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• W2092124807 abstract "Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS. Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa1 Δfaa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS. Hfd1 and ALDH3A2 are aldehyde dehydrogenases involved in sphingolipid metabolism The S1P-to-glycerolipid metabolic pathway is conserved between yeast and mammals Long-chain bases are metabolized to ether-linked glycerolipids in ALDH3A2 null cells Hexadecenal, hexadecenoic acid, and hexadecenoyl-CoA are metabolic products of S1P Sphingosine 1-phosphate (S1P) plays important roles as an intracellular signaling molecule and as a ligand for G protein-coupled receptors (S1P1–S1P5) (Kihara et al., 2007Kihara A. Mitsutake S. Mizutani Y. Igarashi Y. Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate.Prog. Lipid Res. 2007; 46: 126-144Crossref PubMed Scopus (138) Google Scholar, Pyne and Pyne, 2011Pyne S. Pyne N.J. Translational aspects of sphingosine 1-phosphate biology.Trends Mol. Med. 2011; 17: 463-472Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The FDA-approved immunomodulator FTY720 undergoes in vivo phosphorylation, then mimics S1P ligand functions (Pyne and Pyne, 2011Pyne S. Pyne N.J. Translational aspects of sphingosine 1-phosphate biology.Trends Mol. Med. 2011; 17: 463-472Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). S1P also functions as an intermediate in the metabolism of sphingolipids, which function in biological processes and are major components of eukaryotic plasma membranes (Kihara et al., 2007Kihara A. Mitsutake S. Mizutani Y. Igarashi Y. Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate.Prog. Lipid Res. 2007; 46: 126-144Crossref PubMed Scopus (138) Google Scholar). Sphingolipid metabolism involves salvage pathways that generate the sphingolipid backbone ceramide (Cer) and/or its component sphingosine (Sph), which are then utilized to rebuild sphingolipids. Alternatively, the Sph is phosphorylated by a Sph kinase to form S1P (Kihara et al., 2007Kihara A. Mitsutake S. Mizutani Y. Igarashi Y. Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate.Prog. Lipid Res. 2007; 46: 126-144Crossref PubMed Scopus (138) Google Scholar). S1P can be metabolized to glycerolipids through degradation involving a S1P lyase, which cleaves S1P into a fatty aldehyde (hexadecenal) and phosphoethanolamine (Kihara et al., 2007Kihara A. Mitsutake S. Mizutani Y. Igarashi Y. Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate.Prog. Lipid Res. 2007; 46: 126-144Crossref PubMed Scopus (138) Google Scholar) (Figure 1A ). Disorders in sphingolipid metabolism, termed sphingolipidoses, are well known, with ∼40 having been identified thus far (Sillence and Platt, 2003Sillence D.J. Platt F.M. Storage diseases: new insights into sphingolipid functions.Trends Cell Biol. 2003; 13: 195-203Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). However, to date, no such disorder has been associated with S1P or its metabolism. Beyond the generation of hexadecenal, no precise metabolic pathway has been determined for S1P-to-glycerolipid conversion, and little is understood regarding the enzymes involved or any disorders arising from their disruption. Considering that aldehyde molecules are highly reactive, for example with primary amines (James and Zoeller, 1997James P.F. Zoeller R.A. Isolation of animal cell mutants defective in long-chain fatty aldehyde dehydrogenase. Sensitivity to fatty aldehydes and Schiff's base modification of phospholipids: implications for Sjögren-Larsson syndrome.J. Biol. Chem. 1997; 272: 23532-23539Crossref PubMed Scopus (56) Google Scholar), it would be expected for aberrations in S1P metabolism to lead to pathological disorders. Cer comprises a long-chain base (LCB) attached to fatty acid (FA) via an amide bond. Sph, the major LCB in mammals, contains a trans-double bond between C4 and C5, while another LCB, dihydrosphingosine (DHS), has no double bond (Figure 1A). DHS is generated via the de novo sphingolipid synthesis pathway and is metabolized similarly to S1P (Figure 1A). The metabolic pathway of DHS1P has already been identified (Kihara et al., 2007Kihara A. Mitsutake S. Mizutani Y. Igarashi Y. Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate.Prog. Lipid Res. 2007; 46: 126-144Crossref PubMed Scopus (138) Google Scholar) (Figure 1A), yet it is unknown when and how the trans-double bond derived from S1P is converted to the saturated bond. Moreover, the fatty aldehyde dehydrogenase (FALDH), the acyl-CoA synthetase (ACS), and the saturating enzyme involved in S1P/DHS1P metabolic pathway all remain unidentified. ALDH3A2 is an FALDH that exhibits activity toward saturated and unsaturated aliphatic aldehydes of 6–24 carbons in length (Kelson et al., 1997Kelson T.L. Secor McVoy J.R. Rizzo W.B. Human liver fatty aldehyde dehydrogenase: microsomal localization, purification, and biochemical characterization.Biochim. Biophys. Acta. 1997; 1335: 99-110Crossref PubMed Scopus (117) Google Scholar). Mutations in the ALDH3A2 gene cause Sjögren-Larsson syndrome (SLS), the neurocutaneous disorder characterized by congenital ichthyosis, mental retardation, and spasticity (De Laurenzi et al., 1996De Laurenzi V. Rogers G.R. Hamrock D.J. Marekov L.N. Steinert P.M. Compton J.G. Markova N. Rizzo W.B. Sjögren-Larsson syndrome is caused by mutations in the fatty aldehyde dehydrogenase gene.Nat. Genet. 1996; 12: 52-57Crossref PubMed Scopus (230) Google Scholar, Rizzo, 2007Rizzo W.B. Sjögren-Larsson syndrome: molecular genetics and biochemical pathogenesis of fatty aldehyde dehydrogenase deficiency.Mol. Genet. Metab. 2007; 90: 1-9Crossref PubMed Scopus (117) Google Scholar). Although accumulated fatty aldehydes in SLS patients are thought to be responsible for the pathology, the source of the accumulation remains unclear. In the present study, we have identified ALDH3A2 as the FALDH active within the S1P metabolic pathway. ACSs catalyze the conversion of FAs to their activated forms, i.e., acyl-CoAs. The yeast Saccharomyces cerevisiae and mammals have 7 and 26 ACSs, respectively (Black and DiRusso, 2007Black P.N. DiRusso C.C. Yeast acyl-CoA synthetases at the crossroads of fatty acid metabolism and regulation.Biochim. Biophys. Acta. 2007; 1771: 286-298Crossref PubMed Scopus (154) Google Scholar, Watkins et al., 2007Watkins P.A. Maiguel D. Jia Z. Pevsner J. Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome.J. Lipid Res. 2007; 48: 2736-2750Crossref PubMed Scopus (236) Google Scholar). In the present study, we identified Faa1 and Faa4 in yeast and ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6 in mammals as ACSs responsible for S1P/DHS1P metabolic pathways. We also demonstrated that hexadecenoic acid is the intermediate of S1P metabolism. From these results, we determined the entire S1P metabolic pathway. Metabolism of LCBs/LCB 1-phosphates (LCBPs) and the enzymes responsible are mostly conserved from yeast to man. Therefore, we first used the yeast S. cerevisiae for identification of the FALDH involved in the metabolism of LCBs/LCBPs. Wild-type yeast cells and eight yeast strains carrying mutations in genes encoding aldehyde dehydrogenases (Δhfd1, Δald2, Δald3, Δald4, Δald5, Δald6, Δuga2, and Δmsc7), as well as a mutant for the LCBP lyase gene (Δdpl1) (as a control), were labeled with [4,5-3H]DHS. In wild-type cells, DHS was metabolized to both sphingolipids (Cer, inositol phosphorylceramide [IPC], mannosylinositol phosphorylceramide [MIPC], and mannosyldiinositol phosphorylceramide [M(IP)2C]) and glycerolipids (phosphatidylethanolamine [PE], phosphatidylcholine [PC], phosphatidylserine [PS], and phosphatidylinositol [PI]) (Figure 2A and Figure S1A). In contrast, DHS was not converted to glycerolipids in the Δdpl1 cells (Figure 2A), as expected from its function in the LCB/LCBP metabolic pathway (Figure 1B). Of the eight aldehyde dehydrogenase mutants, only the Δhfd1 cells exhibited a deficiency in DHS-to-glycerolipid conversion (Figure 2A), indicating that Hfd1 is the FALDH involved in the metabolism of DHS/DHS1P in yeast. In the Δhfd1 cells, hexadecanol but not hexadecanal accumulated (Figure S1B), suggesting that some unmetabolized hexadecanal was reduced to hexadecanol. However, the accumulated hexadecanol levels were lower than would be expected when considering the sum of glycerolipid levels in wild-type cells (Figure 2A and Figures S1A and S1B). We speculate that most of the unmetabolized hexadecanal might have reacted with several cellular components having primary amines before being converted to hexadecanol. To date, no function for Hfd1 has been identified. Homology searches revealed that, among mammalian aldehyde dehydrogenases, Hfd1 shares the highest sequence similarity with mammalian ALDH3A2 (30.0% identity and 50.6% similarity). The ALDH3A2 gene is known to be responsible for the neurocutaneous disorder SLS (Rizzo, 2007Rizzo W.B. Sjögren-Larsson syndrome: molecular genetics and biochemical pathogenesis of fatty aldehyde dehydrogenase deficiency.Mol. Genet. Metab. 2007; 90: 1-9Crossref PubMed Scopus (117) Google Scholar). ALDH3A2 reportedly exhibits activity toward saturated and unsaturated aliphatic aldehydes ranging from 6–24 carbons in length (Kelson et al., 1997Kelson T.L. Secor McVoy J.R. Rizzo W.B. Human liver fatty aldehyde dehydrogenase: microsomal localization, purification, and biochemical characterization.Biochim. Biophys. Acta. 1997; 1335: 99-110Crossref PubMed Scopus (117) Google Scholar). To establish that ALDH3A2 is involved in the DHS/DHS1P metabolism, we introduced the human ALDH3A2 gene into the Δhfd1 yeast cells and examined the metabolism of [4,5-3H]DHS. Introduction of the ALDH3A2 gene resulted in the recovery of DHS conversion to glycerolipids in cells deficient in Δhfd1 (Figure 2B), indicating that ALDH3A2 and Hfd1 are functional homologs and are involved in DHS/DHS1P metabolism. A lack of radiolabeled S1P or Sph for use as tracers has hampered study into their metabolic pathway. Commercially available [3-3H]Sph can label sphingolipids, but not glycerolipids, since the 3-3H atom is removed during S1P conversion to hexadecenal by S1P lyase (Figures S2A and S2B). In contrast, 4,5-3H-labeled DHS enables monitoring of its metabolism both to sphingolipids and to glycerolipids (Figures S2A and S2B). In a late 1960s study, [7-3H]Sph administered intravenously to a rat was converted in the liver to both sphingolipids and glycerolipids, and the major Sph-derived FA in glycerolipids was palmitic acid (Stoffel and Sticht, 1967Stoffel W. Sticht G. Metabolism of sphingosine bases, I. Degradation and incorporation of (3-14C)erythro-DL-dihydrosphingosine and (7-3H2)erythro-DL-sphingosine into sphingolipids of rat liver.Hoppe Seylers Z. Physiol. Chem. 1967; 348: 941-943PubMed Google Scholar). No further analysis of Sph-to-glycerolipid metabolism has been performed, leaving a gap in our knowledge of this important metabolic pathway. To address this deficit, we prepared traceable [11,12-3H]Sph from [9,10-3H]palmitic acid. When [11,12-3H]Sph was separated by TLC with commercially available [3-3H]Sph and [4,5-3H]DHS, [11,12-3H]Sph presented as a single band that migrated at the position identical to [3-3H]Sph (Figure 3A ). When incubated with Sph kinase, [11,12-3H]Sph was completely converted to S1P (Figure 3B). These results indicate that our [11,12-3H]Sph is highly pure. To date, Sph-to-glycerolipid metabolism has not been examined using mammalian cultured cells, so it remains unclear whether Sph is converted to glycerolipids as efficiently as DHS. To test this, human embryonic kidney (HEK) 293T cells were labeled with [4,5-3H]DHS or [11,12-3H]Sph. DHS and Sph were metabolized to sphingolipids and to glycerolipids with similar efficiency (Figure 3C). In this experiment, half of the samples were treated with alkaline solution. Since ester linkages in glycerolipids are hydrolyzed by alkaline treatment, glycerolipid bands disappeared, while sphingolipid bands remained unchanged (Figure 3C). Using another cell line, mouse embryonic carcinoma F9 cells, we repeated the labeling experiments. We also used F9 SPL−/− cells, which lack the S1P lyase SPL, as a negative control that cannot convert DHS or Sph to glycerolipids (Ikeda et al., 2005Ikeda M. Kihara A. Kariya Y. Lee Y.M. Igarashi Y. Sphingolipid-to-glycerophospholipid conversion in SPL-null cells implies the existence of an alternative isozyme.Biochem. Biophys. Res. Commun. 2005; 329: 474-479Crossref PubMed Scopus (12) Google Scholar). Again, Sph and DHS were similarly metabolized to sphingolipids and to glycerolipids in wild-type F9 cells, but were metabolized only to sphingolipids in the SPL−/− cells (Figure 3D). Cer and glucosylceramide (GlcCer), observed at high levels after a 2 hr labeling period, had decreased by 24 hr. Instead, lactosylceramide (LacCer), which is a further metabolite of GlcCer, and the glycerolipids PE and PS/PI had increased. These results indicate that DHS and Sph are similarly metabolized in mammalian cells. Natural LCBs found in the yeast S. cerevisiae include DHS and phytosphingosine (Figure 1B). Sph is not found in the yeast, yet the yeast can still use exogenously added Sph as a precursor for sphingolipids (Tani et al., 2006Tani M. Kihara A. Igarashi Y. Rescue of cell growth by sphingosine with disruption of lipid microdomain formation in Saccharomyces cerevisiae deficient in sphingolipid biosynthesis.Biochem. J. 2006; 394: 237-242Crossref PubMed Scopus (24) Google Scholar). We labeled yeast cells with [11,12-3H]Sph and examined its metabolism and compared it with that of labeled DHS. In contrast to their conversion in mammalian cells, DHS and Sph were not metabolized similarly in wild-type yeast cells. DHS was metabolized efficiently to sphingolipids and glycerolipids, whereas Sph was converted mainly to glycerolipids, with a small amount converted to sphingolipids (Figure 4A ); it is possible that sphingolipid biosynthetic enzymes such as Cer synthase might have lower activities toward Sph. Again, the DHS-to-glycerolipid conversion was Dpl1 and Hfd1 dependent (Figure 4A). This Sph-to-glycerolipid metabolic pathway was blocked in Δhfd1 cells as in Δdpl1 cells (Figure 4A), indicating that Hfd1 catalyzes not only conversion of hexadecanal derived from DHS1P to palmitic acid, but also that of hexadecenal (more precisely trans-2-hexadecenal) from S1P to hexadecenoic acid (trans-2-hexadecenoic acid). We also observed accumulation of hexadecenol in the Δhfd1 cells (Figure S1B), which might reflect unmetabolized hexadecenal reduced to hexadecenol. Introduction of human ALDH3A2 cDNA into the Δhfd1 cells restored the Sph-to-glycerolipid conversion (Figure 4B), similar to results in the [4,5-3H]DHS labeling experiment (Figure 2B). To date, the enzymatic activity of ALDH3A2 toward 2-trans-hexadecenal has not been investigated. However, our results suggest that ALDH3A2 is active toward 2-trans-hexadecenal. To confirm this, we performed in vitro assays using affinity-purified 3xFLAG-ALDH3A2. ALDH3A2 did indeed exhibit FALDH activities toward both 2-trans-hexadecenal and hexadecanal, although its activity toward hexadecanal was ∼2-fold higher than that toward 2-trans-hexadecenal (Figure 4C). Sph was proven to be metabolized to palmitic acid in glycerolipids in mammals (Stoffel and Sticht, 1967Stoffel W. Sticht G. Metabolism of sphingosine bases, I. Degradation and incorporation of (3-14C)erythro-DL-dihydrosphingosine and (7-3H2)erythro-DL-sphingosine into sphingolipids of rat liver.Hoppe Seylers Z. Physiol. Chem. 1967; 348: 941-943PubMed Google Scholar). However, it was unclear whether in yeast the FA in glycerolipids converted from Sph is also palmitic acid or if it is hexadecenoic acid still containing the 2-trans double bond. To examine the FA species in glycerolipids derived from Sph, lipids were extracted from wild-type yeast cells labeled with [11,12-3H]Sph, treated with alkaline solution to release FAs, and separated by TLC using hexane/diethylether/acetic acid (30:70:1, v/v), a resolving buffer that can separate palmitic acid and hexadecenoic acid. The FA derived from Sph migrated at a position identical to that of palmitic acid, but not hexadecenoic acid (Figure 4D). Similarly, FA derived from [11,12-3H]Sph in mammalian cells and FAs derived from [4,5-3H]DHS in either yeast or mammalian cells all migrated at a position identical to that of palmitic acid, but not hexadecenoic acid (Figure 4D). These results indicate that both Sph and DHS are converted to palmitic acid irrespective of yeast and mammalian cells. Molecular species of FA derived from Sph in yeast was also examined by double bond cleavage assay. Carbon-to-carbon double bonds can be cleaved with KIO4 in the presence of KMnO4 (Scarim et al., 1989Scarim J. Ghanbari H. Taylor V. Menon G. Determination of phosphatidylcholine and disaturated phosphatidylcholine content in lung surfactant by high performance liquid chromatography.J. Lipid Res. 1989; 30: 607-611PubMed Google Scholar). This method can determine whether FA derived from Sph in yeast is saturated palmitic acid or unsaturated hexadecenoic acid. Control palmitic acid was indeed resistant to KIO4/KMnO4 treatment, whereas hexadecenoic acid was sensitive (Figure 4E). Both the FAs metabolized from [11,12-3H]Sph in either HEK293T cells or yeast were resistant to KIO4/KMnO4 treatment, confirming the above results that FA in glycerolipids converted from Sph is palmitic acid both in yeast and in mammals. Taken together, these results affirm that both Sph and DHS are converted to glycerolipids after conversion to palmitoyl-CoA. Thus, the Sph-to-glycerolipid metabolic pathway is highly conserved between yeast and mammals. Aldehyde molecules are toxic to cells due to their high reactivity (James and Zoeller, 1997James P.F. Zoeller R.A. Isolation of animal cell mutants defective in long-chain fatty aldehyde dehydrogenase. Sensitivity to fatty aldehydes and Schiff's base modification of phospholipids: implications for Sjögren-Larsson syndrome.J. Biol. Chem. 1997; 272: 23532-23539Crossref PubMed Scopus (56) Google Scholar). We examined the toxicity of endogenously generated hexadecanal and hexadecenal by adding DHS or Sph to the medium of wild-type, Δhfd1, or Δdpl1 yeast cells. The Δhfd1 cells were more sensitive both to DHS and to Sph, as compared to wild-type cells (Figures S3A and S3B). The Δdpl1 cells were also sensitive to DHS and Sph, but especially to Sph (Figure S3A). However, in the Δdpl1 cells, toxicities were caused by an accumulation of LCBPs, and the more toxic effect of Sph reflects higher accumulation levels of S1P compared to DHS1P in the Δdpl1 cells (Figure 4A) (Zhang et al., 2001Zhang X. Skrzypek M.S. Lester R.L. Dickson R.C. Elevation of endogenous sphingolipid long-chain base phosphates kills Saccharomyces cerevisiae cells.Curr. Genet. 2001; 40: 221-233Crossref PubMed Scopus (50) Google Scholar). In the Δhfd1 cells, both S1P and DHS1P levels were similar to those in wild-type cells (Figure 4A), so the sensitivity to exogenous LCBs observed for these cells may be caused by toxicities of endogenously generated fatty aldehydes. We next investigated the involvement of ALDH3A2 in LCB/LCBP metabolism using mammalian cells. FAA-K1A cells were isolated from CHO-K1 cells as a FALDH mutant cell line; these cells displayed ∼10% of the FALDH activity compared to CHO-K1 cells (James and Zoeller, 1997James P.F. Zoeller R.A. Isolation of animal cell mutants defective in long-chain fatty aldehyde dehydrogenase. Sensitivity to fatty aldehydes and Schiff's base modification of phospholipids: implications for Sjögren-Larsson syndrome.J. Biol. Chem. 1997; 272: 23532-23539Crossref PubMed Scopus (56) Google Scholar). RT-PCR revealed that ALDH3A2 mRNA was barely detectable in the FAA-K1A cells (Figure 5A ). CHO-K1 and FAA-K1A cells were labeled with [4,5-3H]DHS, and lipids were separated by TLC. DHS was metabolized to both sphingolipids and glycerolipids in CHO-K1 cells (Figure 5B), similar to results in the HEK293T and F9 cells described above (Figures 3C and 3D). In contrast, in FAA-K1A cells, the PC level was greatly reduced, but another lipid, which migrated adjacent to and slightly above PC, was produced (Figure 5B, arrowhead). This lipid was determined to be another type of choline glycerophospholipid, ether-linked glycerophospholipid (1-alkyl/alkenyl-2-acyl-glycero-3-phosphocholine), since it was not converted to FA but to 1-alkyl (or alkenyl)-glycerophosphocholine (GPC) by alkaline treatment (Figure 5B, dot), due to an alkaline-resistant ether bond. Little [4,5-3H]DHS was converted to PS or PI in the FAA-K1A cells. PE was also reduced, but again ether-linked ethanolamine glycerophospholipid (1-alkyl/alkenyl-2-acyl-glycero-3-phosphoethanolamine) appeared in FAA-K1A cells (Figure 5B, arrowhead). In mammals, most ether-linked glycerophospholipids contain choline or ethanolamine as a polar group and can be divided into plasmanyl type glycerophospholipids (plasmanylcholine and plasmanylethanolamine) containing a 1-alkyl group, and plasmenyl type glycerophospholipids (plasmenylcholine and plasmenylethanolamine) containing a 1-alkenyl group. The plasmenyl type glycerophospholipids are also called plasmalogens, and these play a variety of cellular functions, including functioning in membrane dynamics, acting as a source of arachidonic acid for generation of lipid mediators, intracellular signaling, and performing as antioxidants (Nagan and Zoeller, 2001Nagan N. Zoeller R.A. Plasmalogens: biosynthesis and functions.Prog. Lipid Res. 2001; 40: 199-229Crossref PubMed Scopus (460) Google Scholar). To determine whether the observed ether-linked glycerophospholipids were plasmanyl or plasmenyl type, we treated the labeled lipids with acid. Plasmenyl glycerophospholipids (plasmalogens) are known to be sensitive to acid due to a reactive vinyl ether double bond (Nagan and Zoeller, 2001Nagan N. Zoeller R.A. Plasmalogens: biosynthesis and functions.Prog. Lipid Res. 2001; 40: 199-229Crossref PubMed Scopus (460) Google Scholar) (Figure S4A). The ether-linked choline glycerophospholipid was resistant to acid, whereas the ether-linked ethanolamine glycerophospholipid was sensitive (Figure 5B), indicating that they were plasmanylcholine and plasmenylethanolamine, respectively. Similar results were obtained when cells were labeled with [11,12-3H]Sph (Figure 5B). In both labeling experiments, the band intensities of 1-alkyl-GPC and 1-alkenyl-glycerophosphoethanolamine (GPE) (Figure 5B, dots) were weaker than those of the original plasmanylcholine and plasmenylethanolamine (Figure 5B, arrowheads). This suggests that LCBs were metabolized both to the sn-1 alkyl/alkenyl groups and the sn-2 acyl group of the plasmanylcholine/plasmenylethanolamine. Our results suggest that fatty aldehyde derived from LCBs that had accumulated due to deficiency in ALDH3A2 was converted to fatty alcohol and utilized in the synthesis of ether-linked glycerolipids. To confirm that ALDH3A2 is indeed responsible for the reduced ester-linked glycerolipid labeling and the generation of label in ether-linked glycerolipids in the FAA-K1A cells, we generated FAA-K1A cells stably expressing 3xFLAG-ALDH3A2. Two independent cell lines (FAA-ALDH3A2.A and FAA-ALDH3A2.B cells) expressed 3xFLAG-ALDH3A2 at similar levels (Figure S4B). A [4,5-3H]DHS labeling experiment revealed that the plasmenylethanolamine and plasmanylcholine observed in FAA-K1A cells both disappeared in these stable clones, and PC and PS/PI levels were restored to the levels found in CHO-K1 cells (Figure S4C). Thus, a lack of ALDH3A2 causes metabolism of LCBs/LCBPs to ether-linked glycerolipids in CHO-K1 cells. FAs must be converted to acyl-CoAs prior to their incorporation into glycerolipids. However, the ACSs involved in LCB/LCBP metabolism have not been identified. Yeasts have seven ACSs (Faa1, Faa2, Faa3, Faa4, Fat1, Fat2, and Acs1). To identify which ACSs are involved in LCB/LCBP metabolism in yeast, a [4,5-3H]DHS labeling experiment was performed using single-deletion mutants or double-deletion mutants of the yeast ACS genes. Of the mutants, only the Δfaa1 Δfaa4 double-deletion mutant was defective in converting DHS to glycerolipids (Figure 6A ), indicating that Faa1 and Faa4 function redundantly in the DHS/DHS1P metabolic pathway. When [11,12-3H]Sph was used for labeling, abundant glycerolipids produced in wild-type cells were greatly reduced in Δfaa1 Δfaa4 cells (Figure 6B), and instead, accumulation of FA, the substrate of the ACSs, was observed (Figure 6B, dot). Thus, Faa1 and Faa4 function not only in DHS/DHS1P metabolism, but also in the Sph/S1P metabolic pathway in yeast. To identify the molecular species of the accumulated FA, the Δfaa1 Δfaa4 cells were labeled with [11,12-3H]Sph, and lipids were separated by TLC using as a solvent system hexane/diethylether/acetic acid (30:70:1, v/v), which can separate palmitic acid and hexadecenoic acid. The FA accumulated in Δfaa1 Δfaa4 cells labeled with [11,12-3H]Sph migrated to a position identical to that of hexadecenoic acid (Figure 6C) and was sensitive to KIO4/KMnO4 treatment (Figure 6D), whereas the FA generated by [4,5-3H]DHS labeling migrated to a position identical to that of palmitic acid (Figure 6C). These results indicate that the Δfaa1 Δfaa4 cells accumulated hexadecenoic acid as a [11,12-3H]Sph metabolite, which is in contrast to wild-type cells, in which [11,12-3H]Sph was metabolized to palmitic acid, a constituent of glycerolipids (Figures 4D and 4E). Considering that hexadecenoic acid is a substrate of the ACSs Faa1 and Faa4, the product should be hexadecenoyl-CoA. Thus, it is most probable that hexadecenoyl-CoA is converted to palmitoyl-CoA before incorporation into glycerolipids. We next investigated the sensitivity of the Δfaa1 Δfaa4 cells to exogenous LCBs and compared it to that of the other tested mutants. In contrast to the Δhfd1 cells, the Δfaa1 Δfaa4 cells were not sensitive to exogenous DHS or Sph, indicating that accumulated hexadecenoic acid is not toxic to these cells (Figure S3B). Thus, toxic fatty aldehydes are detoxified by Hfd1. Of the 26 mammalian ACSs, Faa1 and Faa4 share the highest homology with the ACSL subfamily members ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6, with ACSL4 exhibiting the highest similarity overall (26.6% identity and 47.7% similarity to Faa1 and 30.3% identity and 47.9% similarity to Faa4). When these proteins were expressed in Δfaa1 Δfaa4 cells as N-terminally 3xFLAG-tagged proteins, all restored Sph-to-glycerolipid conversion (Figure 6E). However, two other ACS subfamily members, ACSS1 and ACSM1, exhibited no activity, and restoration was weak for ACSL3, probably due to low expression levels (Figure 6F). These results indicate that ACSL family members can also convert hexadecenoic acid to hexadecenoyl-CoA, in a manner similar to that observed for yeast Faa1 and Faa4. Aside from its well-known function as a bioactive lipid molecule, S1P is also important as an intermediate in the sphingolipid-to-glycerolipid conversion pathway. However, certain steps in this metabolic pathway and the metabolic enzymes involved with those steps remain unresolved. At least three pathways were considered in studying the conversion of hexadecenal (the product of S1P by S1P lyase) to palmitoyl-CoA (Figure S5, Schemes 1 to 3). It is possible that hexadecenal is first converted to hexadecanal, then to palmi" @default.
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• W2092124807 title "The Sjögren-Larsson Syndrome Gene Encodes a Hexadecenal Dehydrogenase of the Sphingosine 1-Phosphate Degradation Pathway" @default.
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