Lysosomal Degradation on Vesicular Membrane Surfaces
1998; Elsevier BV; Volume: 273; Issue: 46 Linguagem: Inglês
10.1074/jbc.273.46.30271
ISSN1083-351X
AutoresGundo Wilkening, Thomas Linke, Konrad Sandhoff,
Tópico(s)Cellular transport and secretion
ResumoAccording to a recent hypothesis (Sandhoff, K., and Kolter, T. (1996) Trends Cell Biol. 6, 98–103), glycolipids, which originate from the plasma membrane, are exposed to lysosomal degradation on the surface of intralysosomal vesicles. Taking the interaction of membrane-bound lipid substrates and lysosomal hydrolases as an experimental model, we studied the degradation of glucosylceramides with different acyl chain lengths by purified glucocerebrosidase in a detergent-free liposomal assay system. Our investigation focused on the stimulating effect induced by lysosomal components such as sphingolipid activator protein C (SAP-C or saposin C), anionic lysosomal lipids, bis(monoacylglycero)phosphate, and dolichol phosphate, as well as degradation products of lysosomal lipids, e.g. dolichols and free fatty acids. The size of the substrate-containing liposomal vesicles was varied in the study.Enzymatic hydrolysis of glucosylceramide carried by liposomes made of phosphatidylcholine and cholesterol was rather slow and only weakly accelerated by the addition of SAP-C. However, the incorporation of anionic lipids such as bis(monoacylglycero)phosphate, dolichol phosphate, and phosphatidylinositol into the substrate carrying liposomes stimulated glucosylceramide hydrolysis up to 30-fold. Dolichol was less effective. SAP-C activated glucosylceramide hydrolysis under a variety of experimental conditions and was especially effective for the increase of enzyme activity when anionic lipids were inserted into the liposomes. Glucosylceramides with short acyl chains were found to be degraded much faster than the natural substrates. Dilution experiments indicated that the added enzyme molecules associate at least partially with the membranes and act there. Surface plasmon resonance experiments demonstrated binding of SAP-C at concentrations up to 1 μm to liposomes. At higher concentrations (2.5 μm SAP-C), liposomal lipids were released from the liposome coated chip. A model for lysosomal glucosylceramide hydrolysis is discussed. According to a recent hypothesis (Sandhoff, K., and Kolter, T. (1996) Trends Cell Biol. 6, 98–103), glycolipids, which originate from the plasma membrane, are exposed to lysosomal degradation on the surface of intralysosomal vesicles. Taking the interaction of membrane-bound lipid substrates and lysosomal hydrolases as an experimental model, we studied the degradation of glucosylceramides with different acyl chain lengths by purified glucocerebrosidase in a detergent-free liposomal assay system. Our investigation focused on the stimulating effect induced by lysosomal components such as sphingolipid activator protein C (SAP-C or saposin C), anionic lysosomal lipids, bis(monoacylglycero)phosphate, and dolichol phosphate, as well as degradation products of lysosomal lipids, e.g. dolichols and free fatty acids. The size of the substrate-containing liposomal vesicles was varied in the study. Enzymatic hydrolysis of glucosylceramide carried by liposomes made of phosphatidylcholine and cholesterol was rather slow and only weakly accelerated by the addition of SAP-C. However, the incorporation of anionic lipids such as bis(monoacylglycero)phosphate, dolichol phosphate, and phosphatidylinositol into the substrate carrying liposomes stimulated glucosylceramide hydrolysis up to 30-fold. Dolichol was less effective. SAP-C activated glucosylceramide hydrolysis under a variety of experimental conditions and was especially effective for the increase of enzyme activity when anionic lipids were inserted into the liposomes. Glucosylceramides with short acyl chains were found to be degraded much faster than the natural substrates. Dilution experiments indicated that the added enzyme molecules associate at least partially with the membranes and act there. Surface plasmon resonance experiments demonstrated binding of SAP-C at concentrations up to 1 μm to liposomes. At higher concentrations (2.5 μm SAP-C), liposomal lipids were released from the liposome coated chip. A model for lysosomal glucosylceramide hydrolysis is discussed. sphingolipid activator protein (saposin) N-((6-(biotinoyl)amino)hexanoyl)-1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine bis(monoacylglycero)phosphate glucosylceramide (glucosyl-d-erythro-2-N-acylsphingosine) [U-14C]glucosyl-d-erythro-2-N-acetylsphingosine [1-14C]glucosyl-d-erythro-2-N-hexanoylsphingosine [1-14C]glucosyl-d-erythro-2-N-dodecanoylsphingosine [1-14C]glucosyl-d-erythro-2-N-octadecanoylsphingosine large unilamellar vesicles 4-methylumbelliferyl-β-d-glucopyranoside phosphatidic acid (egg yolk) phosphatidylcholine (egg yolk) phosphatidylinositol (bovine brain) small unilamellar vesicle, TES,N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid d-glucosyl-N-acylsphingosine glucohydrolase (EC3.2.1.45). The degradation of plasma membrane-derived glycolipids takes place in the acidic compartments of the cells. According to a recently proposed hypothesis on the topology of lysosomal digestion (1Sandhoff K. Kolter T. Trends Cell Biol. 1996; 6: 98-103Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 2Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar), glycolipids of the plasma membrane reach the lysosomal compartment as components of vesicles through the endocytotic route. In the case of glycolipids with short oligosaccharide head groups, two proteins are required for the physiological degradation of each glycolipid substrate, a water-soluble lysosomal exohydrolase and a sphingolipid activator protein (SAP).1 The latter facilitates the interaction between vesicle-bound substrate and water-soluble enzyme. The deficiency of activator proteins SAP-A, -B, -C, and -D in a multiple activator protein deficiency disease (2Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar) or in SAP precursor knockout mice (3Fujita N. Suzuki K. Popko B. Maeda N. Klein A. Henseler M. Sandhoff K. Nakayasu H. Suzuki K. Hum. Mol. Genet. 1996; 5: 711-725Crossref PubMed Scopus (194) Google Scholar) results in an accumulation of glycolipids with a short oligosaccharide chain as well as of vesicle structures within the acidic compartment of the cells (4Bradova V.F. Smid F. Ulrich B. Roggendorf W. Paton B.C. Harzer K. Hum. Genet. 1993; 92: 143-152Crossref PubMed Scopus (115) Google Scholar, 5Burkhardt K.J. Hüttler S. Klein A. Möbius W. Habermann A. Griffiths G. Sandhoff K. Eur. J. Cell Biol. 1997; 73: 10-18PubMed Google Scholar). Endocytosis of the missing protein, the SAP precursor, by cultured mutant cells reverses both, the lipid and the vesicle accumulation (5Burkhardt K.J. Hüttler S. Klein A. Möbius W. Habermann A. Griffiths G. Sandhoff K. Eur. J. Cell Biol. 1997; 73: 10-18PubMed Google Scholar). For the lysosomal degradation of glucosylceramide, the enzyme glucocerebrosidase and the protein cofactor SAP-C (also called saposin C) are required (2Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar, 6Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1447Crossref PubMed Scopus (129) Google Scholar). A series of in vitro studies has been performed to understand the interaction between the enzyme and the activator (7Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Barca A. Scerch C. J. Biol. Chem. 1997; 272: 16862-16867Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Vaccaro A.M. Ciaffoni F. Salvioli R. Barca A. Tognozzi D. Scerch C. J. Biol. Chem. 1995; 270: 30576-30580Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Berend S.L. Radin N.S. Biochim. Biophys. Acta. 1981; 664: 572-582Crossref PubMed Scopus (79) Google Scholar, 10Peters S. Coyle P. Coffee C.J Glew R. J. Biol. Chem. 1977; 252: 563-573Abstract Full Text PDF PubMed Google Scholar, 11Morimoto S. Kishimoto Y. Tomich J. Weiler S. Ohashi T. Barranger J.A. Kretz K.A. O'Brien J.S. J. Biol. Chem. 1990; 265: 1933-1937Abstract Full Text PDF PubMed Google Scholar, 12Prence E.M. Biochem. J. 1995; 310: 571-575Crossref PubMed Scopus (5) Google Scholar, 13Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Maras B. Barca A. FEBS Lett. 1993; 336: 159-162Crossref PubMed Scopus (45) Google Scholar, 14Vabbro D. Grabowski G.A. J. Biol. Chem. 1991; 266: 15021-15027Abstract Full Text PDF PubMed Google Scholar, 15Qui X. Leonova T. Grabowski G.A. J. Biol. Chem. 1994; 269: 16746-16753Abstract Full Text PDF PubMed Google Scholar). Human acid d-glucosyl-N-acylsphingosine glucohydrolase (glucocerebrosidase; EC 3.2.1.45) is a water-soluble lysosomal protein. It cleaves the linkage of its physiological substrate, glucosylceramide (GlcCer) as well as water-soluble synthetic β-glucosides, e.g.4-methylumbelliferyl-β-d-glucoside (MuGlc) (7Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Barca A. Scerch C. J. Biol. Chem. 1997; 272: 16862-16867Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Vaccaro A.M. Ciaffoni F. Salvioli R. Barca A. Tognozzi D. Scerch C. J. Biol. Chem. 1995; 270: 30576-30580Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Berend S.L. Radin N.S. Biochim. Biophys. Acta. 1981; 664: 572-582Crossref PubMed Scopus (79) Google Scholar, 10Peters S. Coyle P. Coffee C.J Glew R. J. Biol. Chem. 1977; 252: 563-573Abstract Full Text PDF PubMed Google Scholar, 11Morimoto S. Kishimoto Y. Tomich J. Weiler S. Ohashi T. Barranger J.A. Kretz K.A. O'Brien J.S. J. Biol. Chem. 1990; 265: 1933-1937Abstract Full Text PDF PubMed Google Scholar, 12Prence E.M. Biochem. J. 1995; 310: 571-575Crossref PubMed Scopus (5) Google Scholar, 13Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Maras B. Barca A. FEBS Lett. 1993; 336: 159-162Crossref PubMed Scopus (45) Google Scholar, 14Vabbro D. Grabowski G.A. J. Biol. Chem. 1991; 266: 15021-15027Abstract Full Text PDF PubMed Google Scholar, 15Qui X. Leonova T. Grabowski G.A. J. Biol. Chem. 1994; 269: 16746-16753Abstract Full Text PDF PubMed Google Scholar, 16Sarmientos F. Schwarzmann G. Sandhoff K. Eur. J. Biochem. 1986; 160: 527-537Crossref PubMed Scopus (49) Google Scholar). Defective activity of the enzyme causes glucosylceramide accumulation in patients suffering from Gaucher disease, of which three clinical variants are known, i.e. types 1, 2, and 3 (17Brady R.O. Kanfer J.N. Shapiro D. Biochem. Biophys. Res. Commun. 1965; 18: 221-225Crossref PubMed Scopus (585) Google Scholar, 18Patrick, A. D. (1965) Biochem. J. 97,17c–18cGoogle Scholar). A known activator of the enzyme is the small glycoprotein, SAP-C, which enhances hydrolysis rates of glucocerebrosidase in vitro(7Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Barca A. Scerch C. J. Biol. Chem. 1997; 272: 16862-16867Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Vaccaro A.M. Ciaffoni F. Salvioli R. Barca A. Tognozzi D. Scerch C. J. Biol. Chem. 1995; 270: 30576-30580Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Berend S.L. Radin N.S. Biochim. Biophys. Acta. 1981; 664: 572-582Crossref PubMed Scopus (79) Google Scholar, 10Peters S. Coyle P. Coffee C.J Glew R. J. Biol. Chem. 1977; 252: 563-573Abstract Full Text PDF PubMed Google Scholar, 11Morimoto S. Kishimoto Y. Tomich J. Weiler S. Ohashi T. Barranger J.A. Kretz K.A. O'Brien J.S. J. Biol. Chem. 1990; 265: 1933-1937Abstract Full Text PDF PubMed Google Scholar, 12Prence E.M. Biochem. J. 1995; 310: 571-575Crossref PubMed Scopus (5) Google Scholar, 13Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Maras B. Barca A. FEBS Lett. 1993; 336: 159-162Crossref PubMed Scopus (45) Google Scholar, 14Vabbro D. Grabowski G.A. J. Biol. Chem. 1991; 266: 15021-15027Abstract Full Text PDF PubMed Google Scholar, 15Qui X. Leonova T. Grabowski G.A. J. Biol. Chem. 1994; 269: 16746-16753Abstract Full Text PDF PubMed Google Scholar) and in vivo (2Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar, 6Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1447Crossref PubMed Scopus (129) Google Scholar). Together with three other small glycoproteins, more specifically the saposins A, B, and D, SAP-C is derived from a single precursor protein, the SAP precursor or prosaposin (2Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar). The in vivo function of SAP-C was demonstrated by the identification of patients with an isolated deficiency of this glycoprotein (2Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar). Its deficiency causes a juvenile type of Gaucher disease but not the severe infantile form (type 2). In addition, feeding of purified SAP-C to fibroblasts from a patient with a SAP precursor deficiency reduced the level of GlcCer storage, whereas SAP-A, -B, and –D were not effective (6Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1447Crossref PubMed Scopus (129) Google Scholar). Glucocerebrosidase also requires negatively charged detergents or phospholipids for its full enzymatic activity in vitro(7Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Barca A. Scerch C. J. Biol. Chem. 1997; 272: 16862-16867Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Vaccaro A.M. Ciaffoni F. Salvioli R. Barca A. Tognozzi D. Scerch C. J. Biol. Chem. 1995; 270: 30576-30580Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Berend S.L. Radin N.S. Biochim. Biophys. Acta. 1981; 664: 572-582Crossref PubMed Scopus (79) Google Scholar, 10Peters S. Coyle P. Coffee C.J Glew R. J. Biol. Chem. 1977; 252: 563-573Abstract Full Text PDF PubMed Google Scholar, 11Morimoto S. Kishimoto Y. Tomich J. Weiler S. Ohashi T. Barranger J.A. Kretz K.A. O'Brien J.S. J. Biol. Chem. 1990; 265: 1933-1937Abstract Full Text PDF PubMed Google Scholar, 12Prence E.M. Biochem. J. 1995; 310: 571-575Crossref PubMed Scopus (5) Google Scholar, 13Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Maras B. Barca A. FEBS Lett. 1993; 336: 159-162Crossref PubMed Scopus (45) Google Scholar, 14Vabbro D. Grabowski G.A. J. Biol. Chem. 1991; 266: 15021-15027Abstract Full Text PDF PubMed Google Scholar, 15Qui X. Leonova T. Grabowski G.A. J. Biol. Chem. 1994; 269: 16746-16753Abstract Full Text PDF PubMed Google Scholar, 16Sarmientos F. Schwarzmann G. Sandhoff K. Eur. J. Biochem. 1986; 160: 527-537Crossref PubMed Scopus (49) Google Scholar). Earlier kinetic studies with GlcCer were mainly performed in the presence of detergents in order to solubilize the lipophilic substrate or in the presence of phosphatidylserine, which is a minor component of the lysosomes (0–3% of the phospholipid composition) (19Wherett J.R. Huterer S. J. Biol. Chem. 1972; 247: 4114-4120Abstract Full Text PDF PubMed Google Scholar, 20Stremmel W. Debuch H. Hoppe-Seylers Z. Physiol. Chem. 1976; 357: 803-810Crossref PubMed Scopus (39) Google Scholar, 21Bleistein J. Heidrich H.G. Debuch H. Hoppe-Seylers Z. Physiol. Chem. 1980; 361: 595-597PubMed Google Scholar). The acidic compartments of the cells contain negatively charged lipids such as bis(monoacylglycero)phosphate (BMP), which has been localized in the intraorganellar vesicular structures of late endosomes (22Koboyashi T. Stang E. Fang K.S. de Moerlose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (652) Google Scholar), and dolichol phosphate (23Cojnacki T. Dallner G. Biochem. J. 1988; 251: 1-9Crossref PubMed Scopus (241) Google Scholar). These molecular species as well as degradation products like dolichol and free fatty acids may influence the degradation of glucosylceramide. To verify the above mentioned model of the topology of lysosomal digestion, the experimental conditions should mimic the lipid composition and size of the vesicles as closely as possible. The convex curvature of the small vesicles (40–100 nm in diameter) (4Bradova V.F. Smid F. Ulrich B. Roggendorf W. Paton B.C. Harzer K. Hum. Genet. 1993; 92: 143-152Crossref PubMed Scopus (115) Google Scholar) favors the spreading of the glycoconjugate head groups on their surface and makes them easy prey for exohydrolases in the presence of activator proteins. To mimic the in vivosituation, we studied the degradation of glucosylceramide-bearing liposomes by glucocerebrosidase in the presence of SAP-C. The liposomes with diameters ranging from 40–100 nm contained various lysosomal anionic lipids. d-[14C]Glucose (specific activity 230 Ci/mol) was from ICN. Phosphatidylcholine (egg yolk), phosphatidic acid (egg yolk), phosphatidylinositol (bovine liver), dolichol (porcine liver), dolichol phosphate (porcine liver), cholesterol, 1,2-dipalmitoyl-sn-glycerol, sodium taurocholate, Triton X-100, phospholipase A2 (pig pancreas), and 4-methylumbelliferyl-β-d-glucopyranoside, were purchased from Sigma. Phenylphosphoryl dichloride was from Fluka, and Biotin-X-DHPE was from Molecular Probes, Inc. (Eugene, OR). SA™ and HPA™ sensor chips were purchased from BiaCore. Fine silica gel Lichroprep Si 60 and Lichroprep RP 18 were obtained from Merck. All other chemicals were of analytical grade or the highest purity available. Glucosylsphingosine and [14C]glucosylsphingosine (230 Ci/mol), as well as the glucosylceramides with different fatty acyl chains (C2-, C6-, C12-, and C18-GlcCer) were synthesized according to the method described by Sarmientos et al. (16Sarmientos F. Schwarzmann G. Sandhoff K. Eur. J. Biochem. 1986; 160: 527-537Crossref PubMed Scopus (49) Google Scholar). The structures of the labeled and unlabeled products were analyzed by mass spectrometry (matrix-assisted laser desorption ionization mass spectrometry). The labeled compounds were diluted with the unlabeled compounds to reach a specific radioactivity of 2.4 Ci/mol. BMP was prepared as described previously (24Dang Q.Q Rogalle P. Salvayre R. Douste-Blazy Lipids. 1982; 17: 798-802Crossref Scopus (9) Google Scholar) with the following modifications. A solution of 250 mg of sn-1,2- dipalmitoylglycerol (0.44 mmol) and 42 mg (0.22 mmol) of phenylphosphoryldichloride was stirred in 2-ml anhydrous pyridine under argon in a screw-capped vial at 37 °C for 24 h. Two volumes of water were gradually added, and the mixture was centrifuged at 3000 rpm. The precipitate was washed three times with water and dried under reduced pressure. The crude solid product was dissolved in a minimum of chloroform and chromatographed on Lichroprep Si 60 (1.2 cm × 100-cm column) with chloroform as eluent. Fractions containing bis-(1,2-dipalmitoyl-sn-glycero-3)-(phenyl)phosphate (compound I) were collected and lyophilized. Bis-(1,2-dipalmitoylglycero)phosphate (compound II) was obtained by hydrogenolysis of compound I in the presence of platinum oxide. The product was applied to a column and eluted at first with chloroform to remove residues of dipalmitoylglycerol and subsequently with chloroform/methanol (2/1, v/v). The pure product compound II was lyophilized from benzene. Bis-(1-palmitoyl-sn-glycero-3)-phosphate was derived by enzymatic hydrolysis of compound II by phospholipase A2 from porcine pancreas in a mixture of diethylether and sodium borate buffer, pH 8.3, as described before (24Dang Q.Q Rogalle P. Salvayre R. Douste-Blazy Lipids. 1982; 17: 798-802Crossref Scopus (9) Google Scholar). The product was purified by column chromatography (Lichroprep Si 60, 1.2 cm × 100 cm) in a chloroform/methanol/water (80/20/1) system. The yield of the lyso compound was 20 mg. Its structure was confirmed by mass spectrometry (fast atom bombardment mass spectrometry, matrix-assisted laser desorption ionization mass spectrometry). The modified lysosomal glucocerebrosidase (Ceredase™), manufactured by Genzyme (Boston) from human placenta, was a gift from Dr. Hans Aerts (Amsterdam). The concentrated enzyme preparation was stored in 1% human serum albumin in order to stabilize the glucocerebrosidase activity and was diluted with deionized water immediately before use. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the hydrolysis of 1 μmol of MuGlc/min in a detergent-containing assay. The final volume of 0.2 ml contained the following substances: 0.1 m citrate, 0.1 phosphate buffer, pH 5.5, 1 mm MuGlc, 0.4% (v/w) Triton X-100, 0.8% (v/w) sodium taurocholate. SAP-C was isolated from the spleen of a patient with Gaucher disease as described previously (6Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1447Crossref PubMed Scopus (129) Google Scholar). The purity of the SAP-C preparation was ensured by polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization mass spectrometry (data not shown). Large unilamellar vesicles (LUVs) were prepared by the following procedure (25MacDonald R.C. MacDonald R.I. Menco B.P.M. Takeshita K. Subbarao N.K. Hu L. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1382) Google Scholar). Phosphatidylcholine, cholesterol, [14C]glucosylceramide, and the other lipids (see Figs. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7) were dissolved in organic solvents (ethanol, chloroform, methanol). Appropriate aliquots of lipid solutions were mixed, dried under nitrogen, and kept under vacuum for at least 1 h. The lipid mixture was hydrated at a concentration of 6.25 mm in deionized water and freeze-thawed 10 times in liquid nitrogen to ensure solute equilibration between trapped and bulk solutions.Figure 2Dilution of the incubation mixture with buffer reduces enzymatic GlcCer hydrolysis (A) only slightly and enzymatic MuGlc hydrolysis (B) drastically in the presence of LUVs. A, the concentration of [14C]glucosylceramide-carrying LUVs doped with 20 mol % PA as stimulating lipid, was altered by the addition of various amounts of buffer. Assay mixtures contained constant concentrations of SAP-C (2 μm) (▪), constant amounts of SAP-C (0.8 μg) (•), or no SAP-C (○). B, The concentration of LUVs, doped with 20 mol % PA, was altered by the addition of various amounts of buffer. Assays with 5 μg of SAP-C (•) or without SAP-C (○) were performed, and the preparation of the LUVs was carried out as reported under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Lysosomal lipids in LUVs stimulate the enzymatic GlcCer hydrolysis in the presence and absence of SAP-C. A, assays were conducted with GlcCer as substrate in the absence (○) and presence of SAP-C (2.5 μm) (•) as described under "Experimental Procedures," using LUVs with various proportions of synthetic BMP (0–40 mol %). B, assays were carried out as described under "Experimental Procedures" with varying concentrations of PI in LUVs, with (•) and without (○) the addition of 2.5 μm SAP-C, keeping the total lipid concentration in the assays constant. C, GlcCer carrying LUVs with or without PA were doped with increasing proportions of dolichol (0–10 mol %) and assayed for enzymatic GlcCer hydrolysis as described under "Experimental Procedures" as follows: without SAP-C (○), with 2.5 μm SAP-C (•), with 10 mol % PA (▵), and with 10 mol % PA and 2.5 μm SAP-C (▴).D, assays were performed with varying concentrations of dolichol phosphate (DP) (0–10 mol %) with 2.5 μm SAP-C (•) or no SAP-C (○) as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Stimulation of the enzymatic GlcCer hydrolysis by oleic acid and hexanoic acid in the presence and in the absence of SAP-C. A suspension of 28 mm oleic acid (OA) or hexanoic acid (HA) (7 mm in the assay mixture), prepared as described previously (10Peters S. Coyle P. Coffee C.J Glew R. J. Biol. Chem. 1977; 252: 563-573Abstract Full Text PDF PubMed Google Scholar), were added to LUVs in the presence and in the absence of 2.5 μmSAP-C. The LUVs contained either 0 or 10 mol % PA. Assays were carried out as described in experimental procedures.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Curvature of vesicles stimulates the enzymatic hydrolysis of GlcCer and MuGlc in the presence of PA and SAP-C. A, LUVs and SUVs, doped with different proportions of PA and 3 mol % [14C]glucosylceramide, were incubated with 50 microunits of glucocerebrosidase in the absence and in the presence of SAP-C (2.5 μm) and assayed as described under "Experimental Procedures" as follows: LUVs (○), LUVs with SAP-C (•), SUVs (▵), and SUVs with SAP-C (▴).B, assay mixtures contained 2 mm MuGlc, unilamellar liposomes (3.125 μm lipid) of different size and varying amounts of PA without and with SAP-C (2.5 μm) as follows: LUVs (○), LUVs with SAP-C (•), SUVs (▵), and SUVs with SAP-C (▴). The enzyme assays were carried out as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Hydrolysis of LUV-bound GlcCer as a function of the fatty acyl chain length. The degradation of the following LUV-bound substrates (3 mol %) were measured: C2-GlcCer, C6-GlcCer, C12-GlcCer, and C18-GlcCer. Assays were conducted in the absence of SAP-C (○), in the presence of 2.5 μm SAP-C (•), in the presence of 20 mol % PA (▵), and in the presence of 20 mol % PA and 2.5 μm SAP-C (▴) as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Interaction of SAP-C and immobilized vesicles. LUVs (50 μl, lipid concentration 0.1 mm) were immobilized to give a response signal of 2500 RU. SAP-C was injected at a flow rate of 20 μl/min in 50 mm sodium citrate buffer (pH 4.5) for 180 s. Then, as indicated by anupward arrow, SAP-C free buffer was injected. Theupper time courses show the association of various concentrations of SAP-C (0–2.5 μm) on vesicles, containing PC and cholesterol bearing 0 mol % (A) or 3 mol % GlcCer (B). The lower time courses display different concentrations of SAP-C interacting with vesicles, spiked with 20 mol % BMP without (C) or with 3 mol % GlcCer (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The multilamellar vesicles were pressed through polycarbonate filters (pore size, 100 nm; Nucleopore) mounted in a miniextruder (Liposo-Fast; Avestin). We subjected samples to 19 passes through two filters in tandem as recommended (25MacDonald R.C. MacDonald R.I. Menco B.P.M. Takeshita K. Subbarao N.K. Hu L. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1382) Google Scholar). Small unilamellar vesicles (SUVs) were produced by sonification of LUVs with a Microtip sonicator (Branson, Danbury, CT) at 0 °C for 40 min (intervals of 15-s sonification, 30-s pause) under a stream of argon to avoid a degradation of the lipids resulting from the high temperatures. Subsequently, they were centrifuged at 100,000 ×g av for 15 min. The concentration of the liposomes (SUVs and LUVs) was proved by measuring their radioactivity. In order to determine the proportion of the accessible substrate, glucosylceramide-containing LUVs and SUVs were exhaustively treated with glucocerebrosidase (500 milliunits) for 2 h. Under these conditions, about 50% of glucosylceramide in LUVs and 65% in SUVs can be degraded (16Sarmientos F. Schwarzmann G. Sandhoff K. Eur. J. Biochem. 1986; 160: 527-537Crossref PubMed Scopus (49) Google Scholar). The liposomes for the assays with MuGlc were prepared as described above, but without the addition of GlcCer. For the BiaCore measurements with the SA™ chip, 0.1 mol % Biotin-X-DHPE was added to the lipid solution before drying it. The liposomes were diluted in TES buffer (final concentration 10 mm TES, 300 mm NaCl, 2 mmCaCl2, pH 7.0) to a lipid concentration of 0.05 mm. The size of the liposomes was controlled by electron microscopy. The LUVs had a diameter of 90–120 nm, and the SUVs were about 40 nm in diameter (data not shown). The standard incubation mixtures contained the following components in a final volume of 40 μl: human serum albumin (20 ng), sodium citrate buffer (50 mm, pH 4.5), unilamellar liposomes (LUVs or SUVs), SAP-C as indicated, and glucocerebrosidase (25–50 microunits, 1–3 ng). Ceredase™ was used as a glucocerebrosidase source for all assays. Comparative experiments of Ceredase™ and human serum albumin free native glucocerebrosidase showed that small amounts of human serum albumin did not effect the activation of glucocerebrosidase by SAP-C and acidic lipids as previously reported by another working group (15Qui X. Leonova T. Grabowski G.A. J. Biol. Chem. 1994; 269: 16746-16753Abstract Full Text PDF PubMed Google Scholar). The standard incubation conditions were 37 °C for 20 min. Incubation time and the amount of enzyme were chosen in such a way tha
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