Saposin B binds and transfers phospholipids
2006; Elsevier BV; Volume: 47; Issue: 5 Linguagem: Inglês
10.1194/jlr.m500547-jlr200
ISSN1539-7262
AutoresFiorella Ciaffoni, Massimo Tatti, Alessandra Boe, Rosa Salvioli, Arvan L. Fluharty, Sandro Sonnino, Anna Maria Vaccaro,
Tópico(s)Cellular transport and secretion
ResumoSaposin 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. 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. Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D, generated in late endosomes/lysosomes from a single precursor, prosaposin (1O'Brien J.S. Kishimoto Y. Saposin proteins: structure, function, and role in human lysosomal storage disorders.FASEB J. 1991; 5: 301-308Crossref PubMed Scopus (290) Google Scholar, 2Kishimoto Y. Hiraiwa M. O'Brien J.S. Saposins: structure, function, distribution, and molecular genetics.J. Lipid Res. 1992; 33: 1255-1267Abstract Full Text PDF PubMed Google Scholar). Because of their ability to modulate the lysosomal enzymatic degradation of several sphingolipids (SLs), the saposins play an important role in the pathogenesis of sphingolipidoses, a group of lysosomal storage disorders characterized by SL accumulation (1O'Brien J.S. Kishimoto Y. Saposin proteins: structure, function, and role in human lysosomal storage disorders.FASEB J. 1991; 5: 301-308Crossref PubMed Scopus (290) Google Scholar, 2Kishimoto Y. Hiraiwa M. O'Brien J.S. Saposins: structure, function, distribution, and molecular genetics.J. Lipid Res. 1992; 33: 1255-1267Abstract Full Text PDF PubMed Google Scholar). Mutations affecting the coding region of Sap B cause a variant form of metachromatic leukodystrophy with lysosomal storage of cerebroside sulfate (CS) (3Wenger D.A. De Gala G. Williams C. Taylor H.A. Stevenson R.E. Pruitt J.R. Miller J. Garen P.D. Balentine J.D. Clinical, pathological, and biochemical studies on an infantile case of sulfatide/GM1 activator protein deficiency.Am. J. Med. Genet. 1989; 33: 255-265Crossref PubMed Scopus (37) Google Scholar, 4Schlote W. Harzer K. Christomanou H. Paton B.C. Kustermann-Kuhn B. Schmid B. Seeger J. Bendt U. Schuster I. Langenbeck U. Sphingolipid activator protein 1 deficiency in metachromatic leukodystrophy with normal arylsulfatase A activity. A clinical, morphological, biochemical and immunological study.Eur. J. Pediatr. 1991; 150: 584-591Crossref PubMed Scopus (60) Google Scholar).The structural and functional properties of Sap B have been the focus of several studies. Sap B, like the other three saposins, consists of ∼80 amino acids, including six cysteines (2Kishimoto Y. Hiraiwa M. O'Brien J.S. Saposins: structure, function, distribution, and molecular genetics.J. Lipid Res. 1992; 33: 1255-1267Abstract Full Text PDF PubMed Google Scholar), forming three disulfide bridges (5Vaccaro A.M. Salvioli R. Barca A. Tatti M. Ciaffoni F. Maras B. Siciliano R. Zappacosta F. Amoresano A. Pucci P. Structural analysis of saposin C and B: complete localization of disulfide bridges.J. Biol. Chem. 1995; 270: 9953-9960Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In solution, Sap B is present as a dimer, which gives rise to higher order aggregates on increasing Sap B concentration (6Fisher G. Jatzkewitz H. The activator of cerebroside sulphatase. Purification from human liver and identification as a protein.Hoppe Seylers Z. Physiol. Chem. 1975; 356: 605-613Crossref PubMed Scopus (97) Google Scholar, 7Fluharty A.L. Meek W.E. Katona Z. Tsay K.K. The cerebroside sulfate activator from pig kidney: derivatization, cerebroside sulfate binding, and metabolic correction.Biochem. Med. Metab. Biol. 1992; 47: 86-96Crossref PubMed Scopus (11) Google Scholar). Crystallization has confirmed the dimer structure of Sap B, which consists of two clasped V-shaped monomers (8Ahn V.E. Faull K.F. Whitelegge J.P. Fluharty A.L. Priveé G.G. Crystal structure of saposin B reveals a dimeric shell for lipid binding.Proc. Natl. Acad. Sci. USA. 2003; 100: 38-43Crossref PubMed Scopus (160) Google Scholar).Because the lack of Sap B leads to the storage of SLs, especially CS (3Wenger D.A. De Gala G. Williams C. Taylor H.A. Stevenson R.E. Pruitt J.R. Miller J. Garen P.D. Balentine J.D. Clinical, pathological, and biochemical studies on an infantile case of sulfatide/GM1 activator protein deficiency.Am. J. Med. Genet. 1989; 33: 255-265Crossref PubMed Scopus (37) Google Scholar, 4Schlote W. Harzer K. Christomanou H. Paton B.C. Kustermann-Kuhn B. Schmid B. Seeger J. Bendt U. Schuster I. Langenbeck U. Sphingolipid activator protein 1 deficiency in metachromatic leukodystrophy with normal arylsulfatase A activity. A clinical, morphological, biochemical and immunological study.Eur. J. Pediatr. 1991; 150: 584-591Crossref PubMed Scopus (60) Google Scholar), the interaction of the saposin with SLs has been investigated extensively. It was shown that Sap B is able to solubilize in vitro several SLs such as CS, ganglioside GM1 (GM1), and globotriaosylceramide, whereas direct interaction with SL hydrolases has never been observed (9Vogel A. Schwarzmann G. Sandhoff K. Glycosphingolipid specificity of the human sulfatide activator protein.Eur. J. Biochem. 1991; 200: 591-597Crossref PubMed Scopus (65) Google Scholar, 10Li S.C. Kihara H. Serizawa S. Li Y.T. Fluharty A.L. Mayes J.S. Shapiro L.J. Activator protein required for the enzymatic hydrolysis of cerebroside sulfate.J. Biol. Chem. 1985; 260: 1867-1871Abstract Full Text PDF PubMed Google Scholar, 11Li S.C. Sonnino S. Tettamanti G. Li Y.T. Characterization of a non-specific activator protein for the enzymatic hydrolysis of glycolipids.J. Biol. Chem. 1988; 263: 6588-6591Abstract Full Text PDF PubMed Google Scholar, 12Wenger D.A. Inui K. Studies on the sphingolipid activator protein for the enzymatic hydrolysis of GM1 ganglioside and sulfatide.in: Brady R.O. Barranger J. Molecular Basis of Lysosomal Storage Disorders. Academic Press, New York1984: 61-78Crossref Google Scholar). It is thus assumed that the physiological function of Sap B is mainly related to its capacity to favor the degradative action of water-soluble SL hydrolases by binding and solubilizing SLs from lysosomal membranes. The resulting Sap B-SL complexes are more accessible substrates for the enzymes.It should be noted that several experiments concerning the in vitro interaction between Sap B and SLs have been performed with micellar dispersions. The crucial point to understand about the actual physiological function(s) of Sap B from lipid binding experiments is that the saposin behaves in vitro as it would in vivo. To elucidate in more detail the lipid binding properties of Sap B, we have reexamined its interaction with lipids by inserting SLs into models of biological membranes such as large unilamellar vesicles (LUVs) composed of phospholipids and cholesterol (Chol), the main components of the membranes where SLs reside. Special attention has been paid to the Sap B interaction with anionic phospholipids, because they are very abundant in the late endosomal/lysosomal compartment (13Kobayashi T. Beuchat M.H. Chevallier J. Makino A. Mayran N. Escola J.M. Lebrand C. Cosson P. Kobayashi T. Gruenberg J. Separation and characterization of late endosomal membrane domains.J. Biol. Chem. 2002; 35: 32157-32164Abstract Full Text Full Text PDF Scopus (283) Google Scholar) where saposins also are localized. Anionic phospholipids have been shown to be the target of two other saposins, Sap C and Sap D (14Vaccaro A.M. Salvioli R. Tatti M. Ciaffoni F. Saposins and their interaction with lipids.Neurochem. Res. 1999; 24: 307-314Crossref PubMed Scopus (100) Google Scholar, 15Vaccaro A.M. Ciaffoni F. Tatti M. Salvioli R. Barca A. Tognozzi D. Scerch C. pH-dependent conformational properties of saposins and their interactions with phospholipid membranes.J. Biol. Chem. 1995; 270: 30576-30580Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 16Ciaffoni F. Tatti M. Salvioli R. Vaccaro A.M. Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids.Biochem. J. 2003; 373: 785-792Crossref PubMed Scopus (15) Google Scholar). Moreover, anionic phospholipids have been reported to inhibit the formation of the Sap B-CS complex, a demonstration of the capacity of Sap B to interact also with phospholipids (17Fluharty C.B. Johnson J. Whitelegge J. Faull K.F. Fluharty A.L. Comparative lipid binding study on the cerebroside sulfate activator (saposin B).J. Neurosci. Res. 2001; 63: 82-89Crossref PubMed Scopus (18) Google Scholar, 18Fisher G. Jatzkewitz H. The activator of cerebroside sulphatase. A model of the activation.Biochim. Biophys. Acta. 1978; 528: 69-76Crossref PubMed Scopus (63) Google Scholar). The results presented here reveal that in the presence of vesicles mimicking the lipid composition of biological membranes, Sap B preferentially interacts with phospholipids, especially anionic phospholipids.MATERIALS AND METHODSPhosphatidylcholine (PC) from egg yolk, GM1 from porcine brain, phosphatidylinositol (PI) from bovine liver, phosphatidylethanolamine (PE) from chicken egg, and cardiolipin (CL) from bovine heart were from Avanti Polar Lipids, Inc. (Alabaster, AL). Chol was from Sigma. PC, 1,2-di[1-14C]palmitoyl (110 mCi/mmol), and Chol[7(n)-3H] (7 Ci/mmol) were from Amersham Biosciences (Buckinghamshire, UK). CS, bovine (stearoyl-sodium salt), and CS (stearoyl 1-14C) (50 mCi/mmol) were from American Radiolabeled Chemicals, Inc. (St. Louis, MO). PI, l-α-[myo-inositol-2-3H(N)] (8.5 Ci/mmol), cholesteryl oleate (oleate-1-14C) (55 mCi/mmol), and cholesteryl oleate [cholesteryl-1,2,6,7-3H(N)] (60–100 Ci/mmol) were from Perkin-Elmer Life Sciences (Boston, MA). [3H]GM1 was prepared by labeling the ganglioside in the terminal galactose moiety (19Ghidoni R. Tettamanti G. Zambotti V. An improved procedure for the in vitro labeling of ganglioside.Ital. J. Biochem. 1974; 23: 320-328PubMed Google Scholar). All other chemicals were of the purest available grade.Sap B preparationHuman Sap B was purified from spleens of patients with type 1 Gaucher's disease according to a previously reported procedure (5Vaccaro A.M. Salvioli R. Barca A. Tatti M. Ciaffoni F. Maras B. Siciliano R. Zappacosta F. Amoresano A. Pucci P. Structural analysis of saposin C and B: complete localization of disulfide bridges.J. Biol. Chem. 1995; 270: 9953-9960Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar); it involved essentially the heat treatment of a water homogenate followed by ion-exchange chromatography on DEAE-Sephacel, gel filtration FPLC on a Superdex 75 HR 10/30 column (Amersham Biosciences), and reverse-phase high-pressure liquid chromatography on a protein C4 column (Vydac). Sap B was also purified from pig kidney according to a published protocol (20Fluharty A.L. Katona Z. Meek W.E. Frei K. Fowler A.V. The cerebroside sulfate activator from pig kidney: purification and molecular structure.Biochem. Med. Metab. Biol. 1992; 47: 66-85Crossref PubMed Scopus (20) Google Scholar). The purity of the Sap B preparations was verified by N-terminal sequence analysis, SDS-PAGE, and electrospray mass spectrometry (5Vaccaro A.M. Salvioli R. Barca A. Tatti M. Ciaffoni F. Maras B. Siciliano R. Zappacosta F. Amoresano A. Pucci P. Structural analysis of saposin C and B: complete localization of disulfide bridges.J. Biol. Chem. 1995; 270: 9953-9960Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar).Vesicle preparationLUVs were prepared as reported previously (15Vaccaro A.M. Ciaffoni F. Tatti M. Salvioli R. Barca A. Tognozzi D. Scerch C. pH-dependent conformational properties of saposins and their interactions with phospholipid membranes.J. Biol. Chem. 1995; 270: 30576-30580Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 16Ciaffoni F. Tatti M. Salvioli R. Vaccaro A.M. Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids.Biochem. J. 2003; 373: 785-792Crossref PubMed Scopus (15) Google Scholar). Briefly, appropriate amounts of lipids dissolved in chloroform were mixed, and the solvent was evaporated under nitrogen. The resulting lipid films were dried overnight in vacuum desiccators. The dry lipids were dispersed by vortex-mixing in buffer A (2 mM l-histidine, 2 mM N-Tris Hydroxymethyl Methyl-2-Aminoethane Sulfonic Acid, 50 mM NaCl, and 1 mM EDTA, pH 7.4) followed by 10 cycles of freeze-thawing; finally, they were passed 21 times through two stacked 0.1 μm diameter pore polycarbonate membranes in a Liposofast-Miniextruder (Avestin, Ottawa, Canada). The vesicles were supplemented with the specified labeled lipids. PC was supplemented with [14C]PC to a specific activity of 8.5 × 103 dpm/nmol. PI, GM1, Chol, and CS were supplemented with [3H]PI, [3H]GM1, [3H]Chol, and [14C]CS, respectively, to a specific activity of ∼24 × 103 dpm/nmol each.Multilamellar vesicles (MLVs) were prepared by mixing appropriate amounts of lipids dissolved in chloroform and evaporating the solvent under nitrogen. The resulting lipid films were kept overnight under vacuum. The dry lipids were dispersed by vortex-mixing in buffer A. The suspension was blended by a vortex mixer until all of the aggregates were dispersed. The vesicles were centrifuged at 40,000 g for 15 min at 4°C. The supernatant was discarded, and the MLV pellet, gently resuspended in buffer A, was used for phospholipid transfer experiments (see below).Small unilamellar vesicles (SUVs) were prepared from MLVs by sonication under nitrogen in a Branson B15 sonifier at 0°C for 10 min with a microtip at a power setting of 30 W (60 s sonication, 30 s pause). The preparation was centrifuged at 100,000 g for 30 min, and the supernatant was used for phospholipid transfer experiments (see below).Gel-permeation experimentsTo test the Sap B-induced solubilization of lipids, LUVs (50 nmol, total lipids) with or without Sap B (2 nmol) were incubated in 100 μl of buffer B (10 mM acetate, 50 mM NaCl, and 1 mM EDTA, pH 4.2) at 37°C for 30 min. The reaction was arrested by adjusting the pH of the solution to 7.4 with diluted NaOH, and the mixture was applied to a Sepharose CL-4B column (1 × 30 cm) preequilibrated and eluted at room temperature with buffer A. The flow rate was 0.5 ml/min. Fractions of 0.5 ml were collected. The lipid distribution was determined by measuring the radioactivity of the fractions. When two labeled lipids were present, double isotope counting conditions were adopted. To confirm the identity of the lipids eluted from the Sepharose CL-4B column, the fractions were extracted with chloroform-methanol (2:1, v/v) and analyzed by TLC on high-performance TLC plates (Silica gel 60; Merck). Plates were developed in chloroform-methanol-water (95:5:5, v/v/v). Radioactive lipids separated on TLC plates were visualized by autoradiography.To determine the pattern of Sap B elution from the Sepharose CL-4B column, the fractions were concentrated with a Microcon-YM-3 centrifugal filter device (molecular mass cutoff, 3 kDa) by centrifuging at 13,000 g until all of the liquid except 100 μl was passed through the filter. The presence of Sap B in each retentate was tested by SDS-PAGE analysis of an aliquot of the concentrated fractions. SDS-PAGE was performed with a 10% polyacrylamide gel (21Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). After electrophoresis, Sap B was detected with a silver-staining kit for proteins (SilverQuest™; Invitrogen, Carlsbad, CA).Assay of Sap B intermembrane phospholipid transfer activityThe phospholipid transfer activity of Sap B was determined by measurement of intermembrane transfer of labeled phospholipids between donor SUVs and acceptor MLVs essentially as described by DiCorleto et al. (22DiCorleto P.E. Fakharzadeh F.F. Searles L.L. Zilversmit D.B. Stimulation by acidic phospholipids of protein-catalyzed phosphatidylcholine transfer.Biochim. Biophys. Acta. 1977; 468: 296-304Crossref PubMed Scopus (19) Google Scholar, 23DiCorleto P.E. Zilversmit D.B. Protein-catalyzed exchange of phosphatidylcholine between sonicated liposomes and multilamellar vesicles.Biochemistry. 1977; 16: 2145-2150Crossref PubMed Scopus (66) Google Scholar, 24DiCorleto P.E. Warach J.B. Zilversmit D.B. Purification and characterization of two phospholipid exchange proteins from bovine heart.J. Biol. Chem. 1979; 16: 7795-7802Abstract Full Text PDF Google Scholar). SUVs consisting of PI/PC (5:95) were supplemented with either [3H]PI or [14C]PC to a specific activity of 24 × 103 or 2 × 103 dpm/nmol, respectively. SUVs also contained a trace of cholesteryl oleate, labeled with either 14C or 3H. MLVs consisted of PC/PE/CL (70:25:5). The phospholipid transfer incubations contained, in a total volume of 400 μl of buffer B, pH 4.5, 0.2 nmol of Sap B (unless specified otherwise), SUVs (20 nmol, total lipids), and a large excess of MLVs (1,600 nmol, total lipids). The phospholipid transfer was arrested by adjusting the pH of the solution to 7.4 with diluted NaOH. MLVs were pelleted by centrifugation at 40,000 g at 4°C for 15 min. SUVs were quantitatively recovered in the supernatant, as judged by the recovery of labeled cholesteryl oleate, a nontransferable marker (>98%). SUVs used for PI transfer evaluation were supplemented with [3H]PI and [14C]cholesteryl oleate. The decrease in the 3H/14C ratio after incubation measured the transfer of PI from SUVs to MLVs. SUVs used for PC transfer evaluation were supplemented with [14C]PC and [3H]cholesteryl oleate. The decrease in the 14C/3H ratio after incubation measured the transfer of PC from SUVs to MLVs. The small amount of transfer ( 5.0 the interaction between lipids and Sap B decreased markedly.Fig. 3Lipid extraction from LUVs as a function of the amount of Sap B, of time, and of pH. Sap B was incubated with LUVs (50 nmol, total lipids) composed of PI/PC/Chol/GM1 (10:60:20:10) and then chromatographed on a Sepharose CL-4B column as described for Fig. 1A. LUVs were supplemented with [14C]PC (closed squares), [3H]PI (closed circles), or [3H]GM1 (closed triangles), and lipid extraction was calculated as shown in Table 1. A: Increasing amounts of Sap B were incubated with LUVs in buffer B, pH 4.2, for 30 min. B: Sap B (2 nmol) was incubated with LUVs in buffer B, pH 4.2, for different periods of time. C: Sap B (2 nmol) was incubated with LUVs in buffer B, appropriately adjusted in the range from pH 4.2 to 5.5, for 30 min. For pH 6.0, the buffer was 10 mM Na-citrate, 1 mM EDTA, and 50 mM NaCl. Each point represents the mean of at least three replicates ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Sap B-induced solubilization of CS, whose lysosomal degradation is modulated by Sap B (3Wenger D.A. De Gala G. Williams C. Taylor H.A. Stevenson R.E. Pruitt J.R. Miller J. Garen P.D. Balentine J.D. Clinical, pathological, and biochemical studies on an infantile case of sulfatide/GM1 activator protein deficiency.Am. J. Med. Genet. 1989; 33: 255-265Crossref PubMed Scopus (37) Google Scholar, 4Schlote W. Harzer K. Christomanou H. Paton B.C. Kustermann-Kuhn B. Schmid B. Seeger J. Bend
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