(Received for publication, July 7, 1995; and in revised form, September 11, 1995)
From the
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.
Saposins (Sap) ()A, B, C, and D are glycoproteins
released by proteolytic processing of a single precursor called
prosaposin(1, 2, 3, 4, 5, 6) .
The mature forms of Sap B and C have been localized in lysosomes by
various biochemical and immunochemical
techniques(7, 8, 9) . There is histochemical
evidence that Sap C is partly bound to the lysosomal
membrane(9) .
The amino acid sequences of saposins are
highly similar. Each saposin consists of 80 amino acids, including
six identically placed cysteine
residues(1, 2, 3, 4) . Recently, we
have established that human Sap B and C have the same disulfide
structure, with a central big loop surrounded by two smaller
ones(10) . Most likely, this disulfide arrangement is present
also in Sap A and D, where the six cysteine residues are at strictly
conserved positions. Consistently with their structural similarity,
saposins show common physicochemical characteristics; they are
heat-stable, acidic proteins resistant to most
proteinases(1, 2, 3, 4) .
One assessed function of saposins is the promotion of the enzymatic degradation of specific sphingolipids in lysosomes(1, 2, 3, 4) . Sap C appears to be required in vivo by glucosylceramidase for the hydrolysis of glucosylceramide since a genetic defect of Sap C causes a juvenile variant of Gaucher's disease with glucosylceramide accumulation in tissues(11) . A defect of Sap B results in a variant form of metachromatic leukodystrophy with sulfatide accumulation in tissues(12, 13) . The fact that the absence of one or another saposin causes different pathologies indicates that each saposin has distinct biological functions. When all four saposins are absent due to a genetic defect that prevents the synthesis of prosaposin(14) , accumulation of several sphingolipids has been observed in lysosomes with symptoms similar to those of Gaucher's and Farber's diseases (15) .
Several attempts have been made to elucidate the role of saposins in sphingolipid enzymatic degradation. Sap C was originally purified on the basis of its ability to activate the lysosomal enzyme glucosylceramidase in the presence of phosphatidylserine (PS)(16) . The enzyme activation was thought to be caused by a direct interaction of the saposin with the enzyme(16, 17) . In previous studies, we reinvestigated the role of Sap C, and the use of model lipid membranes provided us information on the mechanism whereby glucosylceramidase is stimulated by Sap C and PS. We found that, in order to express its activity, glucosylceramidase must be bound to acidic phospholipid-containing membranes (18, 19) and that Sap C promoted the enzyme binding by interacting with the bilayer(20) . We next examined the effects of Sap C on the physical properties of PS bilayers and showed that the saposin induced destabilization at acidic pH values (21) . The finding that the glucosylceramidase-stimulating ability of Sap C primarily depended on its interaction with acidic phospholipids prompted us to further investigate the mechanism of the saposin membrane action. In the present study, we examined 1) whether the pH-dependent binding of Sap C to PS-containing bilayers depended on a pH-dependent change of conformational state; 2) whether the other three saposins (A, B, and D), which possess similar structure, shared the Sap C ability to act on phospholipid membranes; and 3) whether a relation could be found between conformation of saposins, membrane binding, and bilayer perturbation. Finally, the specificity of Sap C in promoting the binding of glucosylceramidase to PS-containing membranes was evaluated.
For
glucosylceramidase binding studies, the enzyme (1000 units) was
incubated at 37 °C for 10 min with PS-containing LUV (200 µg),
individual saposins (10 µg), and albumin (100 µg) in 0.4 ml of
buffer A, pH 5.0, supplemented with 10 mM dithioerythritol.
The mixture was then centrifuged at 130,000 g for 1 h.
The amount of glucosylceramidase in the supernatant was determined by
measuring the enzyme activity according to the standard assay (see
above). The amount of liposome-bound glucosylceramidase was expressed
relative to the amount of enzyme in the supernatant centrifuged in the
absence of saposins.
Figure 1: Effect of pH on the partitioning of saposins in Triton X-114. Each saposin was incubated at 37 °C in Triton X-114 solutions at the indicated pH values. The separation of the resulting aqueous (a) and detergent (b) phases and their analysis by SDS-polyacrylamide electrophoresis and immunoblotting were performed as reported under ``Experimental Procedures.'' Each saposin was visualized with the corresponding anti-saposin antiserum.
Figure 2:
Binding of saposins to liposomes as a
function of pH and lipid composition. Each saposin was incubated with
LUV composed of either cholesterol/PC(- - -) or
cholesterol/PC/PS (-) in buffer A adjusted to the
indicated pH values. The percentage of saposin bound to liposomes was
measured as reported under ``Experimental Procedures.''
and
, Sap A;
and
, Sap B;
and
, Sap C;
and
, Sap D.
Figure 3: Leakage of liposomes induced by saposins. Shown is the time course of release of calcein entrapped in LUV composed of either cholesterol/PC (left) or cholesterol/PC/PS (right) induced by saposins at different pH values. Each saposin was injected into a stirred cuvette thermostated at 37 °C and containing a 1-ml solution of liposomes (75 µM total lipid) in buffer A adjusted to the indicated pH values. The final concentration of saposins in the mixtures was 100 nM. A, Sap A; B, Sap B; C, Sap C; D, Sap D.
The extent of membrane destabilization depended on the amount of saposin C or D added (Fig. 4). At concentrations as low as 20 nM (protein/lipid molar ratio of 1:3750), the two saposins were still able to induce >40% leakage from PS-containing LUV after 100 s at pH 4.5.
Figure 4: Dependence of leakage on the amount of added saposins. Different amounts of either Sap C (C) or Sap D (D) were injected into a stirred cuvette thermostated at 37 °C and containing a 1-ml solution of LUV composed of cholesterol/PC/PS (75 µM total lipid) in buffer A, pH 4.5. The final concentrations of saposins in the mixtures are indicated.
Figure 5:
Effect of saposins on the binding of
glucosylceramidase to PS-containing LUV. Glucosylceramidase was
incubated in buffer A, pH 5.0, with LUV composed of cholesterol/PC/PS
and individual saposins. The percentage of glucosylceramidase bound to
liposomes was measured as reported under ``Experimental
Procedures.'' , Sap A;
, Sap B;
, Sap C;
, Sap D.
The fact that Sap A was unable to promote enzyme binding prompted us to re-evaluate the capacity of Sap A to stimulate the enzyme activity in the presence of PS-containing LUV. Table 1shows that while Sap C markedly increased the enzyme activity, Sap A, B, and D had no effect on glucosylceramidase under our experimental conditions.
Saposins presumably exert their biological activities in lysosomes (1, 2, 3, 4) , where the environment is maintained at pH values in the range 4.0-5.5(36, 37) . By studying the partitioning of these proteins between aqueous and detergent (Triton X-114) phases, we have presently found that a low pH induces changes in the hydrophobicity of Sap A, C, and D. In contrast, an acidic environment does not affect the apparent hydrophilicity of Sap B, which, also at pH 4.0, segregates into the aqueous phase.
The observation that mildly acidic conditions mimicking the interior pH of lysosomes promote an increase in the superficial hydrophobicity of saposins A, C, and D suggests that this mechanism might be relevant to their mode of action. The induction of hydrophobic properties by low pH values has been observed for several proteins. This is the salient characteristic of carboxypeptidase E(32) , influenza virus hemagglutinin(33) , colicin(34) , and clathrin(35) . These proteins at low pH undergo conformational changes that cause protein-membrane interaction and consequent perturbation of the bilayer.
The pH-triggered increases in the hydrophobicity of Sap C and D clearly correlated with their binding to vesicles composed of cholesterol and phospholipids, the predominant lipids in biological membranes. Of the four saposins, Sap C shows the highest affinity for phospholipid membranes. Its binding to PS vesicles at low pH had been previously assessed(20) . We have now found that, although the presence of an acidic phospholipid such as PS has a positive effect, Sap C can also associate with neutral phospholipid-containing vesicles. The binding properties of Sap C are consistent with the fact that Sap C is localized, at least in part, on the lysosomal membrane(9) , where high levels of phospholipids are present(38) .
In contrast with Sap C and D, Sap A and B show a poor binding efficiency for phospholipid vesicles. For Sap B, this finding is consistent with its apparent hydrophilicity both at neutral and acidic pH. The lack of interaction of Sap A with phospholipid membranes, in spite of its increased hydrophobicity at low pH, might be explained by the higher carbohydrate content of this saposin. In fact, two oligosaccharide side chains are present in the Sap A molecule instead of one as in the other saposins(23) .
The pH-dependent binding of saposins C and D to liposomes dramatically affects the integrity of the phospholipid bilayer. The membrane destabilization is favored by the same conditions that promote the binding of the two saposins, namely the presence of PS and acidic pH. Sap A and B, which poorly interact with phospholipid vesicles, have a minimal effect on the permeability of the bilayers. Although these results suggest that saposin-induced destabilization involves saposin binding, other factors also appear to influence the rate of leakage from the liposomes. Sap C, which is completely bound to PS-containing liposomes both at pH 4.0 and 5.0, induces a very rapid calcein release at pH 4.0, but not at pH 5.0 (see Fig. 2and Fig. 3). Most likely, the degree of Sap C embedding into the bilayer is different at the two pH values. According to Papahadjopoulos et al.(39) , the simple binding of proteins to the surface of lipid bilayers without penetration has minimal effects on membrane permeability, while the surface binding of proteins followed by their penetration into the bilayers results in a large increase in the permeability of the membranes.
It is well known that Sap C is required by glucosylceramidase to fully express its activity(16) . We have recently found that the Sap C requirement is related to the saposin ability to mediate the interaction between glucosylceramidase and PS-containing liposomes (20) . By comparing the effects of the four saposins, we have now found that Sap C is the only saposin able to promote the association of the enzyme with the lipid surface. Sap A, which has been also reported as a glucosylceramidase activator(23) , has no effect on the enzyme binding and, under our experimental conditions (Table 1), on the enzyme activity. The specificity of Sap C may depend on either a specific Sap C-induced perturbation of the bilayer or/and a specific interaction between Sap C and glucosylceramidase consequent to a saposin conformational change after insertion into the bilayer. Based on the present findings, we speculate that the exposure to the low lysosomal pH triggers a change necessary for Sap C to penetrate into acidic phospholipid-containing membranes, perturb the lipid organization, and finally promote the binding and activation of glucosylceramidase. According to this hypothesis, Sap C might play a major role in favoring the association of glucosylceramidase with the lysosomal membrane.
Recently, it was found that Sap D stimulates the lysosomal enzymatic degradation of ceramide(40) . It was proposed that the stimulation of acid ceramidase activity by Sap D depends on the direct association of the saposin with the enzyme and not with the lipid substrate(41) . Our present results provide the first experimental evidence that Sap D has the capacity to bind to PS-containing vesicles and perturb the bilayer. It would be of interest to investigate if the pH-dependent destabilizing activity of Sap D directly or indirectly affects the lysosomal ceramide degradation.
Presumably, saposins play several critical roles in lysosomes. A well documented function is that of stimulating the lysosomal degradation of sphingolipids(1, 2, 3, 4) . In addition, it has been reported by Hiraiwa et al.(42) that all four saposins and also their precursor, prosaposin, form high affinity complexes with gangliosides and transport them from one membrane to the other. This finding prompted the authors to propose that prosaposin and saposins might serve in vivo as transport proteins for gangliosides. Our present results showing that saposins C and D possess a potentiality to modify the phospholipid assembly at low pH values suggest that, independently of the presence of sphingolipids, these two saposins might have a further role in the organization of lysosomal and intralysosomal membranes. When all saposins are absent for a genetic defect in the start codon of the prosaposin gene(14) , the accumulation of intralysosomal vesicles has been observed(43) . This finding suggested that saposins are essential for the degradation of intralysosomal vesicles(4) ; it is conceivable that the pH-dependent membrane-destabilizing activities of Sap C and D are relevant in this lysosomal process.