phenotype: apparently, mutations involving cysteine residues lead to a neurological variant of GD.
Human Molecular Genetics 08/2010; 19(15):2987-97. DOI:10.1093/hmg/ddq204 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The notion that prosaposin (Prosap) is likely involved in brain development and regeneration led us to explore its expression in stem/progenitor neural cells and its fate after cell differentiation. The expression of procathepsin-cathepsin D (proCath-Cath D), an endoprotease that plays an important role in the processing and sorting of Prosap, has been concomitantly examined. Our data evidenced that in embryonic human neural progenitor cells (eHNPCs) intact and high molecular weight intermediate forms of Prosap and intermediate forms of Cath D accumulated inside the cells, while the formation of saposins and mature Cath D was impaired. Furthermore, neither Prosap nor proCath D were secreted from eHNPCs. The block of the processing and secretion shared by Prosap and proCath D was overcome during the course of differentiation of eHNPCs into a mixed population of astrocytes and neuronal cells. Upon differentiation, large amounts of Prosap and proCath D were secreted from the cells, while saposins and mature Cath D were produced inside the cells. The dramatic accumulation of Prosap (an antiapoptotic factor) and reduction of mature Cath D (a proapoptotic factor) in the undifferentiated eHNPCs most likely play a role in the molecular mechanisms regulating the resistance to apoptotic signals of these cells and might represent a critically important issue in HNPCs biology.
[Show abstract][Hide abstract] ABSTRACT: Gaucher disease is generally caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The degradation of glycosphingolipids requires also the participation of sphingolipid activator proteins. The prosaposin PSAP gene codes for a single protein which undergoes post-translational cleavage to yield four proteins named saposins A, B, C and D. Saposin (SAP-) C is required for glucosylceramide degradation, and its deficiency results in a variant form of Gaucher disease. In this report, we present clinical, biochemical, and molecular findings in a 36-year-old man and his 30-year-old sister with non-neuronopathic Gaucher disease due to SAP-C deficiency. Very high levels of chitotriosidase activity, chemokine CCL18, and increased concentration of glucosylceramide in plasma and normal beta-glucosidase activity in skin fibroblasts were observed in the patients. A molecular genetics study of the PSAP gene enabled the identification of one missense mutation, p.L349P, located in the SAP-C domain and another mutation, p.M1L, located in the initiation codon of the prosaposin precursor protein. The presented findings describe the first cases where the non-neuronopathic Gaucher disease has been definitely demonstrated to be a consequence of SAP-C deficiency. Three previously described cases in the literature displayed a Gaucher type 3 phenotype.
[Show abstract][Hide abstract] ABSTRACT: Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known. Using large unilamellar vesicles (LUVs) as membrane models, we have now found that Sap B simultaneously extracts from the lipid surface neutral [phosphatidylcholine (PC)] and anionic [phosphatidylinositol (PI)] phospholipids, fewer SLs [ganglioside GM1 (GM1) or cerebroside sulfate (CS)], and no Chol. More PI than SL (GM1 or CS) was solubilized from LUVs containing equal amounts of PI and SLs. An increase in PI level had a poor effect on the Sap B-induced solubilization of GM1 or CS but strongly inhibited that of PC. Sap B was able not only to bind, but also to transfer phospholipids between lipid surfaces. Both the phospholipid binding and transfer activities were optimal at low pH values. These results represent the first biochemical analysis of the Sap B interaction with phospholipids. The capacity of Sap B to bind and transfer phospholipids occurs under conditions mimicking the interior of the late endosomal/lysosomal compartment and thus might have physiological relevance.
The Journal of Lipid Research 06/2006; 47(5):1045-53. DOI:10.1194/jlr.M500547-JLR200 · 4.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370-->Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (approximately 20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo.
[Show abstract][Hide abstract] ABSTRACT: Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability.
[Show abstract][Hide abstract] ABSTRACT: Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles. The Sap D-particle complexes formed in an acidic milieu can be dissociated by an increase in pH, suggesting a dynamic association of Sap D with membranes. The presence of anionic phospholipids is required also for the Sap D-induced perturbation and solubilization of membranes containing a neutral sphingolipid such as ceramide or a ganglioside such as G(M1). At appropriate Sap D concentrations Cer and G(M1) are solubilized as constituents of small phospholipid particles. Our findings imply that most functions of Sap D are dependent on its interaction with anionic phospholipids, which mediate the Sap D effect on other components of the membrane such as sphingolipids. On consideration of the properties of Sap D we propose that Sap D might have a role in the definition of the structure and function of membranes, such as the intra-endolysosomal membranes, that are rich in anionic phospholipids.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The reconstitution of the activity of the lysosomal enzyme glucosylceramidase requires anionic phospholipids and, at least, a protein factor, saposin C (Sap C). We have previously proposed a mechanism for the glucosylceramidase activation [Vaccaro et al. (1993) FEBS Lett. 336, 159-162] which implies that Sap C promotes the association of the enzyme with anionic phospholipid-containing membranes, thus favoring the contact between the enzyme and its lipid substrate, glucosylceramide. We have further investigated the properties of Sap C using a fluorescent hydrophobic probe such as 4, 4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS). The binding between bis-ANS and Sap C was pH-dependent, indicating that protonation leads to increased exposure of hydrophobic surfaces of Sap C. The interaction of Sap C with membranes, triggered by the development of hydrophobic properties at low pH values, was affected by the content of anionic phospholipids, such as phosphatidylserine or phosphatidylinositol, suggesting that anionic phospholipids have the potential to modulate the insertion of Sap C in the hydrophobic environment of lysosomal membranes. We previously showed that Sap C and anionic phospholipids are both required for the binding of glucosylceramidase to large vesicles. We have presently observed that Sap C is able to promote the association of glucosylceramidase with the lipid surface only when anionic phospholipids exceed a concentration of 5-10%. This level can be reached by summing lower amounts of individual anionic phospholipids, since they have additive effects. The present data extend and refine our model of the mechanism of glucosylceramidase activation and stress the key role of pH, Sap C and anionic phospholipids in promoting the interaction of the enzyme with membranes.
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Biochemistry 08/1999; 263(2):486-94. DOI:10.1046/j.1432-1327.1999.00521.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.
[Show abstract][Hide abstract] ABSTRACT: The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines. These saposin-like (SAPLIP) proteins are surfactant protein B (SP-B), 'Entamoeba histolytica' pore-forming peptide, NK-lysin, acid sphingomyelinase and acyloxyacyl hydrolase. The structural homology and the conserved disulfide bridges suggest for all SAPLIPs a common fold, called 'saposin fold'. Up to now a precise fold, comprising five alpha-helices, has been established only for NK-lysin. Despite their similar structure each saposin promotes the degradation of specific sphingolipids in lysosomes, e.g. Sap B that of sulfatides and Sap C that of glucosylceramides. The different activities of the saposins must reside within the module of the alpha-helices and/or in additional specific regions of the molecule. It has been reported that saposins bind to lysosomal hydrolases and to several sphingolipids. Their structural and functional properties have been extensively reviewed and hypotheses regarding their molecular mechanisms of action have been proposed. Recent work of our group has evidenced a novel property of saposins: some of them undergo an acid-induced change in hydrophobicity that triggers their binding to phospholipid membranes. In this article we shortly review recent findings on the structure of saposins and on their interactions with lipids, with special attention to interactions with phospholipids. These findings offer a new approach for understanding the physiological role of saposins in lysosomes.
Neurochemical Research 03/1999; 24(2):307-14. DOI:10.1023/A:1022530508763 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The degradation of glucosylceramide in lysosomes is accomplished by glucosylceramidase with the assistance of, at least, another protein, saposin C (Sap C), which is generated from a large precursor together with three other similar proteins, saposins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydrolysis of glucosylceramide inserted in large and small phospholipid liposomes. The glucosylceramide contained in large unilamellar vesicles (LUV) was degraded by glucosylceramidase at a rate 7-8-fold lower than glucosylceramide inserted in small unilamellar vesicles (SUV). The separate addition of either Sap A or Sap C to the LUV system partially stimulated the sphingolipid degradation while saposins B and D had no effect. In the presence of both Sap A and Sap C, the rate of sphingolipid degradation was higher than the sum of the rates with the two saposins individually, indicating synergism in their actions. The stimulatory effect of the two saposins depended on the incorporation of an acidic phospholipid such as phosphatidylserine (PS) into LUV. The characteristics of glucosylceramidase activation by Sap C were different from those of Sap A. Sap C increased the rate of hydrolysis of both the artificial water soluble substrate, 4-methylumbelliferyl-beta-D-glucopyranoside, and the lipid substrate, glucosylceramide, while Sap A only stimulated degradation of the sphingolipid. Also the binding properties of Saps A and C were markedly different. At acidic pH values, Sap C bound to PS-containing LUV and promoted the association of glucosylceramidase with the membrane. In contrast, Sap A had poor affinity for the membrane even in the presence of glucosylceramide; moreover, Sap A did not potentiate the capacity of Sap C to mediate glucosylceramidase binding. In conclusion, our results show that both Sap A and Sap C are required for maximal hydrolysis of glucosylceramide inserted in PS-containing LUV, that their effects are synergistic, and that their mode of action is different. Sap C is responsible for the membrane binding of glucosylceramidase, while Sap A stimulation is possibly related to its effect on the conformation of the enzyme. It can be envisaged that Sap A in conjunction with Sap C might have a physiological role in glucosylceramide degradation.
[Show abstract][Hide abstract] ABSTRACT: Saposins A, B, C, and D are small lysosomal glycoproteins released by proteolysis from a single precursor polypeptide, prosaposin. We have presently investigated the conformational states of saposins and their interaction with membranes at acidic pH values similar to those present in lysosomes. With the use of phase partitioning in Triton X-114, experimental evidence was provided that, upon acidification, saposins (Sap) A, C, and D acquire hydrophobic properties, while the hydrophilicity of Sap B is apparently unchanged. The pH-dependent exposure of hydrophobic domains of Sap C and D paralleled their pH-dependent binding to large unilamellar vesicles composed of phosphatidylcholine, phosphatidylserine, and cholesterol. In contrast, the binding of Sap A to the vesicles was very restricted, in spite of its increased hydrophobicity at low pH. A low affinity for the vesicles was also shown by Sap B, a finding consistent with its apparent hydrophilicity both at neutral and acidic pH. At the acidic pH values needed for binding, Sap C and D powerfully destabilized the phospholipid membranes, while Sap A and B minimally affected the bilayer integrity. In the absence of the acidic phospholipid phosphatidylserine, the induced destabilization markedly decreased. Of the four saposins, only Sap C was able to promote the binding of glucosylceramidase to phosphatidylserine-containing membranes. This result is consistent with the notion that Sap C is specifically required by glucosylceramidase to exert its activity. Our finding that an acidic environment induces an increased hydrophobicity in Sap A, C, and D, making the last two saposins able to interact and perturb phospholipid membranes, suggests that this mechanism might be relevant to the mode of action of saposins in lysosomes.
[Show abstract][Hide abstract] ABSTRACT: Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures. Electrospray analysis showed that deglycosylated saposins B and C are mainly present as 79- and 80-residue monomeric polypeptides, respectively. Fast atom bombardment mass analysis of peptide mixtures obtained by a combination of chemical and enzymatic cleavages demonstrated that the pairings of the three disulfide bridges present in each saposin are Cys4-Cys77, Cys7-Cys71, Cys36-Cys47 for saposin B and Cys5-Cys78, Cys8-Cys72, Cys36-Cys47 for saposin C. We have recently shown that saposin C interacts with phosphatidylserine-containing vesicles inducing destabilization of the lipid surface (Vaccaro, A. M., Tatti, M., Ciaffoni, F., Salvioli, R., Serafino, A., and Barca, A. (1994) FEBS Lett. 349, 181-186); this perturbation promotes the binding of the lysosomal enzyme glucosylceramidase to the vesicles and the reconstitution of its activity. It was presently found that the effects of saposin C on phosphatidylserine liposomes and on glucosylceramidase activity are markedly reduced when the three disulfide bonds are irreversibly disrupted. These results stress the importance of the disulfide structure for the functional properties of the saposin.
Journal of Biological Chemistry 05/1995; 270(17):9953-60. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that saposin C (Sap C), a glucosylceramidase activator protein, interacts with phosphatidylserine (PS) large unilamellar vesicles (LUV), promoting the glucosylceramidase binding to the bilayer [(1993) FEBS Lett. 336, 159-162]. In the present paper the consequences of the Sap C interaction on the lipid organization of the vesicles are reported. It was found that Sap C perturbs the PS bilayer as shown by the release of an encapsulated fluorescent dye. Three different procedures, resonance energy transfer, gel filtration and electron microscopy, indicated that the activator protein is also able to make PS liposomes fuse. The effects of Sap C on PS vesicles were observed at low but not at neutral pH. The lipid composition of the bilayer also affected the Sap C-induced destabilization; in fact, the presence of PS in mixed LUV was essential for significant leakage to occur. These results demonstrate for the first time that Sap C is a protein capable of destabilizing and fusing acidic phospholipid-containing membranes in a pH-dependent fashion.
[Show abstract][Hide abstract] ABSTRACT: The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.
[Show abstract][Hide abstract] ABSTRACT: The influence of phosphatidylserine (PS) liposome size on their capacity to activate and bind purified glucosylceramidase was investigated. Gel filtration and flotation experiments showed that large unilamellar vesicles (LUV) of either pure PS or PS in admixture with phosphatidylcholine (PC) are unable to tightly bind purified glucosylceramidase, and thus, to fully stimulate its activity. By contrast, small unilamellar vesicles (SUV) of PS adsorb glucosylceramidase can either be favoured or inhibited by factors affecting the bilayer curvature of PS liposomes. An increase of PS vesicle size induced by a fusogenic agent such as poly(ethylene glycol) (PEG), decreased enzyme binding and activity. On the contrary, the reduction of PS LUV size by sonication increased their stimulating ability. Enzyme association with PS SUV is reversible. In fact, glucosylceramidase bound to PS SUV was released from the lipid surface when the SUV were transformed into larger vesicles by PEG; dissociation from the vesicles resulted in a dramatic decrease of enzyme activity. Although PS LUV are unable to reconstitute glucosylceramidase, their association with oleic acid (OA) promotes the interaction with glucosylceramidase. This phenomenon is best explained in terms of OA-induced surface defects of PS LUV, with consequent exposure of the more hydrophobic part of the membrane and hence the improved binding of hydrophobic region/s of glucosylceramidase. Our data indicate that the physical organization of the PS-containing liposomes is of critical importance of glucosylceramidase reconstitution. The observation that physical changes of the lipid surface can markedly affect the enzyme activity offers a new approach to the study of glucosylceramidase regulation.
[Show abstract][Hide abstract] ABSTRACT: Studies were conducted to investigate the mechanism by which acidic phospholipid-containing vesicles stimulate purified placental glucosylceramidase activity towards the water-soluble substrate 4-methylumbelliferyl-beta-D-glucopyranoside (MUGlc). Vesicles composed of pure phosphatidic acid (PA) or pure phosphatidylserine (PS) stimulated the activity of the enzyme about 20-fold. The inclusion of cholesterol and phosphatidylcholine (PC), beside PA, into the vesicles slightly improved their stimulatory effect. Further addition of oleic acid (OA) markedly increased the stimulation (50-fold). By ultracentrifugation and gel permeation procedures it was shown that, under optimal conditions for stimulation of the MUGlc hydrolysis by acidic phospholipid-containing vesicles, purified glucosylceramidase spontaneously binds to their surface. Interestingly, the molar fraction of the acidic phospholipid into the mixed vesicles, rather than its concentration in the assay, is the crucial parameter for activation and binding of the enzyme. The importance of glucosylceramidase association with appropriate vesicles for enzyme activation was indicated by observing that the presence of 0.2 M citrate-phosphate buffer (pH 5.5), that prevented the binding to PA-containing surfaces, also inhibited the enzyme activity. Our results indicate that the reconstitution of glucosylceramidase activity occurs through the spontaneous tight association of the enzymatic protein with preformed acidic phospholipid-containing vesicles.
[Show abstract][Hide abstract] ABSTRACT: We have found that, under some experimental conditions, the placental glucosylceramidase shows an anomalous behaviour on gel filtration chromatography. At pH 5.6, the optimal pH of the enzymatic assay, the purified enzyme remains bound to either Superose 6 or TSK-40-XL HPLC columns, while the interaction of the crude glucosylceramidase contained in the water extract of the lysosome-mitochondrial fraction of placenta with the two HPLC gel matrices is much weaker. The quite different behaviour of the crude compared to the purified enzyme may be explained by the formation in the crude preparation of associated form(s) of glucosylceramidase with suitable endogenous compound(s), which compete with the gel matrices for the binding to the enzyme. The most likely one component of the enzyme complex is the placental activating factor, previously reported by us (Vaccaro et al. (1985) Biochim. Biophys. Acta 836, 157-166), as indicated by the negligible stimulation of the crude enzyme activity on addition of the factor, either before or after passage through the HPLC columns. On the assumption that the behaviour of crude glucosylceramidase on gel filtration becomes similar to that of the purified enzyme when its interaction with endogenous substance(s) is impaired, we have identified some conditions which prevent the formation of the enzyme associated form(s): (a) the addition of guanidine chloride (0.2 M), a cahotropic agent, to the crude preparation; and (b) the increase of pH up to 8. In conclusion, taking advantage of the anomalous behaviour of glucosylceramidase on gel filtration chromatography, evidence has been obtained that placental glucosylceramidase may occur under several forms which had not been previously reported; a difference in experimental conditions can promote the formation of one or another form, by possibly affecting the composition and/or the stoichiometry and/or the stability of the enzyme complex.