Glucosylceramidase Mass and Subcellular Localization Are Modulated by Cholesterol in Niemann-Pick Disease Type C
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m313517200
ISSN1083-351X
AutoresRosa Salvioli, Susanna Scarpa, Fiorella Ciaffoni, Massimo Tatti, Carlo Ramoni, Marie T. Vanier, Anna Maria Vaccaro,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoNiemann-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. 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. Niemann-Pick disease type C (NPC) 1The abbreviations used are: NPC, Niemann-Pick disease type C; Chol, unesterified lipoprotein-derived cholesterol; SL, sphingolipids; GC, glucosylceramide; GM2, GalNAcβ4(Neu5Acα3)Galβ4Glc-ceramide; GCase, glucosylceramidase; DMEM, Dulbecco's modified Eagle's medium; LPDS, lipoprotein-deficient bovine serum; FBS, fetal bovine serum; LAMP1, lysosome-associated membrane protein type 1; LBPA, lysobisphosphatidic acid. is an autosomal-recessive neurovisceral lipid storage disorder (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Book Co., New York2001: 3611-3633Google Scholar). Most cases of NPC are caused by mutations in the NPC1 gene (2Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie J. Dwyer N.K. Neufeld E.B. Chang T. Liscum L. Strauss III, J.F. Ohno K. Zeigler M. Carmi R. Sokol J. Markie D. O'Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle D.A. Science. 1997; 277: 228-231Crossref PubMed Scopus (1250) Google Scholar) encoding a protein which possesses a sterol-sensing domain (3Davies J.P. Ioannou Y.A. J. Biol. Chem. 2000; 275: 24367-24374Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The putative function of NPC1 protein is to facilitate the recycling of lipids from late endosomes/lysosomes to other cellular membranes (4Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A.M. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 5Cruz J.C. Sugii S. Yu C. Chang T.Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 6Lange Y. Rigney M. Steck T. J. Biol. Chem. 2000; 275: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). High levels of unesterified lipoprotein-derived cholesterol (Chol) accumulate in NPC1-deficient cells. Although alterations of Chol metabolism play a key role in the pathogenesis of NPC, there is also a more general dysfunction of the intracellular metabolism of lipids such as sphingolipids (SL) (7Vanier M.T. Suzuki K. Brain Pathol. 1998; 8: 163-174Crossref PubMed Scopus (111) Google Scholar, 8Vanier M.T. Neurochem. Res. 1999; 24: 481-489Crossref PubMed Scopus (168) Google Scholar, 9Watanabe Y. Akaboshi S. Ishida G. Takeshima T. Yano T. Taniguchi M. Onho K. Nakashima K. Brain Dev. 1998; 20: 95-97Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 10Taniguchi M. Shinoda Y. Ninomiya H. Vanier M.T. Onho K. Brain Dev. 2001; 23: 414-421Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Spleen and liver of NPC patients accumulate not only Chol, but also glucosylceramide (GC), lactosylceramide, and sphingomyelin. Normal concentrations of Chol, but pathological levels of GC, lactosylceramide, GM2-ganglioside and asialo-GM2 in brain are typical findings (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Book Co., New York2001: 3611-3633Google Scholar). These observations indicate that the NPC1 protein may function in Chol and SL homeostasis. In normal cells, the SL are degraded in late endosomes/lysosomes by specific hydrolases. Some of these enzymes need the assistance of activator proteins such as saposins to exert their function (11O'Brien J. Kishimoto Y. FASEB J. 1991; 5: 301-308Crossref PubMed Scopus (309) Google Scholar, 12Kishimoto Y. Hiraiwa M. O'Brien J.S. J. Lipid Res. 1992; 33: 1255-1267Abstract Full Text PDF PubMed Google Scholar, 13Furst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (250) Google Scholar). Saposins are a group of four similar small glycoproteins, Sap A, B, C, and D, each of them stimulating the enzymatic degradation of specific SL. In fact, Sap B is required for the degradation of sulfatides by arylsulfatase A, and Sap C is required for the degradation of GC by glucosylceramidase (GCase) (14Wenger D.A. De Gala G. Williams C. Taylor H.A. Stevenson R.E. Pruitt J.R. Miller J. Garen P.D. Balentine J.D. Am. J. Med. Genet. 1989; 33: 255-265Crossref PubMed Scopus (42) Google Scholar, 15Schlote W. Harzer K. Christomanou H. Paton B.C. Kustermann-Kuhn B. Schmid B. Seeger J. Bendt U. Schuster I. Langenbeck U. Eur. J. Pediatr. 1991; 150: 584-591Crossref PubMed Scopus (60) Google Scholar, 16Christomanou H. Chabas A. Pampols T. Guardiola A. Klin. Wochenschr. 1989; 67: 999-1003Crossref PubMed Scopus (84) Google Scholar). The physiological role of saposins has been unequivocally demonstrated by the observation that SL storage diseases can be caused either by the deficiency of a specific hydrolase or of an individual saposin. For instance, Gaucher disease, a genetic disorder characterized by an extensive GC accumulation within the lysosomes of cells of monocyte/macrophage origin, can be caused by a deficit of either GCase or Sap C (16Christomanou H. Chabas A. Pampols T. Guardiola A. Klin. Wochenschr. 1989; 67: 999-1003Crossref PubMed Scopus (84) Google Scholar). In the Sap C-deficient cases of Gaucher disease, normal levels of GCase are unable to degrade GC. The role of Sap C in the enzymatic GC degradation has been examined in detail. In the past, we have provided compelling evidence that Sap C, at low pH values mimicking the acidic lysosomal environment, tightly binds to and destabilizes anionic phospholipid-containing membranes (17Vaccaro A.M. Ciaffoni F. Tatti M. 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). Upon affecting the physical organization of these membranes, Sap C promotes the association of GCase with the lipid surface, thus favoring the contact between the enzyme and its lipid substrate, GC (18Vaccaro 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, 19Salvioli R. Tatti M. Ciaffoni F. Vaccaro A.M. FEBS Lett. 2000; 472: 17-21Crossref PubMed Scopus (29) Google Scholar). Anionic phospholipids play a key role in the Sap C-promoted interaction of GCase with membranes; changes in the level and organization of these lipids can affect the topology and activity of GCase (18Vaccaro 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, 19Salvioli R. Tatti M. Ciaffoni F. Vaccaro A.M. FEBS Lett. 2000; 472: 17-21Crossref PubMed Scopus (29) Google Scholar). Markedly increased amounts of GC have been found not only in Gaucher disease, but also in visceral tissues and in brains from NPC patients (8Vanier M.T. Neurochem. Res. 1999; 24: 481-489Crossref PubMed Scopus (168) Google Scholar, 20Vanier M.T. Biochim. Biophys. Acta. 1983; 750: 178-184Crossref PubMed Scopus (154) Google Scholar). Because SLs such as GC are believed to be centrally involved in the pathogenesis of NPC disease (21Zervas M. Somers K.L. Thrall M.A. Walkley S.U. Curr. Biol. 2001; 11: 1283-1287Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), the mechanism of their accumulation and the properties of the hydrolases involved in the SL degradation have been extensively investigated. For instance, it has been found that the activities of GCase and sphingomyelinase are markedly reduced in NPC fibroblasts (22Besley G.T.N. Moss S.E. Biochim. Biophys. Acta. 1983; 752: 54-64Crossref PubMed Scopus (21) Google Scholar). Chol-mediated regulation of sphingomyelinase activity has been investigated (23Thomas G.H. Tuck-Muller C.M. Miller C.S. Reynolds L.W. J. Inherited Metab. Dis. 1989; 12: 139-151Crossref PubMed Scopus (38) Google Scholar, 24Reagan J.W. Hubbert M.L. Shelness G. J. Biol. Chem. 2000; 275: 38104-38110Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), whereas informations on the regulation mechanism of GCase in NPC cells are not available. It is important to fill this gap, because the accumulation of GC is very pronounced in several NPC tissues. The aim of our present work was to investigate the factors that might influence the GCase activity and stability in NPC cells. The possibility that the function of Sap C, a required cofactor for the enzymatic degradation of GC, might be altered in these cells was also taken into consideration and investigated. Materials—Complete™ (protease inhibitor mixture) was obtained from Roche Applied Science. Dulbecco's modified Eagle's medium (DMEM) was obtained from Euroclone Ltd, UK. [35S]methionine (Tran35S-Label™, 1175 Ci/mmol) and methionine/cysteine-deficient DMEM were obtained from ICN Biomedicals, Inc., Costa Mesa, CA. Lipoprotein-deficient bovine serum (LPDS) was obtained from Cocalico Biologicals, Inc. Filipin and protein A-Sepharose CL-4B were obtained from Sigma. Kodak X-Omat Blue films were from PerkinElmer Life Sciences. Prolong anti-fade kit was obtained from Molecular Probes (Eugene, OR). SDS-PAGE reagents were from Bio-Rad. ECL Western blotting reagents were from Amersham Bioscience, Buckinghamshire, UK. Cell Cultures—Two human fibroblast lines with previously described severe NPC1 mutations were used (25Millat G. Marçais C. Tomasetto C. Chikh K. Fensom A.H. Harzer K. Wenger D.A. Ohno K. Vanier M.T. Am. J. Hum. Genet. 2001; 68: 1373-1378Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The NPC1a cell line (81057) was homozygous for a Q775P mutation located in the sterol-sensing domain and shown to produce no detectable NPC1 protein by Western blot analysis. The NPC1b cell line (90089, affected sib of reported 87024) was homozygous for a V282fs mutation. Normal and NPC1 fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mm glutamine, 100 units/ml of penicillin, and 100 μg/ml streptomycin. For specific experiments, NPC1 cells were first grown in DMEM supplemented with 10% FBS and then subcultured in fresh medium containing 10% LPDS for the indicated periods of time. GCase Assay—To measure the GCase activity, lipid substrate GC, purified from Gaucher spleens, was utilized (26Vaccaro A.M. Muscillo M. Salvioli R. Tatti M. Gallozzi E. Suzuki K. FEBS Lett. 1987; 216: 190-194Crossref PubMed Scopus (6) Google Scholar). GC was labeled with tritium in the glucose moiety (27McMaster Jr., M.C. Radin N.S. J. Labelled Comp. Radiopharm. 1977; 13: 353-357Crossref Scopus (30) Google Scholar). The assay mixture contained in a final volume of 0.1 ml: 0.1/0.2 M citrate/phosphate buffer, pH 5.6, 10 μg of cell homogenate, 20 μg of GC supplemented with the 3H-labeled compound to a specific activity of 3000 dpm/nmol, 0.25% taurocholate, and 0.05% oleic acid. The assay mixtures were incubated for 1 h at 37 °C. The incubation was terminated by the addition of 0.4 ml of chloroform/methanol (2:1) and 50 μl of a 0.1% glucose solution. After shaking and centrifugation at 4000 rpm, the enzymatically released [3H]glucose present in the aqueous phase was estimated by radioactivity measurements. Antibodies—Mouse monoclonal (8E4) and rabbit polyclonal anti-GCase antibodies were kindly provided by Dr. H. Aerts, E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands. Rabbit anti-human Sap C antibody was prepared in our laboratory (17Vaccaro A.M. Ciaffoni F. Tatti M. 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). Mouse monoclonal anti-human lysosome-associated membrane protein type 1 (LAMP1) antibody, developed by Dr. J. T. August, was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa (Iowa City, IA). Mouse monoclonal anti-lysobisphosphatidic acid (LBPA) antibody (6C4) was a generous gift of Dr. J. Gruenberg, Department of Biochemistry, University of Geneva, Switzerland. The anti-actin monoclonal antibody was obtained from Sigma. Western Blotting—SDS-PAGE was performed with 10% acrylamide gels (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212847) Google Scholar). After electrophoresis, the proteins were electroblotted to polyvinylidene difluoride membranes (Bio-Rad), and GCase was detected with anti-GCase monoclonal antibody 8E4 using an ECL Western blotting kit, according to the manufacturer's instructions (Amersham Bioscience, Buckinghamshire, UK). Metabolic Labeling and Immunoprecipitation of GCase or Sap C— Skin fibroblast cultures were grown until they almost reached confluency. Prior to being labeled, the cells were washed twice with ice-cold PBS supplemented with 1 mm MgCl2 and 0.1 mm CaCl2 and starved for 2 h in methionine and cysteine-free medium containing 4% dialyzed FBS. This medium was replaced with the labeling medium (DMEM lacking methionine and cysteine and supplemented with [35S]methionine, 150 μCi/ml, and 4% dialyzed FBS). After a 1-h incubation, the cells were washed three times with DMEM and non-radioactive chase medium was added (DMEM containing 4% FBS). The cells were chased for the indicated periods and then harvested and disrupted in lysis buffer (0.5% Triton X-100 and a protease inhibitor mixture tablet/50 ml in 50 mm phosphate buffer, pH 6.5). The suspensions were subjected to brief sonication and centrifuged at 20,000 × g for 30 min. For removing DNA and histones, the supernatants were incubated with 0.03% protamine sulfate for 45 min at 4 °C and centrifuged as above. Constant values of total 35S-labeled cellular proteins were utilized for each experimental point. After the addition of 0.1% BSA, the cell lysates were incubated with rabbit preimmune serum overnight at 4 °C, and nonspecific complexes were precipitated with protein A-Sepharose CL-4B. The clarified supernatants were then incubated either with anti-GCase or anti-Sap C antiserum. Cross-reacting material was precipitated with protein A-Sepharose CL-4B. The immunocomplexes were washed four times with PBS containing 1% BSA, 1% Triton X-100, 1% SDS, 0.4% sodium deoxycholate, and then with only PBS. The washed precipitates were separated by SDS-PAGE. Labeled proteins were detected by fluorography. Fluorescence Microscopy—For fluorescence microscopy, the cells were grown on Labteck chamber slides (Nunc, Naperville, IL) and fixed with 4% paraformaldehyde in PBS for 30 min. Cells were then rinsed with PBS, permeabilized with 0.05% saponin for 7 min, and incubated with 3% bovine serum albumin for 2 h. For intracellular free unesterified Chol staining, fixed cells were incubated with filipin solution (0.05% in PBS) for 30 min. The cells were observed with a UV 330–380 filter. For double immunostaining, the cells were incubated for 1 h with a specific rabbit polyclonal primary antibody (anti-Sap C or polyclonal anti-GCase), rinsed twice with PBS, and incubated for 1 h with the secondary anti-rabbit antibody conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR). The cells were then rinsed twice with PBS, incubated for 1 h with a specific mouse monoclonal primary antibody (anti-GCase (8E4), anti-LAMP1, or anti-LBPA), rinsed twice with PBS, and incubated with the secondary anti-mouse antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Finally, the cells were mounted with ProLong antifade reagent (Molecular Probes) and observed with an Olympus BX52 fluorescence microscope equipped with appropriate filters. The images were acquired using the IAS 2000 software. When specified, the fluorescence was viewed by confocal laser-scanning microscopy using a Leica TCS 4D apparatus equipped with an argon-krypton laser, double-dichroic splitters (488/568 nm), 520-nm barrier filter for Alexa Fluor-488 (green), and 590-nm barrier filter for Alexa Fluor-594 (red) observations. Image acquisition and processing were conducted by using SCANware, Multicolor Analysis (Leica Lasertechnik, GmbH, Heidelberg, Germany), and Adobe Photoshop software programs. Signals from different fluorescent probes were taken in parallel, and colocalization was detected in yellow. Primary antibodies were used at the following dilutions: anti-Sap C (1:300), monoclonal anti-GCase (1:300), polyclonal anti-GCase (1:100), anti-LAMP1 (1:200), and anti-LBPA (1:80). GCase Activity and Mass Are Reduced in NPC Fibroblasts— GCase activity has been reported to be markedly diminished in NPC cells (22Besley G.T.N. Moss S.E. Biochim. Biophys. Acta. 1983; 752: 54-64Crossref PubMed Scopus (21) Google Scholar). As shown in Fig. 1, the GCase activity was ∼400 nmol/h/mg of protein in normal fibroblasts, whereas it was reduced to 75–100 nmol/h/mg protein (about 20% of the normal value) in cell lines from two NPC patients who lacked the NPC1 protein (NPC1a and NPC1b). Thus, much less functional GCase is present in NPC1 fibroblasts as a consequence of either a reduction or inactivation of the enzyme protein. To examine the first possibility, the GCase mass was analyzed by Western blotting using a monoclonal anti-GCase antibody. The intensity of the enzyme bands in both the NPC1 cell lines was ∼80% weaker than in normal fibroblasts (Fig. 1, inset), indicating that much less protein was present. Thus, the difference in activity between the control and the mutated cells correlates well with differences in the enzyme mass. GCase Activity and Mass Are Modulated by Chol Accumulation—To address the possibility that the decreased amount of GCase is related to the accumulation of endolysosomal free Chol, the NPC1 cells were grown in LPDS medium. It is known that NPC cells no longer accumulate Chol when cultured for more than 2 days in lipoprotein-free medium (1Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E. Neufeld E.B. Blanchette-Mackie J.E. Pentchev P.G. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Book Co., New York2001: 3611-3633Google Scholar). Accordingly, the free Chol level was dramatically reduced upon removal of low density lipoproteins, as indicated by the cytochemical filipin-staining of the NPC1 cells (data not shown). The GCase activity increased 3–4 times after 7 days of subculture with LPDS (Fig. 2A), and a parallel increase of the GCase protein was observed (Fig. 2B). When normal fibroblasts cells are grown in lipoprotein-free medium for 7 days, we observed a ∼20% increase of the GCase activity (from about 400 to 450–480 nmol/h/mg), whereas in NPC1 cells, the increase of activity was ∼250% (from about 100 to 300–350 nmol/h/mg). Thus, the level of free Chol in the endolysosomal system is able to modulate the level of GCase protein. Maturation of GCase in NPC1 Cells—To investigate at which step of maturation the amount of GCase decreased in the NPC1 cells, the biosynthesis and processing of the enzyme was examined by pulse-chase experiments (Fig. 3). According to previous findings (29Erickson A.H. Ginns E.I. Barranger J.A. J. Biol. Chem. 1985; 260: 14319-14324Abstract Full Text PDF PubMed Google Scholar, 30Jonsson L.M.V. Murray G.J. Sorrell S.H. Strijland A. Aerts J.F.G.M. Ginns E.I. Barranger J.A. Tager J.M. Schram A.W. Eur. J. Biochem. 1987; 164: 171-179Crossref PubMed Scopus (71) Google Scholar), a GCase precursor form (about 62 kDa) in normal fibroblasts was observed after a pulse of 1 h. A band at higher molecular mass (about 65 kDa) appeared after a 24-h chase. The fibroblast chased for 72 h contained an additional 58-kDa band of mature GCase. A similar pattern was observed in NPC1 fibroblasts. The densitometric quantitation of the intensity of the bands revealed that the amount of the 62-kDa precursor formed during a 1-h pulse was essentially the same in both control and NPC1 cells. In contrast, after a 72-h chase, much less GCase was detected in NPC1 than in control fibroblasts. These results indicate that the stability of the mature forms of GCase is markedly decreased in NPC1 cells. Maturation of Sap C in NPC1 Cells—Sap C, a small glycoprotein (about 10 kDa) derived from a large molecular mass precursor, prosaposin (65–70 kDa) (31Fujibayashi S. Wenger D.A. J. Biol. Chem. 1986; 261: 15339-15343Abstract Full Text PDF PubMed Google Scholar), is the specific activating and stabilizing factor of GCase (11O'Brien J. Kishimoto Y. FASEB J. 1991; 5: 301-308Crossref PubMed Scopus (309) Google Scholar). A possible cause of the GCase instability in NPC1 cells might be a reduced amount of Sap C. To test this hypothesis, the biosynthesis and maturation of Sap C have been examined. As shown in Fig. 4, the amount of the prosaposin 65- to 70-kDa forms detected after pulse-labeling for 1 h and the amount of Sap C generated after 72 h of chase were nearly the same in normal and NPC1 fibroblasts. Thus, the instability of GCase in NPC1 cells simply cannot be ascribed to a lack of Sap C. Nevertheless, it must be noted that during a chase of 5 h, about 50% of prosaposin was converted to the mature saposin in normal fibroblasts, although only 5–15% was cleaved in NPC1 cells. This finding, consistent upon repetition, indicates that the prosaposin processing was retarded in the mutated cells. Subcellular Localization of GCase, Sap C, and LBPA—Our previous findings showed that the activity of GCase is efficiently expressed only when the enzyme is bound to membranes containing anionic phospholipids (19Salvioli R. Tatti M. Ciaffoni F. Vaccaro A.M. FEBS Lett. 2000; 472: 17-21Crossref PubMed Scopus (29) Google Scholar, 32Vaccaro A.M. Tatti M. Ciaffoni F. Salvioli R. Maras B. Barca A. FEBS Lett. 1993; 336: 159-162Crossref PubMed Scopus (45) Google Scholar). Sap C, which preferentially interacts with these lipids (17Vaccaro A.M. Ciaffoni F. Tatti M. 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), in turn promotes the association of GCase with the lipid surface. According to this model, it can be expected that GCase, Sap C, and LBPA, the most abundant anionic phospholipid of the endolysosomal compartment (33Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (670) Google Scholar), colocalize in the same regions of the late endosomal/lysosomal membranes in control fibroblasts. As shown in Fig. 5, double-immunostaining revealed a complete colocalization of GCase and Sap C, as evident in the merged images. Moreover, all of the vesicular structures that contained GCase and Sap C also contained the anionic phospholipid LBPA. The late endosomal/lysosomal localization of GCase and Sap C was confirmed by the complete colocalization of the two proteins with LAMP1, a typical endolysosomal marker (Fig. 6).Fig. 6GCase and Sap C colocalize with LAMP 1 in normal fibroblasts. Normal human fibroblasts were double-immunostained for LAMP1 and either Sap C (top panels) or GCase (bottom panels), as described under "Experimental Procedures." All vesicles containing LAMP1 were also GCase-positive and Sap C-positive. Note that GCase was visualized with a polyclonal antibody (red, bottom left panel). Bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate whether an altered subcellular distribution might be responsible of the GCase instability, we performed the same immunofluorescence tests in NPC1 cells. As shown in Fig. 7, extensive storage of free Chol was observed in the two NPC1 cell lines (NPC1a and NPC1b), as visualized by the characteristic staining with filipin. Fig. 7 also shows that some cells staining for Chol were almost devoid of GCase. This observation was quantified by scoring NPC1a and NPC1b cells for GCase staining (n = 20 fields for each cell line). The enzyme was nearly absent in about 50% of the cells, an observation in keeping with the low amount of GCase found in the fibroblast homogenates (see Fig. 1). Immunofluorescence microscopy furthermore revealed that GCase distributed toward the periphery of vesicles in enlarged rings containing a heavy burden of Chol. The segregation of GCase toward the periphery of vesicular structures was constantly observed in NPC1 fibroblasts. In cells in which a significant amount of GCase was present, double-immunostaining showed the non-coincidence of the GCase distribution with those of LBPA and Sap C (Fig. 8). Also, the colocalization of Sap C with LBPA was impaired (Fig. 8). The intracellular distribution of GCase and Sap C was further defined by Laser scanning confocal microscopy. As shown in Fig. 9, the contact among GCase and its activating and stabilizing factor, Sap C, is rare in NPC1 cells, whereas the two proteins completely colocalize in control fibroblasts.Fig. 9Comparison of the GCase and Sap C localization in normal and NPC1 fibroblasts. NPC1 (top panels) and normal (bottom panels) fibroblasts were double-immunostained for GCase (green) and Sap C (red) and observed by laser scanning microscopy as described under "Experimental Procedures." The right panels show an enlargement of the regions outlined by the boxes in the overlaid panels. The comparison of the overlaid images clearly shows that, in normal fibroblasts, the intracellular vesicles are yellow, indicating that each contains both GCase and Sap C, whereas in NPC1 cells, most of the vesicles are either green or red, indicating that the two proteins reside in distinct vesicles. Bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the depletion of Chol results in a dramatic increase of both the GCase mass and activity (see Fig. 2), we have investigated whether a reduction of the Chol level could also re-establish the colocalization of GCase with Sap C. The NPC1 cells were cultured for 7 days in medium containing LPDS. After this time, the morphology of the cells changed, and the filipin staining was no more detectable. As shown in Fig. 10, the decrease of Chol storage actually restored the colocalization of GCase with Sap C. In addition to an impairment in Chol trafficking, the NPC cells are characterized by an extensive endolysosomal accumulation of SL. Previous studies have shown that the activity of sphingomyelinase and GCase, which are responsible of the degradation of two SLs present at high concentrations in NPC tissues, namely sphingomyelin and GC respectively, are markedly reduced (22Besley G.T.N. Moss S.E. Biochim. Biophys. Acta. 1983; 752: 54-64Crossref PubMed Scopus (21) Google Scholar). Our present results show that the reduction of GCase activity is paralleled by a decrease of th
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