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Stimulation of lysosomal sphingomyelin degradation by sphingolipid activator proteins
Chemistry and Physics of Lipids 102 (1999) 35 – 43 www.elsevier.com/locate/chemphyslip
Stimulation of lysosomal sphingomyelin degradation by sphingolipid activator proteins Klaus Ferlinz, Thomas Linke, Oliver Bartelsen, Manfred Weiler, Konrad Sandhoff * Kekule´ -Institut fu¨r Organische Chemie und Biochemie, Uni6ersita¨t Bonn, Gerhard-Domagk-Str. 1, D 53121 Bonn, Germany
Abstract Lysosomal breakdown of glycosphingolipids with short hydrophilic carbohydrate headgroups is achieved by the simultaneous action of specific hydrolases and sphingolipid activator proteins (SAPs). Activator proteins are considered to facilitate the enzyme/substrate interaction between water-soluble enzymes and membrane-bound substrates. Sphingomyelin, containing the small hydrophilic phosphorylcholine moiety, is hydrolysed by acid sphingomyelinase (acid SMase). Recent experimental data on the in vivo and in vitro role of activator proteins in sphingomyelin breakdown by acid SMase are reviewed. These data combined with the results using homogenous protein preparations as well as a liposomal assay system mimicking the physiological conditions suggest that lysosomal sphingomyelin degradation is not critically dependent on any of the known activator proteins. Moreover, evidence is provided that the assumed intramolecular activator domain of acid SMase and especially the presence of negatively charged lipids in the lysosomes are sufficient for sphingomyelin turnover. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acid sphingomyelinase; Sphingolipid activator proteins; Intra-lysosomal vesicles
1. Lysosomal sphingolipid catabolism Glycosphingolipids and sphingomyelin are major constituents of the outer leaflet of eukaryotic membranes. They consist of a hydrophobic ceramide moiety which anchors the sphingolipid in the cell membrane and a hydrophilic headgroup extruding into the extracellular space. More than 300 different glycosphingolipids which differ in * Corresponding author. Tel.: +49-228-73-5346; fax: +49228-73-7778. E-mail address: [email protected] (K. Sandhoff)
number, type and linkage of individual sugar residues, have yet been identified from natural sources. Although the molecular structure as well as their biosynthetic and catabolic pathways have been intensively studied, the physiological role of these complex cell surface compounds is still matter of debate. The glycosphingolipid pattern appears to be dynamic and highly dependent on cell type, cell growth and differentiation, ontogenesis and transformation. Several lines of evidence suggest that glycosphingolipids are essentially involved in cell recognition and adhesion processes during embryogenesis and development as well as
0009-3084/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 9 9 ) 0 0 0 7 3 - 0
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K. Ferlinz et al. / Chemistry and Physics of Lipids 102 (1999) 35–43
certain signal transduction mechanisms (Hakomori and Igarashi, 1995). In contrast, sphingomyelin, the predominant sphingolipid in the outer leaflet of plasma membranes, has originally been considered to be particularly involved in the maintenance of biophysical properties of the plasma membrane. During the past years interest in sphingomyelin metabolism has increased since it turned out that sphingomyelin is the immediate educt for the generation of the signalling molecule ceramide (Spiegel et al., 1996; Hannun and Obeid, 1997). The involvement of sphingomyelinases, ceramide as well as sphingosin and its metabolites in regulation of cell death and differentiation is also extensively reviewed in this edition. The bulk membrane components of eukaryotic cells are continuously internalized by endocytotic processes and either degraded and or recycled back to the membrane. A recent model of topology for sphingolipid hydrolysis suggests that plasma membrane components are selectively internalized by vesicular flow and trafficked toward the acidic compartments (Fig. 1). Maturation of endosomal membranes results in the formation of intra-endo/lysosomal vesicles and other membrane structures which finally serve as substrate for acidic hydrolases (Fu¨rst and Sandhoff, 1992). The lysosomal perimeter membrane itself seems to be protected against hydrolysis by a highly inert glycoprotein derived glycocalix which covers the lumenal surface of the limiting lysosomal membrane. Lysosomal degradation of glycosphingolipids and sphingomyelin is achieved by the action of a variety of substrate-specific exohydrolases which sequentially cleave off terminal sugars or, in the case of sphingomyelin, phosphorylcholine (Fig. 2) (Kolter and Sandhoff, 1997).
protein) (see Fig. 2). Some SAPs are thought to be particularly involved in the solubilization of respective sphingolipids from the lipophilic membrane making them susceptible to the water soluble hydrolases (Giehl et al., 1998; Wilkening et al., 1999). Long hydrophilic carbohydrate structures which sufficiently protrude into the aqueous phase are readily cleaved by the respective enzyme independently of SAPs. Four of the five different SAPs A–D which have been characterized to date are encoded by a common gene. Translation results in the biosynthesis of a SAP precursor (prosaposin) which is proteolytically processed to the active SAP A–D inside the acidic organelles (Vielhaber et al., 1996; Hiesberger et al., 1998). Although these co-factors exhibit several highly conserved structural features, e.g. the array of disulphide bridges and a conserved site of N-glycosylation, their ligand and/or enzyme binding
2. Sphingolipid activator proteins (SAPs) in sphingolipid breakdown In vivo hydrolysis of sphingolipids with short hydrophilic headgroups (5 3 sugar residues in a row) requires additional non-enzymatic co-factors of low molecular weight, so called SAPs (or saposins), of which five different have yet been characterized (SAPs A – D and the GM2-activator
Fig. 1. Topology of lysosomal sphingoipid degradation. Maturation of internalized membrane structures results in the formation of intra-endo/lysosomal vesicles and other membrane structures which finally serve as substrates for acidic hydrolases. The lysosomal perimeter membrane itself seems to be protected against hydrolysis by a highly inert glycoprotein derived glycocalix which covers the lumenal surface of the limiting lysosomal membrane.
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Fig. 2. Scheme of major sphingolipid hydrolysis. Exohydrolytic breakdown of sphingolipids with short hydrophilic headgroups requires non-enzymatic co-factors, sphingolipid activator proteins (SAPs). Both, inherited deficiencies of the respective enzyme as well as of the corresponding activator protein causes lysosomal lipid storage and results in the expression of various sphingolipidoses.
specificity is quite different but not absolute (see Fig. 2). The physiological substrate specificity of lysosomal sphingolipid hydrolases as well as of SAPs has most extensively been investigated taking advantage of the naturally occurring lysosomal sphingolipidoses. Deficiency of any of the numerous sequentially acting exohydrolases leads to a sphingolipid storage disease which is characterized by a massive and specific lysosomal sphingolipid accumulation (Gieselmann, 1995). Rare variants of sphingolipidoses are caused by molecular deficiency of SAPs which are predominantly accompanied by a mild juvenile clinical phenotype
(Sandhoff et al., 1995). The sphingolipid storage pattern resembles those of patient cells with the respective enzyme deficiency directing to the physiological role of the individual activator proteins. However, lipid storage in activator protein deficient cells is restricted to sphingolipids with short hydrophilic headgroups (Fig. 2). More recently, several activator protein deficient knock-out mouse strains have been generated in order to get detailed information on the development and onset of respective lipid storage diseases and furthermore, to develop strategies for future therapeutic approaches (Fujita et al., 1996; Liu et al., 1997).
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3. Lysosomal sphingomyelin breakdown The bulk of membrane sphingomyelin is hydrolysed by the lysosomal enzyme acid sphingomyelinase (acid SMase) (Quintern et al., 1989). Sphingomyelin consists of a ceramide membrane anchor which is linked to a short hydrophilic phosphorylcholine moiety suggesting that in vivo degradation by acid SMase in principle could also depend on the co-action of any of these activator proteins. However, the importance of the SAPs for sphingomyelin degradation remained unclear due to contradictory results obtained from in vivo and in vitro experiments published earlier. Those in vivo results are based on the lipid analysis of a patient and his fetal sibling with a complete absence of SAPs due to a homoallelic mutation in the initiation codon of the SAP-precursor (prosaposin) gene (multiple SAP deficiency) (Harzer et al., 1989; Paton et al., 1990, 1992; Schnabel et al., 1992). Densitometric analysis of kidney and liver lipids of the fetus revealed no significant elevation of sphingomyelin levels compared to fetal control kidneys. Furthermore, these findings were corroborated in a mouse model, in which the prosaposine gene was knocked out (SAP knock-out) (Fujita et al., 1996). Again, no significant abnor-
malities in sphingomyelin levels were found in kidney and liver of SAP deficient mice. The apparent lack for sphingomyelin storage in human multiple SAP deficient tissues was supported by detection of normal acid SMase activity levels in fibroblasts from both the patient and his fetal sibling compared to healthy normal controls when tested in a detergent-free, liposomal assay system (Bradova` et al., 1993). Acid SMase activity levels in SAP knock-out mice were obviously reduced by about 40% compared to healthy controls by so far unknown reasons (Fujita et al., 1996). In contrast to the in vivo findings the stimulation of acid SMase by SAP B, C and D in in vitro detergent-based assay systems has been repeatedly reported (Poulos et al., 1984; Christomanou and Kleinschmidt, 1985; Christomanou et al., 1986; Morimoto et al., 1988). Only one study by Tayama et al. (1993) has been performed to date in which sphingomyelin hydrolysis catalysed by acid SMase and the different SAPs is analysed in more detail. The effect of SAP-D on acid SMase activity in a micellar assay system was found to be dependent on the concentration of the detergent Triton X-100: increasing amounts of SAP-D were shown to stimulate detergent containing, crude acid SMase (concentration of Triton X-100: \
Fig. 3. Effect of the four different sphingolipid activator proteins (SAPs) on sphingomyelin degradation. Acid sphingomyelinase (acid SMase) was assayed with liposomal sphingomyelin in the presence of the different activator proteins (2.5 mg each). The liposomes (70 mol% phosphatidylcholine, 20 mol% cholesterol and 10 mol% sphingomyelin) were prepared by repeated extrusion through a polycarbonate membrane with a pore size of 100 nm. The assay mixture contained a 10 mM sodium acetate buffer, pH 4.5, 150 mM sodium chloride and a total lipid concentration of 1 mM in an incubation volume of 100 ml. The assay mixture was incubated for 2 h at 37°C.
K. Ferlinz et al. / Chemistry and Physics of Lipids 102 (1999) 35–43
0.04% w/v), whereas partially purified, detergentfree acid SMase was inhibited by presence of SAP-D (concentration of Triton X-100: B 0.04% w/v). Contrary to the influence of SAP-D, SAP-A, B and C were rather inhibitory or without any effect on the SMase activity (Tayama et al., 1993). When sphingomyelin was presented in liposomes in absence of Triton X-100 acid SMase activity was stimulated in the order SAP-C \SAP-D \ SAP-A. SAP-B exhibited an inhibitory effect on sphingomyelin degradation; furthermore, the degree of stimulation by SAP A, C and D was decreased by the simultaneous addition of SAP-B (Tayama et al., 1993). Recent computational analysis of the acid SMase sequence in terms of structural homology comparison suggests that the enzyme possibly contains its own intramolecular activator protein domain (Ponting, 1994; Munford et al., 1995). The N-terminal portion of acid SMase polypeptide (amino acids 89 – 165) closely resembles sequence data obtained from SAPs, including positions of cystein residues and several hydrophobic amino acid domains, pointing to a related secondary structure which may well function as a built-in activator protein domain. This would explain — at least in part — the unusually high specific molecular acid SMase in vitro activity on its endogenous lipid substrate (see also Fig. 3). Regarding these putative intramolecular activating properties, one would also expect that in vivo sphingomyelin breakdown by the acid SMase does not critically depend on any additional activator protein which is in line with the lipid data from SAP knock-out mice.
4. Experimental data Earlier studies on activator protein assisted sphingomyelin breakdown, as discussed above, were generally conducted using either crude acid SMase preparations or partially purified enzyme from human urine (Christomanou and Kleinschmidt, 1985) or liver (Morimoto et al., 1988) and do not exactly reflect the physiological role of SAP stimulated sphingomyelin breakdown. Therefore, it was decided to test the highly purified SAPs from human spleen in combination
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with recombinant human acid SMase (rSMase) in a detergent-free liposomal sphingomyelin assay system. The limiting factor of these studies turned out to be the availability of preparative amount of homogenous, enzymatically active acid SMase. In order to circumvent the low natural abundance of acid SMase as well as any critical contaminants a baculovirus expression system for the polyhedrin promotor controlled expression of recombinant human acid SMase in Spodoptera frugiperda (Sf 21) insect cells was established (Bartelsen et al., 1998). Acid SMase-cDNA was inserted into the genome of an AcMNPV baculovirus by allelic replacement. After selection and amplification of recombinant virus, acid SMase was immunochemically and enzymatically detected in cell lysates and media. Almost 80% of recombinant enzyme was secreted. Purification from the culture media was achieved by conventional column chromatography as described recently (Lansmann et al., 1996) and led to a rSMase preparation of apparent homogeneity. Enzymatic comparison of rSMase with immune-affinity purified homogenous placental enzyme indicated that the specific activity of the insect cell derived protein was lower by the factor of 2 whereas the km value was almost identical. Edman sequencing of the N-terminal peptide sequence revealed that the recombinant N-terminus is extended by 23 amino acid residues compared to the lysosomal placenta enzyme (Lansmann et al., 1996). Thus, both proteins, the recombinant and the native placental contain the complete SAP-like N-terminal domain. SAPs used in the studies were purified from human spleen of Gaucher patients essentially as described by Sano et al. in order to obtain sufficient amounts of the individual activator protein (Sano et al., 1988). The purification and separation of individual SAPs A–D is only achieved with considerable difficulty due to their high degree of homology and similar biophysical and biochemical properties. In order to exclude any cross-contamination of SAPs the purity of the final activator protein preparation was tested by Western blotting and MALDI mass spectrometry.
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Fig. 4. Relationship between the rate of sphingomyelin hydrolysis and the molecular percentage of phosphatidylinositol (PI) in the liposomes. Liposomes were prepared and the assay conducted as described in the legend of Fig. 3. Phosphatidylcholine was replaced by increasing amounts of PI.
Homogenous recombinant acid SMase was assayed in two different liposomal sphingomyelin assay systems in the presence of the individual activator proteins. Preliminary results presented herein are means of two independent duplicates (Fig. 3). It was found that SAP-A and SAP-C were the strongest acid SMase stimulators under the above conditions, while SAP-B and SAP-D showed neither a high degree of stimulation nor inhibition of acid sphingomyelin breakdown activity (Fig. 3) (Linke et al., 1998). However, it was observed that both SAP-C and D became more effective stimulators of acid SMase activity, when the liposomes were prepared by ultrasonic irradiation thus producing smaller sized liposomes with a higher degree of curvature (Linke et al., 1998). In order to render the assay conditions more physio-
logically substrate liposomes were designed which mimic intra-endo/lysosomal membrane structures. The lipid composition of late endosomal and lysosomal compartments are characterized by the presence of negatively charged phospholipids such as bis(monoacylglycero)phosphate (BMP) and phosphatidylinositol (PI) (Kobayashi et al., 1998; Wilkening et al., 1999). It was therefore tested whether such lipids could stimulate sphingomyelin breakdown. The overall rate of sphingomyelin hydrolysis was increased dramatically when an acidic phospholipid such as PI was included in the liposomal preparation even without addition of any activator protein (Fig. 4). As illustrated in Fig. 4 the rate of sphingomyelin breakdown increases with increasing negative surface charge of the liposomes. The stimulation of sphingomyelin degradation activity
K. Ferlinz et al. / Chemistry and Physics of Lipids 102 (1999) 35–43
by SAP-D also becomes more pronounced in the presence of anionic lipids, but never reaching the degree of stimulation as observed by SAP-C. The above results suggest that SAPs may indeed stimulate sphingomyelin breakdown by acid SMase under liposomal assay conditions. The observed lack of specificity of the activators on the other hand suggest that the SAPs may affect the physical nature of the liposome bound substrate rather than interacting specifically with acid SMase.
5. Discussion Data obtained from SAP deficient knock-out mice and multiple SAP deficient human tissue combined with the experimental data discussed in this article suggest that presumably the combination of all, the proposed intramolecular SMase activator domain and the SAPs as well as the presence of lysosomal anionic phospholipids (e.g. BMP) maximally stimulate enzymatic sphingomyelin degradation. Even without additional SAPs, sphingomyelin turnover by acid SMase was increased by the factor of approximately 10 using negatively charged substrate liposomes. On the other hand, stimulation by individual SAPs in the absence of acid phospholipids was approximately 5-fold for SAP-C and only 2-fold for SAP-D. Therefore, complete SAP deficiency is obviously not leading to impaired sphingomyelin turnover due to the compensatory effect of the remaining two factors, intramolecular activator domain and negatively charged vesicle surface, which still allow normal sphingomyelin catabolism. In this context it should be noted that none of the known mutations causing Niemann – Pick disease, the inherited acid SMase deficiency, is located within the predicted activator domain whereas several different mutations have been detected downstream the sequence (Schuchman and Desnick, 1995). One hypothesis would be that, opposite to the multiple SAP deficiency, defects within the intramolecular activator domain can be overcome in vivo by the synergistic action of SAPs and acid phospholipids, thus avoiding lysosomal sphingomyelin storage. Possible mutations in the acti-
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vator-like domain encoding sequence which do not result in a pathological phenotype may escape molecular analysis. Ida et al. (1993) published the identification of an acid SMase cDNA which was cloned from a placental cDNA library and contained a single base change within the sequence encoding the putative activator domain of the mature polypeptide. Intriguingly, this presumed cloning artefact resulted in the substitution of arginine for the normal cystein, one of the homologous cystein residues conserved through all activator protein(-like) domains and reduced in vitro activity of mutant sphingomyelinase almost completely. However, there is no in vivo evidence that a non-functional acid SMase activator domain causes lysosomal sphingomyelin storage. Taking these observations together it can be concluded that lysosomal sphingomyelin degradation by acid SMase is stimulated on the one hand by negatively charged intra-endo/lysosomal membrane structures as well as by intramolecular and/ or by separate activator proteins (most potently by SAP-C) which may be capable of substituting each other. Further studies including mutational in vivo analysis of the acid SMase SAP-like domain as well as studies on the sphingomyelin binding capacity of recombinant acid SMase are in progress to thoroughly define the factors required for lysosomal sphingomyelin turnover.
Acknowledgements We thank H. Moczall for excellent technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 400, A5).
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Linke, T., Wilkening, G., Moczall, H., Lansmann, S., Bartelsen, O., Sandhoff, K., 1999. Influence of lysosomal components on sphingomyelin hydrolysis in a detergentfree system (in preparation). Liu, Y., Hoffmann, A., Grinberg, A., Westphal, H., McDonald, M.P., Miller, K.M., Crawley, J.N., Sandhoff, K., Suzuki, K., Proia, R.L., 1997. Mouse model of GM2 activator deficiency manifests cerebellar pathology and motor impairment. Proc. Natl. Acad. Sci. USA 94, 8138 – 8143. Morimoto, S., Martin, M., Kishimoto, Y., O’Brien, J.S., 1988. Saposin D: a sphingomyelinase activator. Biochem. Biophys. Res. Commun. 156, 503 – 510. Munford, R.S., Sheppard, P.O., O’Hara, P.J., 1995. Saposinlike proteins (SAPLIP) carry out diverse functions on a common backbone structure. J. Lipid Res. 36, 1653 – 1663. Paton, B.C., Hughes, J.L., Harzer, K., Poulos, A., 1990. Immunocytochemical localization of sphingolipid activator protein 2 (SAP-2) in normal and SAP-deficient fibroblasts. Eur. J. Cell Biol. 51, 157 – 164. Paton, B.C., Schmid, B., Kustermann-Kuhn, B., Poulos, A., Harzer, K., 1992. Additional biochemical findings in a patient and fetal sibling with a genetic defect in the sphingolipid activator protein (SAP) precursor, prosaposin. Biochem. J. 285, 481 – 488. Ponting, C.P., 1994. Acid sphingomyelinase possesses a domain homologous to its activator proteins B and D. Protein Sci. 3, 359 – 361. Poulos, A., Ranieri, E., Shankaran, P., Callahan, J.W., 1984. Studies on the activation of the enzymatic hydrolysis of sphingomyelin liposomes. Biochem. Biophys. Acta 793, 141 – 148. Quintern, L.E., Schuchman, E.H., Levran, O., Suchi, M., Ferlinz, K., Reinke, H., Sandhoff, K., Desnick, R.J., 1989. Isolation of cDNA clones encoding human acid sphingomyelinase: occurrence of alternatively processed transcripts. EMBO J. 8, 2469 – 2473. Sandhoff, K., Harzer, K., Fu¨rst, W., 1995. In: Scriver, C., Beaudet, A.L., Sly, W.S., Valle, D. (Eds.), The Metabolic and Molecular Bases of Inherited Disease. McGraw Hill, New York, pp. 2427 – 2441. Sano, A., Radin, N.S., Johnson, L.L., Tarr, G.E., 1988. The activator protein for glucosylceramide beta-glucosidase from guinea pig liver. Improved isolation method-and complete amino acid sequence. J. Biol. Chem 263, 19597 – 19601. Schnabel, D., Schro¨der, M., Fu¨rst, W., Klein, A., Hurwitz, R., Zenk, T., Weber, J., Harzer, K., Paton, B.C., Suzuki, K., Sandhoff, K., 1992. Simultaneous deficiency of sphingolipid activator proteins 1 and 2 is caused by a mutation in the initiation codon of their common gene. Biol. Chem. 267, 3312 – 3315. Schuchman, E.H., Desnick, R.J., 1995. Niemann – Pick disease types A and B: acid sphingomyelinase deficiencies. In: Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D. (Eds.), The Metabolic and Molecular Bases of Inherited Disease, 7th edn. McGraw Hill, New York, pp. 2601 – 2624.
K. Ferlinz et al. / Chemistry and Physics of Lipids 102 (1999) 35–43 Spiegel, S., Foster, D., Kolesnick, R., 1996. Signal transduction through lipid second messengers. Curr. Opin. Cell Biol. 8, 159 – 168. Tayama, M., Soeda, S., Kishimoto, Y., Martin, B., Callahan, J.W., Hiraiwa, M., O’Brien, J.S., 1993. Effect of saposins on acid sphingomyelinase. Biochem. J. 290, 401–404. Vielhaber, G., Hurwitz, R., Sandhoff, K., 1996. Biosynthesis, processing and targeting of SAP precursor in cultured
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Stimulation of lysosomal sphingomyelin degradation by sphingolipid activator proteins Klaus Ferlinz, Thomas Linke, Oliver Bartelsen, Manfred Weiler, Konrad Sandhoff * Kekule´ -Institut fu¨r Organische Chemie und Biochemie, Uni6ersita¨t Bonn, Gerhard-Domagk-Str. 1, D 53121 Bonn, Germany
Abstract Lysosomal breakdown of glycosphingolipids with short hydrophilic carbohydrate headgroups is achieved by the simultaneous action of specific hydrolases and sphingolipid activator proteins (SAPs). Activator proteins are considered to facilitate the enzyme/substrate interaction between water-soluble enzymes and membrane-bound substrates. Sphingomyelin, containing the small hydrophilic phosphorylcholine moiety, is hydrolysed by acid sphingomyelinase (acid SMase). Recent experimental data on the in vivo and in vitro role of activator proteins in sphingomyelin breakdown by acid SMase are reviewed. These data combined with the results using homogenous protein preparations as well as a liposomal assay system mimicking the physiological conditions suggest that lysosomal sphingomyelin degradation is not critically dependent on any of the known activator proteins. Moreover, evidence is provided that the assumed intramolecular activator domain of acid SMase and especially the presence of negatively charged lipids in the lysosomes are sufficient for sphingomyelin turnover. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acid sphingomyelinase; Sphingolipid activator proteins; Intra-lysosomal vesicles
1. Lysosomal sphingolipid catabolism Glycosphingolipids and sphingomyelin are major constituents of the outer leaflet of eukaryotic membranes. They consist of a hydrophobic ceramide moiety which anchors the sphingolipid in the cell membrane and a hydrophilic headgroup extruding into the extracellular space. More than 300 different glycosphingolipids which differ in * Corresponding author. Tel.: +49-228-73-5346; fax: +49228-73-7778. E-mail address: [email protected] (K. Sandhoff)
number, type and linkage of individual sugar residues, have yet been identified from natural sources. Although the molecular structure as well as their biosynthetic and catabolic pathways have been intensively studied, the physiological role of these complex cell surface compounds is still matter of debate. The glycosphingolipid pattern appears to be dynamic and highly dependent on cell type, cell growth and differentiation, ontogenesis and transformation. Several lines of evidence suggest that glycosphingolipids are essentially involved in cell recognition and adhesion processes during embryogenesis and development as well as
0009-3084/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 9 9 ) 0 0 0 7 3 - 0
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K. Ferlinz et al. / Chemistry and Physics of Lipids 102 (1999) 35–43
certain signal transduction mechanisms (Hakomori and Igarashi, 1995). In contrast, sphingomyelin, the predominant sphingolipid in the outer leaflet of plasma membranes, has originally been considered to be particularly involved in the maintenance of biophysical properties of the plasma membrane. During the past years interest in sphingomyelin metabolism has increased since it turned out that sphingomyelin is the immediate educt for the generation of the signalling molecule ceramide (Spiegel et al., 1996; Hannun and Obeid, 1997). The involvement of sphingomyelinases, ceramide as well as sphingosin and its metabolites in regulation of cell death and differentiation is also extensively reviewed in this edition. The bulk membrane components of eukaryotic cells are continuously internalized by endocytotic processes and either degraded and or recycled back to the membrane. A recent model of topology for sphingolipid hydrolysis suggests that plasma membrane components are selectively internalized by vesicular flow and trafficked toward the acidic compartments (Fig. 1). Maturation of endosomal membranes results in the formation of intra-endo/lysosomal vesicles and other membrane structures which finally serve as substrate for acidic hydrolases (Fu¨rst and Sandhoff, 1992). The lysosomal perimeter membrane itself seems to be protected against hydrolysis by a highly inert glycoprotein derived glycocalix which covers the lumenal surface of the limiting lysosomal membrane. Lysosomal degradation of glycosphingolipids and sphingomyelin is achieved by the action of a variety of substrate-specific exohydrolases which sequentially cleave off terminal sugars or, in the case of sphingomyelin, phosphorylcholine (Fig. 2) (Kolter and Sandhoff, 1997).
protein) (see Fig. 2). Some SAPs are thought to be particularly involved in the solubilization of respective sphingolipids from the lipophilic membrane making them susceptible to the water soluble hydrolases (Giehl et al., 1998; Wilkening et al., 1999). Long hydrophilic carbohydrate structures which sufficiently protrude into the aqueous phase are readily cleaved by the respective enzyme independently of SAPs. Four of the five different SAPs A–D which have been characterized to date are encoded by a common gene. Translation results in the biosynthesis of a SAP precursor (prosaposin) which is proteolytically processed to the active SAP A–D inside the acidic organelles (Vielhaber et al., 1996; Hiesberger et al., 1998). Although these co-factors exhibit several highly conserved structural features, e.g. the array of disulphide bridges and a conserved site of N-glycosylation, their ligand and/or enzyme binding
2. Sphingolipid activator proteins (SAPs) in sphingolipid breakdown In vivo hydrolysis of sphingolipids with short hydrophilic headgroups (5 3 sugar residues in a row) requires additional non-enzymatic co-factors of low molecular weight, so called SAPs (or saposins), of which five different have yet been characterized (SAPs A – D and the GM2-activator
Fig. 1. Topology of lysosomal sphingoipid degradation. Maturation of internalized membrane structures results in the formation of intra-endo/lysosomal vesicles and other membrane structures which finally serve as substrates for acidic hydrolases. The lysosomal perimeter membrane itself seems to be protected against hydrolysis by a highly inert glycoprotein derived glycocalix which covers the lumenal surface of the limiting lysosomal membrane.
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Fig. 2. Scheme of major sphingolipid hydrolysis. Exohydrolytic breakdown of sphingolipids with short hydrophilic headgroups requires non-enzymatic co-factors, sphingolipid activator proteins (SAPs). Both, inherited deficiencies of the respective enzyme as well as of the corresponding activator protein causes lysosomal lipid storage and results in the expression of various sphingolipidoses.
specificity is quite different but not absolute (see Fig. 2). The physiological substrate specificity of lysosomal sphingolipid hydrolases as well as of SAPs has most extensively been investigated taking advantage of the naturally occurring lysosomal sphingolipidoses. Deficiency of any of the numerous sequentially acting exohydrolases leads to a sphingolipid storage disease which is characterized by a massive and specific lysosomal sphingolipid accumulation (Gieselmann, 1995). Rare variants of sphingolipidoses are caused by molecular deficiency of SAPs which are predominantly accompanied by a mild juvenile clinical phenotype
(Sandhoff et al., 1995). The sphingolipid storage pattern resembles those of patient cells with the respective enzyme deficiency directing to the physiological role of the individual activator proteins. However, lipid storage in activator protein deficient cells is restricted to sphingolipids with short hydrophilic headgroups (Fig. 2). More recently, several activator protein deficient knock-out mouse strains have been generated in order to get detailed information on the development and onset of respective lipid storage diseases and furthermore, to develop strategies for future therapeutic approaches (Fujita et al., 1996; Liu et al., 1997).
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3. Lysosomal sphingomyelin breakdown The bulk of membrane sphingomyelin is hydrolysed by the lysosomal enzyme acid sphingomyelinase (acid SMase) (Quintern et al., 1989). Sphingomyelin consists of a ceramide membrane anchor which is linked to a short hydrophilic phosphorylcholine moiety suggesting that in vivo degradation by acid SMase in principle could also depend on the co-action of any of these activator proteins. However, the importance of the SAPs for sphingomyelin degradation remained unclear due to contradictory results obtained from in vivo and in vitro experiments published earlier. Those in vivo results are based on the lipid analysis of a patient and his fetal sibling with a complete absence of SAPs due to a homoallelic mutation in the initiation codon of the SAP-precursor (prosaposin) gene (multiple SAP deficiency) (Harzer et al., 1989; Paton et al., 1990, 1992; Schnabel et al., 1992). Densitometric analysis of kidney and liver lipids of the fetus revealed no significant elevation of sphingomyelin levels compared to fetal control kidneys. Furthermore, these findings were corroborated in a mouse model, in which the prosaposine gene was knocked out (SAP knock-out) (Fujita et al., 1996). Again, no significant abnor-
malities in sphingomyelin levels were found in kidney and liver of SAP deficient mice. The apparent lack for sphingomyelin storage in human multiple SAP deficient tissues was supported by detection of normal acid SMase activity levels in fibroblasts from both the patient and his fetal sibling compared to healthy normal controls when tested in a detergent-free, liposomal assay system (Bradova` et al., 1993). Acid SMase activity levels in SAP knock-out mice were obviously reduced by about 40% compared to healthy controls by so far unknown reasons (Fujita et al., 1996). In contrast to the in vivo findings the stimulation of acid SMase by SAP B, C and D in in vitro detergent-based assay systems has been repeatedly reported (Poulos et al., 1984; Christomanou and Kleinschmidt, 1985; Christomanou et al., 1986; Morimoto et al., 1988). Only one study by Tayama et al. (1993) has been performed to date in which sphingomyelin hydrolysis catalysed by acid SMase and the different SAPs is analysed in more detail. The effect of SAP-D on acid SMase activity in a micellar assay system was found to be dependent on the concentration of the detergent Triton X-100: increasing amounts of SAP-D were shown to stimulate detergent containing, crude acid SMase (concentration of Triton X-100: \
Fig. 3. Effect of the four different sphingolipid activator proteins (SAPs) on sphingomyelin degradation. Acid sphingomyelinase (acid SMase) was assayed with liposomal sphingomyelin in the presence of the different activator proteins (2.5 mg each). The liposomes (70 mol% phosphatidylcholine, 20 mol% cholesterol and 10 mol% sphingomyelin) were prepared by repeated extrusion through a polycarbonate membrane with a pore size of 100 nm. The assay mixture contained a 10 mM sodium acetate buffer, pH 4.5, 150 mM sodium chloride and a total lipid concentration of 1 mM in an incubation volume of 100 ml. The assay mixture was incubated for 2 h at 37°C.
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0.04% w/v), whereas partially purified, detergentfree acid SMase was inhibited by presence of SAP-D (concentration of Triton X-100: B 0.04% w/v). Contrary to the influence of SAP-D, SAP-A, B and C were rather inhibitory or without any effect on the SMase activity (Tayama et al., 1993). When sphingomyelin was presented in liposomes in absence of Triton X-100 acid SMase activity was stimulated in the order SAP-C \SAP-D \ SAP-A. SAP-B exhibited an inhibitory effect on sphingomyelin degradation; furthermore, the degree of stimulation by SAP A, C and D was decreased by the simultaneous addition of SAP-B (Tayama et al., 1993). Recent computational analysis of the acid SMase sequence in terms of structural homology comparison suggests that the enzyme possibly contains its own intramolecular activator protein domain (Ponting, 1994; Munford et al., 1995). The N-terminal portion of acid SMase polypeptide (amino acids 89 – 165) closely resembles sequence data obtained from SAPs, including positions of cystein residues and several hydrophobic amino acid domains, pointing to a related secondary structure which may well function as a built-in activator protein domain. This would explain — at least in part — the unusually high specific molecular acid SMase in vitro activity on its endogenous lipid substrate (see also Fig. 3). Regarding these putative intramolecular activating properties, one would also expect that in vivo sphingomyelin breakdown by the acid SMase does not critically depend on any additional activator protein which is in line with the lipid data from SAP knock-out mice.
4. Experimental data Earlier studies on activator protein assisted sphingomyelin breakdown, as discussed above, were generally conducted using either crude acid SMase preparations or partially purified enzyme from human urine (Christomanou and Kleinschmidt, 1985) or liver (Morimoto et al., 1988) and do not exactly reflect the physiological role of SAP stimulated sphingomyelin breakdown. Therefore, it was decided to test the highly purified SAPs from human spleen in combination
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with recombinant human acid SMase (rSMase) in a detergent-free liposomal sphingomyelin assay system. The limiting factor of these studies turned out to be the availability of preparative amount of homogenous, enzymatically active acid SMase. In order to circumvent the low natural abundance of acid SMase as well as any critical contaminants a baculovirus expression system for the polyhedrin promotor controlled expression of recombinant human acid SMase in Spodoptera frugiperda (Sf 21) insect cells was established (Bartelsen et al., 1998). Acid SMase-cDNA was inserted into the genome of an AcMNPV baculovirus by allelic replacement. After selection and amplification of recombinant virus, acid SMase was immunochemically and enzymatically detected in cell lysates and media. Almost 80% of recombinant enzyme was secreted. Purification from the culture media was achieved by conventional column chromatography as described recently (Lansmann et al., 1996) and led to a rSMase preparation of apparent homogeneity. Enzymatic comparison of rSMase with immune-affinity purified homogenous placental enzyme indicated that the specific activity of the insect cell derived protein was lower by the factor of 2 whereas the km value was almost identical. Edman sequencing of the N-terminal peptide sequence revealed that the recombinant N-terminus is extended by 23 amino acid residues compared to the lysosomal placenta enzyme (Lansmann et al., 1996). Thus, both proteins, the recombinant and the native placental contain the complete SAP-like N-terminal domain. SAPs used in the studies were purified from human spleen of Gaucher patients essentially as described by Sano et al. in order to obtain sufficient amounts of the individual activator protein (Sano et al., 1988). The purification and separation of individual SAPs A–D is only achieved with considerable difficulty due to their high degree of homology and similar biophysical and biochemical properties. In order to exclude any cross-contamination of SAPs the purity of the final activator protein preparation was tested by Western blotting and MALDI mass spectrometry.
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Fig. 4. Relationship between the rate of sphingomyelin hydrolysis and the molecular percentage of phosphatidylinositol (PI) in the liposomes. Liposomes were prepared and the assay conducted as described in the legend of Fig. 3. Phosphatidylcholine was replaced by increasing amounts of PI.
Homogenous recombinant acid SMase was assayed in two different liposomal sphingomyelin assay systems in the presence of the individual activator proteins. Preliminary results presented herein are means of two independent duplicates (Fig. 3). It was found that SAP-A and SAP-C were the strongest acid SMase stimulators under the above conditions, while SAP-B and SAP-D showed neither a high degree of stimulation nor inhibition of acid sphingomyelin breakdown activity (Fig. 3) (Linke et al., 1998). However, it was observed that both SAP-C and D became more effective stimulators of acid SMase activity, when the liposomes were prepared by ultrasonic irradiation thus producing smaller sized liposomes with a higher degree of curvature (Linke et al., 1998). In order to render the assay conditions more physio-
logically substrate liposomes were designed which mimic intra-endo/lysosomal membrane structures. The lipid composition of late endosomal and lysosomal compartments are characterized by the presence of negatively charged phospholipids such as bis(monoacylglycero)phosphate (BMP) and phosphatidylinositol (PI) (Kobayashi et al., 1998; Wilkening et al., 1999). It was therefore tested whether such lipids could stimulate sphingomyelin breakdown. The overall rate of sphingomyelin hydrolysis was increased dramatically when an acidic phospholipid such as PI was included in the liposomal preparation even without addition of any activator protein (Fig. 4). As illustrated in Fig. 4 the rate of sphingomyelin breakdown increases with increasing negative surface charge of the liposomes. The stimulation of sphingomyelin degradation activity
K. Ferlinz et al. / Chemistry and Physics of Lipids 102 (1999) 35–43
by SAP-D also becomes more pronounced in the presence of anionic lipids, but never reaching the degree of stimulation as observed by SAP-C. The above results suggest that SAPs may indeed stimulate sphingomyelin breakdown by acid SMase under liposomal assay conditions. The observed lack of specificity of the activators on the other hand suggest that the SAPs may affect the physical nature of the liposome bound substrate rather than interacting specifically with acid SMase.
5. Discussion Data obtained from SAP deficient knock-out mice and multiple SAP deficient human tissue combined with the experimental data discussed in this article suggest that presumably the combination of all, the proposed intramolecular SMase activator domain and the SAPs as well as the presence of lysosomal anionic phospholipids (e.g. BMP) maximally stimulate enzymatic sphingomyelin degradation. Even without additional SAPs, sphingomyelin turnover by acid SMase was increased by the factor of approximately 10 using negatively charged substrate liposomes. On the other hand, stimulation by individual SAPs in the absence of acid phospholipids was approximately 5-fold for SAP-C and only 2-fold for SAP-D. Therefore, complete SAP deficiency is obviously not leading to impaired sphingomyelin turnover due to the compensatory effect of the remaining two factors, intramolecular activator domain and negatively charged vesicle surface, which still allow normal sphingomyelin catabolism. In this context it should be noted that none of the known mutations causing Niemann – Pick disease, the inherited acid SMase deficiency, is located within the predicted activator domain whereas several different mutations have been detected downstream the sequence (Schuchman and Desnick, 1995). One hypothesis would be that, opposite to the multiple SAP deficiency, defects within the intramolecular activator domain can be overcome in vivo by the synergistic action of SAPs and acid phospholipids, thus avoiding lysosomal sphingomyelin storage. Possible mutations in the acti-
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vator-like domain encoding sequence which do not result in a pathological phenotype may escape molecular analysis. Ida et al. (1993) published the identification of an acid SMase cDNA which was cloned from a placental cDNA library and contained a single base change within the sequence encoding the putative activator domain of the mature polypeptide. Intriguingly, this presumed cloning artefact resulted in the substitution of arginine for the normal cystein, one of the homologous cystein residues conserved through all activator protein(-like) domains and reduced in vitro activity of mutant sphingomyelinase almost completely. However, there is no in vivo evidence that a non-functional acid SMase activator domain causes lysosomal sphingomyelin storage. Taking these observations together it can be concluded that lysosomal sphingomyelin degradation by acid SMase is stimulated on the one hand by negatively charged intra-endo/lysosomal membrane structures as well as by intramolecular and/ or by separate activator proteins (most potently by SAP-C) which may be capable of substituting each other. Further studies including mutational in vivo analysis of the acid SMase SAP-like domain as well as studies on the sphingomyelin binding capacity of recombinant acid SMase are in progress to thoroughly define the factors required for lysosomal sphingomyelin turnover.
Acknowledgements We thank H. Moczall for excellent technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 400, A5).
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