Sphingomyelinase Activity Causes Transbilayer Lipid Translocation in Model and Cell Membranes

Abstract

Ceramide is known to induce structural rearrangements in membrane bilayers, including the formation of ceramide-rich and -poor domains and the efflux of aqueous solutes. This report describes a novel effect of ceramide, namely the induction of transbilayer lipid movements. This effect was demonstrated in both model (large unilamellar vesicles) and cell (erythrocyte ghost) membranes in which ceramide generation took place in situ through the action of an externally added sphingomyelinase. Two different novel assays were developed to detect transbilayer lipid movement. One of the assays required the preparation of vesicles containing a ganglioside only in the outer monolayer and entrapped neuraminidase. Sphingomyelinase activity induced ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles. The second assay involved the preparation of liposomes or erythrocyte ghosts labeled with a fluorescent energy donor in their inner leaflets. Sphingomyelin hydrolysis was accompanied by fluorescence energy transfer to an impermeable acceptor in the outer aqueous medium. Ceramide-induced transbilayer lipid movement is explained in terms of another well known property of ceramide, namely the facilitation of lamellar to non-lamellar lipid-phase transitions. Thus, sphingomyelinase generates ceramide on one side of the membrane; ceramide then induces the transient formation of non-lamellar structural intermediates, which cause the loss of lipid asymmetry in the bilayer, i.e. the transbilayer movement of ceramide together with other lipids. As direct targets for ceramide tend to be intracellular, these observations may be relevant to the mechanism of transmembrane signaling by means of the sphingomyelin pathway. Ceramide is known to induce structural rearrangements in membrane bilayers, including the formation of ceramide-rich and -poor domains and the efflux of aqueous solutes. This report describes a novel effect of ceramide, namely the induction of transbilayer lipid movements. This effect was demonstrated in both model (large unilamellar vesicles) and cell (erythrocyte ghost) membranes in which ceramide generation took place in situ through the action of an externally added sphingomyelinase. Two different novel assays were developed to detect transbilayer lipid movement. One of the assays required the preparation of vesicles containing a ganglioside only in the outer monolayer and entrapped neuraminidase. Sphingomyelinase activity induced ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles. The second assay involved the preparation of liposomes or erythrocyte ghosts labeled with a fluorescent energy donor in their inner leaflets. Sphingomyelin hydrolysis was accompanied by fluorescence energy transfer to an impermeable acceptor in the outer aqueous medium. Ceramide-induced transbilayer lipid movement is explained in terms of another well known property of ceramide, namely the facilitation of lamellar to non-lamellar lipid-phase transitions. Thus, sphingomyelinase generates ceramide on one side of the membrane; ceramide then induces the transient formation of non-lamellar structural intermediates, which cause the loss of lipid asymmetry in the bilayer, i.e. the transbilayer movement of ceramide together with other lipids. As direct targets for ceramide tend to be intracellular, these observations may be relevant to the mechanism of transmembrane signaling by means of the sphingomyelin pathway. Cell membranes are asymmetric in the steady state, i.e. lipid and protein compositions of the inner and outer leaflets are not the same. Far from being a static phenomenon, this asymmetry arises from a series of constant and concerted transmembrane movements leading to an apparently invariant distribution of bilayer components. Lipid asymmetry in particular is known to be altered in physiological or pathological events such as recognition by phagocytes, blood coagulation, or apoptosis (see Ref. 1Bevers E.M. Comfurius P. Dekkers D.W.C. Zwaal R.F.A. Biochim. Biophys. Acta. 1999; 1439: 317-330Crossref PubMed Scopus (363) Google Scholar for a review). The collapse of lipid asymmetry is often known as “lipid scrambling.” Sphingomyelin is a phospholipid localized mainly at the outer leaflet of the plasma membrane, where it was originally conceived to act in a structural manner to provide a semipermeable barrier to the extracellular environment (2Kolesnick R.N. Prog. Lipid Res. 1991; 30: 1-38Crossref PubMed Scopus (258) Google Scholar). In 1987, however, it was reported that 1,2-diacylglycerols induce sphingomyelinase activation in GH3 pituitary cells (3Kolesnick R.N. Paley A.E. J. Biol. Chem. 1987; 262: 9204-9210Abstract Full Text PDF PubMed Google Scholar), and it was suggested for the first time that this response may activate a sphingomyelin-based signaling pathway (4Kolesnick R.N. J. Biol. Chem. 1989; 264: 7617-7623Abstract Full Text PDF PubMed Google Scholar, 5Kolesnick R.N. Clegg S. J. Biol. Chem. 1988; 263: 6534-6537Abstract Full Text PDF PubMed Google Scholar). Subsequently, Okazaki et al. (6Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar, 7Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar) confirmed this notion, demonstrating that receptor binding of vitamin D3 induced sphingomyelinase activation and that the generated ceramide served as a second messenger in a differentiation pathway of HL-60 cells. Over the past decade, ceramide and other sphingolipids have been identified as primary regulators of signal transduction involved in multiple biological functions (8Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (443) Google Scholar, 9Mathias S. Peña L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (621) Google Scholar). Recent studies have suggested that much of the sphingomyelin in the plasma membrane exists in preformed microdomains in mammalian cells (10Simons K. Ikonen E. Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1078) Google Scholar) called rafts and that this is the site of ceramide generation and signal transmission (11Kolesnick R. J. Clin. Invest. 2002; 110: 3-8Crossref PubMed Scopus (383) Google Scholar). Ceramide was originally conceived of as a classic second messenger, directly and stoichiometrically activating a set of target proteins (see below) (8Hannun Y.A. Luberto C. Argraves K.M. Biochemistry. 2001; 40: 4893-4903Crossref PubMed Scopus (443) Google Scholar, 9Mathias S. Peña L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (621) Google Scholar). However, recent data suggest that ceramide may act in part by physically altering the structure and properties of membrane rafts. In this context, it has been suggested that ceramide generated in rafts induces their coalescence into larger platforms and that these macrodomains serve as a site for oligomerization of cell surface receptors such as Fas and CD40 (12Grassme H. Jendrossek V. Bock J. Riehle A. Gulbins E. J. Immunol. 2002; 168: 298-307Crossref PubMed Scopus (220) Google Scholar, 13Cremesti A. Paris F. Grassme H. Holler N. Tschopp J. Fuks Z. Gulbins E. Kolesnick R. J. Biol. Chem. 2001; 276: 23954-23961Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 14Grassme H. Schwarz H. Gulbins E. Biochem. Biophys. Res. Commun. 2001; 284: 1016-1030Crossref PubMed Scopus (161) Google Scholar) and the internalization of bacteria such as Pseudomonas aeruginosa and Neisseria gonorrhoeae (15Grassme H. Jendrossek V. Riehle A. Von Kurthy G. Berger J. Schwarz H. Weller M. Kolesnick R. Gulbins E. Nat. Med. 2003; 9: 322-330Crossref PubMed Scopus (465) Google Scholar, 16Grassme H. Gulbins E. Brenner B. Ferlinz K. Sandhoff K. Harzer K. Lang F. Meyer T.F. Cell. 1997; 91: 605-615Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). In this paradigm, acid sphingomyelinase is initially translocated onto the outer leaflet of the plasma membrane where it catalyzes the hydrolysis of raft-associated sphingomyelin. Ceramide affects a number of membrane properties in model membrane systems, which may impact platform formation in biological membranes; ceramide increases lipid order, gives rise to ceramide-rich separate domains, and destabilizes the lamellar structures, inducing membrane permeabilization and membrane fusion (see Ref. 17Kolesnick R.N. Goñi F.M. Alonso A. J. Cell. Physiol. 2000; 184: 285-300Crossref PubMed Scopus (381) Google Scholar for a review). As a consequence, treating defined model membrane systems containing sphingomyelin with sphingomyelinase has been found to produce, via ceramide formation, vesicle efflux (18Ruiz-Argüello M.B. Basañez G. Goñi F.M. Alonso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 19Montes L.R. Goñi F.M. Alonso A. J. Biol. Chem. 2002; 277: 11788-11794Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), membrane fusion (20Basañez G. Ruiz-Argüello M.B. Alonso A. Goñi F.M. 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Chem. 2002; 277: 18037-18045Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) demonstrated that ceramide binds to the C1b of protein kinase in and its to the membrane Ceramide a the C1b the binding of the ceramide such as kinase suppressor of Ras and C1b domains, the C1b targets these proteins to of ceramide such as plasma membrane is However, these targets are intracellular, and it is ceramide generated on the outer leaflet of the plasma membrane their The was to the of sphingomyelin hydrolysis on one side of a bilayer induce transbilayer lipid have developed novel systems to transfer a membrane bilayer, one of which have to large unilamellar vesicles and the other to both vesicles and erythrocyte In transmembrane lipid movements in with sphingomyelinase sphingomyelinase from was from and was in the of to of C studies that not sphingomyelinase in with data H. M. T. R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). from and from were from sphingomyelin large unilamellar vesicles. and were from and was from and were from and protein were from were from A neuraminidase was in to Ref. E. A was from large unilamellar vesicles were by the Biochim. Biophys. Acta. PubMed Scopus Google Scholar) of at as Goñi F.M. Alonso A. Biochemistry. 1989; 28: PubMed Scopus Google Scholar). of the vesicles that the not their were in neuraminidase was added to the In this neuraminidase was by through of ganglioside to the outer monolayer of liposomes as the in were and in a of that was of the vesicle were added to this that was of the lipids. were to for at Alonso A. C. I. U. Goñi F.M. FEBS Lett. 1999; PubMed Scopus Google Scholar). of in neuraminidase was from by a protein of the was through the and the was with a to was from the with a were at lipid was and were at for sphingomyelinase on for sphingomyelinase on and for neuraminidase. Sphingomyelinase activity was by C. Acta. 24: Scopus Google Scholar) in the aqueous of an by the of from the at different acid of liposomes containing with neuraminidase was by a of the acid assay S. R.M. Biochemistry. 1996; PubMed Scopus Google Scholar). neuraminidase activity was with an by containing entrapped neuraminidase were as and to The vesicle was with to the of the was then and the was to for was and the of acid was A in the of was erythrocyte membranes were by a and PubMed Scopus Google Scholar, R. S. A. J. T. J. Cell Biol. 91: PubMed Scopus (258) Google Scholar). The membranes from of erythrocyte by the blood were by in and the was with for at and In the the was in of containing and then membranes were and to by for at by in the the membrane was in and for at L. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The ghosts on of the were and in the this the ghosts were labeled with on both of the the outer leaflet which outer and its was in an at with a and were and A was to from ghosts were from by The was in the in the with were by the including the outer leaflet the for membranes was that to separate from a was were at lipid was for assays and for and sphingomyelinase was at and for and or for or were from the at and with of in to a of transfer was by an under the conditions in Lipid of were with ganglioside in to vesicles containing of on the outer leaflet Alonso A. C. I. U. Goñi F.M. FEBS Lett. 1999; PubMed Scopus Google Scholar). These vesicles neuraminidase and at the vesicles were for at no of was in this of of sphingomyelinase to a of these vesicles induced which of been of membrane hydrolysis not at i.e. sphingomyelinase that this not sphingomyelinase or neuraminidase. of the vesicle were at the of sphingomyelinase and for the of neuraminidase was in with that no was the of was by neuraminidase. One of these data is as a of sphingomyelinase was to the inner to neuraminidase. However, it was to the of neuraminidase from the vesicles as a of sphingomyelinase membrane (18Ruiz-Argüello M.B. Basañez G. Goñi F.M. Alonso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 19Montes L.R. Goñi F.M. Alonso A. J. Biol. Chem. 2002; 277: 11788-11794Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, L. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and an efflux of to has been in vesicles L.R. Goñi F.M. Alonso A. J. Biol. Chem. 2002; 277: 11788-11794Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). has a of this neuraminidase activity the vesicles was with the this of the vesicle were through which the of neuraminidase. activity in the is in in the of sphingomyelinase activity of the activity was in the This is of vesicle to stress the containing not sphingomyelinase or neuraminidase were with neuraminidase at the found in the was added to the vesicle from the to In this in the time of hydrolysis is different from the one on sphingomyelinase Thus, the in be explained on the of neuraminidase efflux from the vesicles. was in which neuraminidase have been by a this a was in that the at neuraminidase activity at The of no effect on sphingomyelinase the in was with in the no were found the of in the and of for of data that hydrolysis was by neuraminidase the sphingomyelinase activity to to the lipid in Lipid different assay was developed to the transmembrane movement of in a in which sphingomyelinase activity was to cause membrane permeabilization to of This novel was on fluorescence energy transfer and transfer and is well known and often in membrane of to an large in it not the vesicles. were as that was in the lipid the vesicle was with the impermeable that in the outer was and its fluorescence was was by labeled with only in their inner lipid leaflets were then with sphingomyelinase. activity was the as in were from the at and with in As the sphingomyelinase the outer and energy transfer to the fluorescence of was its with and the of The data are in in the that in the of fluorescence invariant with Thus, fluorescence energy transfer that sphingomyelinase activity induces the transmembrane movement of lipids. in was to erythrocyte ghosts to the transbilayer movement of induced by sphingomyelinase in a cell ghosts were with which into both membrane leaflets. were then and with to outer leaflet was by and Sphingomyelinase activity in erythrocyte membranes much in liposomes and The in were by i.e. of the with The for this of sphingomyelin in blood cells are the of a separate in this of the ceramide fluorescence energy transfer was in ghosts a time The of ceramide and energy transfer and that hydrolysis is the transmembrane lipid movement. was to that sphingomyelinase not transfer of the erythrocyte membrane that ghosts were containing and with sphingomyelinase for The ghosts were then through a protein to that The fluorescence of the ghosts was the as that of an preparation with that no loss of as a of ghosts with were through the the fluorescence was of the in the of the loss of the containing was with a In this the from the and ghosts no the from the ghosts a fluorescence signal not erythrocyte membranes as a of sphingomyelinase fluorescence in a to the of sphingomyelinase activity induces transbilayer lipid movement is to the of ceramide to non-lamellar has been found P. B. R. R.E. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) that a role in the transmembrane distribution of these of may to an altered ceramide have been to transbilayer movements much J. R.E. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). This that the may the In ceramide the of other lipids. studies (18Ruiz-Argüello M.B. Basañez G. Goñi F.M. Alonso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, Goñi F.M. Alonso A. Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) have demonstrated that ceramide the at that are with in systems through sphingomyelinase which were to by Ruiz-Argüello et al. (18Ruiz-Argüello M.B. Basañez G. Goñi F.M. Alonso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), a is lamellar and non-lamellar an lamellar only a bilayer the of As by in Ruiz-Argüello et al. (18Ruiz-Argüello M.B. Basañez G. Goñi F.M. Alonso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), the formation of in a lamellar environment well the loss of A movement and the formation of non-lamellar lipid was suggested by and B. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The observations by Basañez et al. (20Basañez G. Ruiz-Argüello M.B. Alonso A. Goñi F.M. Karlsson G. Edwards K. Biophys. J. 1997; 72: 2630-2637Abstract Full Text PDF PubMed Scopus (92) Google Scholar) are in this These large unilamellar vesicles containing with i.e. the in the and in were no that be to or non-lamellar intermediates be This may be intermediates were However, the data by Basañez et al. (20Basañez G. Ruiz-Argüello M.B. Alonso A. Goñi F.M. Karlsson G. Edwards K. Biophys. J. 1997; 72: 2630-2637Abstract Full Text PDF PubMed Scopus (92) Google Scholar) under the lipid is sphingomyelinase In suggest that sphingomyelinase generates ceramide and that ceramide induces the transient formation of non-lamellar structures, the leading to the loss of asymmetry in the transbilayer lipid from the One is the in and that transbilayer movement on ceramide has the neuraminidase the of ceramide increases and by of increases by In the of ghosts only of phospholipid is and energy transfer as the in increases by This the different of the under hydrolysis and lipid transbilayer In is by the of and it is no in or be transbilayer lipid however, the of ceramide that non-lamellar is the is of the in ceramide is the lipid in erythrocyte which is to the hydrolysis from the ceramide and transbilayer the to observations from to a E.M. Comfurius P. Dekkers D.W.C. Zwaal R.F.A. Biochim. Biophys. Acta. 1999; 1439: 317-330Crossref PubMed Scopus (363) Google Scholar, T. Biochemistry. 2001; 40: PubMed Scopus Google Scholar, D.W.C. Comfurius P. E.M. Zwaal R.F.A. Biochem. J. 2002; PubMed Scopus Google Scholar). These that of treating blood cells with and an in the sphingomyelinase assay and the activity the erythrocyte membranes (18Ruiz-Argüello M.B. Basañez G. Goñi F.M. Alonso A. J. Biol. Chem. 1996; 271: 26616-26621Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), it be that in activating the However, the of transbilayer movements has on the of T. Biochemistry. 2001; 40: PubMed Scopus Google Scholar, D.W.C. Comfurius P. E.M. Zwaal R.F.A. Biochem. J. 2002; PubMed Scopus Google Scholar) in This large of in and protein are known to be in cell membranes H. van P. van G. Nat. Mol. Cell Biol. 2001; PubMed Scopus Google Scholar). The that ceramide formation by sphingomyelinase to transbilayer movements of lipid may be to a number of physiological events and properties not well at one is the apparently distribution of in the outer monolayer of the cell plasma membrane, ceramide receptor proteins are distribution may in the are ceramide and receptor both together of the ceramide signaling are with the that the of the acid sphingomyelinase be translocated into on the outer leaflet of the plasma membrane to and signaling to which in the activation of signaling in this may be through platform formation (12Grassme H. Jendrossek V. Bock J. Riehle A. Gulbins E. J. Immunol. 2002; 168: 298-307Crossref PubMed Scopus (220) Google Scholar, 13Cremesti A. Paris F. Grassme H. Holler N. Tschopp J. Fuks Z. Gulbins E. Kolesnick R. J. Biol. Chem. 2001; 276: 23954-23961Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 14Grassme H. Schwarz H. Gulbins E. Biochem. Biophys. Res. Commun. 2001; 284: 1016-1030Crossref PubMed Scopus (161) Google Scholar). The ceramide, originally generated in the outer is both being to proteins the plasma membrane inner as in and ceramide be accompanied by the transbilayer transfer of other that in to may be as a of ceramide formation, a for the a mechanism for transmembrane signaling distinct from that reported by et al. P. T. J. van J. Cell Biol. 2000; PubMed Scopus Google Scholar). In their the in the loss of membrane asymmetry of a membrane to the inner leaflet and ceramide formation in a time of to However, as the physiological of cell signaling is in a of to a time with the of transbilayer demonstrated in this with the