The Multigenic Sphingomyelin Synthase Family

Sphingomyelin (SM) 3The abbreviations used are: SM, sphingomyelin; DAG, diacylglycerol; GlcCer, glucosylceramide; EPC, ethanolamine phosphorylceramide; ER, endoplasmic reticulum; IPC, inositol phosphorylceramide; LPP, lipid phosphate phosphatase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SMS, sphingomyelin synthase; SAM, sterile α motif. 3The abbreviations used are: SM, sphingomyelin; DAG, diacylglycerol; GlcCer, glucosylceramide; EPC, ethanolamine phosphorylceramide; ER, endoplasmic reticulum; IPC, inositol phosphorylceramide; LPP, lipid phosphate phosphatase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SMS, sphingomyelin synthase; SAM, sterile α motif. is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the enzymatic transfer of a phosphocholine head group from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The enzyme catalyzing this reaction, SM synthase, thus occupies a central position in sphingolipid and glycerophospholipid metabolism and has considerable biological potential as a regulator of pro-apoptotic factor ceramide and mitogenic factor diacylglycerol. Recent identification of the enzyme uncovered a multiplicity of SM synthase genes in each organism where SM synthesis is known to occur. This has shed new light on the pathways, reaction mechanism, regulation, phylogenetic distribution, and biological significance of SM synthesis. The first committed step in SM synthesis is the condensation of l-serine and palmitoyl-CoA. This reaction is catalyzed by serine palmitoyltransferase and yields 3-keto-dihydrosphingosine, which is reduced to dihydrosphingosine. Dihydrosphingosine undergoes N-acylation followed by desaturation to generate ceramide, a central molecule in sphingolipid metabolism (1Dickson R.C. Annu. Rev. Biochem. 1998; 67: 27-48Crossref PubMed Scopus (236) Google Scholar, 2Pewzner-Jung Y. Ben-Dor S. Futerman A.H. J. Biol. Chem. 2006; 281: 25001-25005Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). These reactions occur on the cytosolic surface of the endoplasmic reticulum (ER) (3Mandon E.C. Ehses I. Rother J. van Echten G. Sandhoff K. J. Biol. Chem. 1992; 267: 11144-11148Abstract Full Text PDF PubMed Google Scholar). Subsequently, ceramide is delivered to the Golgi apparatus where it is converted to SM or glucosylceramide (GlcCer). GlcCer synthesis is mediated by GlcCer synthase, which catalyzes the transfer of glucose from UDP-glucose to ceramide. This enzyme resides in the cis Golgi and has its active site oriented toward the cytosol (4Jeckel D. Karrenbauer A. Burger K.N. van Meer G. Wieland F. J. Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (259) Google Scholar). After translocation to the Golgi lumen, GlcCer is converted to more complex glycosphingolipids. However, in most mammalian cell types the bulk of ceramide is converted to SM by a SM synthase in the lumen of the trans Golgi (5Futerman A.H. Stieger B. Hubbard A.L. Pagano R.E. J. Biol. Chem. 1990; 265: 8650-8657Abstract Full Text PDF PubMed Google Scholar, 6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar, 7Yamaoka S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). This enzyme, named SMS1, catalyzes the transfer of phosphocholine from phosphatidylcholine (PC) to ceramide, yielding diacylglycerol (DAG) as a side product (Fig. 1) (8Ullman M.D. Radin N.S. J. Biol. Chem. 1974; 249: 1506-1512Abstract Full Text PDF PubMed Google Scholar, 9Voelker D.R. Kennedy E.P. Biochemistry. 1982; 21: 2753-2759Crossref PubMed Scopus (160) Google Scholar). A second SM synthase, SMS2, resides at the plasma membrane (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar, 10van Helvoort A. van't Hof W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar), but it is unclear whether this enzyme participates in the de novo synthesis of SM. An alternative pathway of SM synthesis has been postulated in which ceramide is first converted to ethanolamine phosphorylceramide (EPC) via transfer of the head group from phosphatidylethanolamine (PE) (11Muehlenberg B.A. Sribney M. Duffe M.K. Can J. Biochem. 1972; 50: 166-173Crossref PubMed Scopus (13) Google Scholar). EPC is then converted to SM by stepwise methylation in a reaction analogous to the S-adenosylmethionine-dependent conversion of PE to PC (Fig. 1). Even though this pathway has been demonstrated in isolated membrane fractions from rat brain and liver (12Malgat M. Maurice A. Baraud J. J. Lipid Res. 1986; 27: 251-260Abstract Full Text PDF PubMed Google Scholar, 13Malgat M. Maurice A. Baraud J. J. Lipid Res. 1987; 28: 138-143Abstract Full Text PDF PubMed Google Scholar), its precise contribution to the de novo synthesis of SM remains to be established. Several lines of evidence indicate that SM formation is critical for cell growth and survival. Chinese hamster ovary mutant cells with a thermolabile serine palmitoyltransferase, the rate-limiting enzyme in sphingolipid synthesis, die in the absence of exogenously added sphingoid base when shifted to the restrictive temperature (14Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar). The mutant cells could be rescued by added SM, but not by GlcCer, the precursor of complex glycosphingolipids. Moreover, a mouse lymphoid cell line with diminished SM synthase activity ceases growth when cultured under serum-free conditions; growth could be restored by heterologous expression of SMS1 or supplementation with exogenous SM (7Yamaoka S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Finally, up- and down-regulation of SM synthase activity has been linked to mitogenic and pro-apoptotic signaling in a variety of mammalian cell types (15Luberto C. Hannun Y.A. J. Biol. Chem. 1998; 273: 14550-14559Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 16Riboni L. Viani P. Bassi R. Giussani P. Tettamanti G. J. Biol. Chem. 2001; 276: 12797-12804Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). How SM synthesis contributes to cell growth and survival is unclear, but any of the following scenarios may explain its critical function. (i) SM accumulates in the exoplasmic leaflet of the plasma membrane where its high packing density and affinity for sterols help create a rigid barrier to the extracellular environment. (ii) The SM pool in the plasma membrane acts as a reservoir of lipid signaling molecules, the liberation of which is catalyzed by acidic or neutral SMases in response to a variety of biological stimuli (18Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (675) Google Scholar, 19Adam-Klages S. Adam D. Wiegmann K. Struve S. Kolanus W. Schneider-Mergener J. Kronke M. Cell. 1996; 86: 937-947Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). SM metabolites like ceramide, sphingosine, and sphingosine 1-phosphate are emerging as critical regulators of cell proliferation, differentiation, and apoptosis (20Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1756) Google Scholar, 21Ogretmen B. Hannun Y.A. Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1006) Google Scholar). (iii) As SM has a strong, inherent capacity to form microdomains, its production in the trans Golgi may affect the lateral organization of other membrane molecules and thus provide a physical basis for sorting events that help establish the compositional and functional differences between the ER, plasma membrane, and Golgi itself (22Holthuis J.C. Pomorski T. Raggers R.J. Sprong H. Van Meer G. Physiol. Rev. 2001; 81: 1689-1723Crossref PubMed Scopus (254) Google Scholar). (iv) SM synthesis in the trans Golgi may create a local pool of DAG, which provides a cue for protein kinase D recruitment and the formation of secretory vesicles (23Baron C.L. Malhotra V. Science. 2002; 295: 325-328Crossref PubMed Scopus (339) Google Scholar). (v) By regulating the cellular levels of pro-apoptotic factor ceramide and mitogenic factor DAG in opposite directions, SM synthesis may have a direct impact on cell proliferation and life span (17Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 24Hampton R.Y. Morand O.H. Science. 1989; 246: 1050Crossref PubMed Scopus (48) Google Scholar). Initial studies revealed that mammalian SM synthases are tightly membrane-bound enzymes that readily lose activity upon solubilization with various detergents (8Ullman M.D. Radin N.S. J. Biol. Chem. 1974; 249: 1506-1512Abstract Full Text PDF PubMed Google Scholar, 9Voelker D.R. Kennedy E.P. Biochemistry. 1982; 21: 2753-2759Crossref PubMed Scopus (160) Google Scholar). This severely hampered their identification by classical biochemical approaches. Purification of a soluble SM synthase released by Pseudomonas aeruginosa (25Luberto C. Stonehouse M.J. Collins E.A. Marchesini N. El-Bawab S. Vasil A.I. Vasil M.L. Hannun Y.A. J. Biol. Chem. 2003; 278: 32733-32743Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) provided no clues on the identity of its mammalian counterparts. Complementary efforts focused on the isolation of SM synthase mutants by screening Chinese hamster ovary cells for resistance to a SM-directed cytolysin (26Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Instead of yielding mutants with a primary defect in SM synthesis, this approach led to the discovery of CERT, a ceramide transfer protein mediating non-vesicular transport of ceramide from the ER to the site of SM synthesis in the trans Golgi (27Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (823) Google Scholar). Using an expression cloning strategy in yeast, Huitema et al. (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar) identified a family of integral membrane proteins exhibiting all features previously ascribed to mammalian SM synthases. This approach exploited structural information available for an enzyme catalyzing inositol phosphorylceramide (IPC) synthesis in yeast, a reaction analogous to SM production in which the head group of phosphatidylinositol is transferred to ceramide (Fig. 1). IPC synthesis requires the product of the AUR1 gene (28Nagiec M.M. Nagiec E.E. Baltisberger J.A. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1997; 272: 9809-9817Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), a protein containing the C2 and C3 active site motifs characteristic for members of the lipid phosphate phosphatase (LPP) superfamily (29Heidler S.A. Radding J.A. Biochim. Biophys. Acta. 2000; 1500: 147-152Crossref PubMed Scopus (54) Google Scholar, 30Waggoner D.W. Xu J. Singh I. Jasinska R. Zhang Q.X. Brindley D.N. Biochim. Biophys. Acta. 1999; 1439: 299-316Crossref PubMed Scopus (114) Google Scholar). BLAST searches for novel sequences encoding integral membrane proteins containing active site motifs common to Aur1p and LPPs identified three families of candidate SM synthase genes with homologues throughout the animal kingdom. Several members of each family were cloned and analyzed for their ability to mediate SM synthesis upon expression in yeast, an organism lacking SM synthase activity. Two of the human proteins tested, SMS1 and SMS2, were active in these assays and localized to the trans Golgi and plasma membrane (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar), the two principle sites of SM synthesis in mammalian cells (5Futerman A.H. Stieger B. Hubbard A.L. Pagano R.E. J. Biol. Chem. 1990; 265: 8650-8657Abstract Full Text PDF PubMed Google Scholar, 9Voelker D.R. Kennedy E.P. Biochemistry. 1982; 21: 2753-2759Crossref PubMed Scopus (160) Google Scholar, 10van Helvoort A. van't Hof W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar). Consistent with these findings, a subsequent study reported the expression cloning of human SMS1 employing a mouse lymphoid cell line with severely diminished SM synthase activity and susceptible to methyl β-cyclodextrin-induced cell death (7Yamaoka S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Moreover, this work provided evidence that SMS1 represents a major SM synthase activity in mammalian cells with a critical role in cell growth. Like most LPPs (30Waggoner D.W. Xu J. Singh I. Jasinska R. Zhang Q.X. Brindley D.N. Biochim. Biophys. Acta. 1999; 1439: 299-316Crossref PubMed Scopus (114) Google Scholar, 31Sigal Y.J. McDermott M.I. Morris A.J. Biochem. J. 2005; 387: 281-293Crossref PubMed Scopus (143) Google Scholar), SMS1 and SMS2 have a six times membrane-spanning core domain topology with both termini facing the cytosol and the C2 and C3 active site residues facing the exoplasmic leaflet (Fig. 2A) (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar), the side of the membrane where SM synthesis is known to occur (5Futerman A.H. Stieger B. Hubbard A.L. Pagano R.E. J. Biol. Chem. 1990; 265: 8650-8657Abstract Full Text PDF PubMed Google Scholar, 10van Helvoort A. van't Hof W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar). This strongly suggests that SM synthases adapted an LPP-type reaction chemistry to catalyze the choline phosphotransferase reaction. As outlined in Fig. 3, this reaction is bi-directional and likely proceeds through the following steps: (i) binding of a two-chain choline phospholipid, PC or SM, to a single binding site; (ii) nucleophilic attack on the lipid-phosphate ester bond by the histidine in C3 assisted by the conserved aspartate in this motif; (iii) formation of a choline phosphohistidine intermediate and release of DAG or ceramide, facilitated by the histidine in C2 acting as a base; (iv) nucleophilic attack of the primary hydroxyl of ceramide or DAG on the choline phosphohistidine intermediate assisted by the histidine in C2; (v) release of SM or PC from the active site to allow another round of catalysis.FIGURE 3Putative reaction mechanism of SMS-mediated SM synthesis. As the simplest model, we propose an LPP-like reaction mechanism that involves a single lipid binding site and proceeds via formation of a choline phosphohistidine intermediate along the following steps. 1, PC binds to the enzyme; 2, the phosphocholine head group is transferred to a conserved histidine residue in the enzyme's active site; 3, while the head group stays bound to the enzyme, DAG is replaced by ceramide; 4, the phosphocholine head group is transferred to ceramide forming SM, which is then released from the enzyme. All steps in this reaction mechanism are reversible, thus satisfying the experimental observation that SM and DAG also can be converted to PC and ceramide (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Consistent with the reported enzymatic characteristics of mammalian SM synthases (10van Helvoort A. van't Hof W. Ritsema T. Sandra A. van Meer G. J. Biol. Chem. 1994; 269: 1763-1769Abstract Full Text PDF PubMed Google Scholar, 32Marggraf W.D. Kanfer J.N. Biochim. Biophys. Acta. 1984; 793: 346-353Crossref PubMed Scopus (48) Google Scholar), SMS1 and SMS2 function as bi-directional lipid choline phosphotransferases capable of converting PC and ceramide into SM and DAG and vice versa (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar). Directionality of the reaction would be primarily determined by the relative concentrations of the phosphocholine acceptors ceramide and DAG in the membrane. However, because the latter compounds are potent modulators of cell behavior, SMS enzymes are likely subject to additional levels of control. For example, SMS1 contains a predicted SAM (sterile α motif) domain at its N terminus that might provide a means for the enzyme to interact with regulatory proteins. SAM domains can bind to SH2 (Src homology 2) domains or other SAM domains and are often in proteins in Biochem. 2003; 28: Full Text Full Text PDF PubMed Scopus Google Scholar). the mouse SMS1 gene is subject to alternative and to SMS1 as as two proteins that the SAM domain and the first two of the six domains G. C. 2005; PubMed Scopus Google Scholar). be of to whether these SMS1 which an active in the of SM synthesis. Consistent with the of SM in SMS homologues are throughout the animal from mammals and to like the each organism capable of SM production a multiplicity of SMS genes in its Fig. two SMS sequences have been identified in P. and the contains SMS at two of which function as SM synthases (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google of SM, EPC, IPC, and SMS family EPC (11Muehlenberg B.A. Sribney M. Duffe M.K. Can J. Biochem. 1972; 50: 166-173Crossref PubMed Scopus (13) Google M. Maurice A. Baraud J. J. Lipid Res. 1986; 27: 251-260Abstract Full Text PDF PubMed Google in are M. J. Biochem. PubMed Scopus Google not K. K. M. H. F. 28: PubMed Scopus Google EPC H. A. Biochim. Biophys. Acta. 1972; PubMed Scopus Google A. S. K. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google K. J. Cell Biol. 1994; PubMed Scopus Google (1Dickson R.C. Annu. Rev. Biochem. 1998; 67: 27-48Crossref PubMed Scopus (236) Google J.A. 2004; PubMed Scopus Google in are not in a new to SMS1 and SMS2, the human contains a gene of function. This gene is homologues occur in and the (Fig. SMS1 and SMS2 homologues and not SM. this organism the SM EPC A. S. K. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). EPC is no EPC synthase has been identified to SM and EPC synthesis a reaction mechanism (12Malgat M. Maurice A. Baraud J. J. Lipid Res. 1986; 27: 251-260Abstract Full Text PDF PubMed Google Scholar, 13Malgat M. Maurice A. Baraud J. J. Lipid Res. 1987; 28: 138-143Abstract Full Text PDF PubMed Google Scholar). SMS1, SMS2, and are and conserved motifs with active site residues (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar), proteins are for the EPC Fig. the in this the of SM synthesis and their potential impact on cellular The of SMS1 and SMS2 in mammals (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar, G. C. 2005; PubMed Scopus Google Scholar) that most mammalian cell types two SM SMS1 in the trans Golgi and SMS2 at the plasma membrane. SMS1 is to SMS2 with to ceramide from the ER, may that SMS1 is primarily for the bulk of cellular SM. cells to to ceramide from the plasma membrane. This transport which may to of the pool of ceramide with the signaling pool of ceramide at the plasma membrane, is by the of GlcCer synthase on the Golgi SMS1 in the Golgi lumen, and ceramide transfer protein in the cytosol with the latter that no ceramide on the ER surface can the in the SMS2 not to the de novo SM synthesis, role it Like SMS1, SMS2 is a phosphocholine that PC or SM as and ceramide or DAG as acceptors in any (6Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar). the of PC and SM at the plasma membrane, the reaction catalyzed by SMS2 in the first be by the relative concentrations of ceramide and an is that SMS2 to the local of DAG and ceramide by and SMases in its and a role in mitogenic and pro-apoptotic lipid signaling at the plasma membrane. However, the of ceramide and DAG on cell proliferation and life not to that cells to the SMS-mediated of these their relative concentrations in the membrane. The of SM synthases in animal cells a of with potential for SM formation is for cell growth and then SMS1 and SMS2 in these these enzymes or biological because of their with cellular How is the site of SM synthesis for membrane and the organization of For example, would SMS1 would be from the Golgi to the the EPC is this enzyme EPC as postulated more then (11Muehlenberg B.A. Sribney M. Duffe M.K. Can J. Biochem. 1972; 50: 166-173Crossref PubMed Scopus (13) Google Scholar), to the de novo SM can cells or with EPC methylation as the pathway for SM that EPC and no SM, can EPC synthesis for SM synthesis in mammalian cells it would occur at the is the SMS family in and in each SMS family in these organisms as SM synthase, or catalyze a How cells SM synthesis in with their are the by which cells the and of the reactions catalyzed by SMS a of these would have not to However, with the identification of a SM synthase family in a has been the of which can be used to the pathways, biological and of SM synthesis in for on the