Sphingolipids, a major lipid class in eukaryotic cells, are characterized by a sphingoid base backbone, often 18-carbon sphingosine. Their biosynthesis occurs through de novo synthesis and via sphingoid base salvage and recycling into the sphingolipid synthesis pathway (1Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics., 2Sphingolipids and their metabolism in physiology and disease., 3Sandhoff R. Schulze H. Sandhoff K. Ganglioside Metabolism in Health and Disease.). De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER) and is initiated by serine palmitoyltransferase. Subsequent enzyme reactions in the ER produce ceramide, composed of a sphingoid base and a fatty acid. Ceramide is modified to generate plasma membrane sphingolipids—sphingomyelin and glycosphingolipids—by the addition of hydrophilic head groups. In the salvage process, sphingoid bases derived from sphingolipids are recycled for the synthesis of ceramide.Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that is transported into the circulation and regulates key physiological functions through interactions with five G protein—coupled receptors (4Emerging biology of sphingosine-1-phosphate: its role in pathogenesis and therapy., 5Green C.D. Maceyka M. Cowart L.A. Spiegel S. Sphingolipids in metabolic disease: the good, the bad, and the unknown.). Changes in sphingolipid metabolic enzymes and transporters that modify extracellular S1P levels have dramatic physiological effects due to altered S1P receptor signaling (6Fifty years of lyase and a moment of truth: sphingosine phosphate lyase from discovery to disease., 7Olivera A. Allende M.L. Proia R.L. Shaping the landscape: metabolic regulation of S1P gradients., 8Baeyens A.A.L. Schwab S.R. Finding a way out: S1P signaling and immune cell migration.). The rapid clearance of S1P from blood in vivo suggests that cellular-uptake mechanisms regulate its levels (9Peest U. Sensken S.C. Andreani P. Hanel P. Van Veldhoven P.P. Graler M.H. S1P-lyase independent clearance of extracellular sphingosine 1-phosphate after dephosphorylation and cellular uptake., 10Venkataraman K. Lee Y.M. Michaud J. Thangada S. Ai Y. Bonkovsky H.L. et al.Vascular endothelium as a contributor of plasma sphingosine 1-phosphate., 11Salous A.K. Panchatcharam M. Sunkara M. Mueller P. Dong A. Wang Y. et al.Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice.). Indeed, a blood S1P clearance pathway mediated by hepatocytes has been described, which leads to its degradation by S1P lyase to form phosphoethanolamine and a long-chain aldehyde and subsequently results in its removal from the sphingolipid synthesis pathway (12Kharel Y. Huang T. Salamon A. Harris T.E. Santos W.L. Lynch K.R. Mechanism of sphingosine 1-phosphate clearance from blood.). However, the other possible metabolic fate of S1P taken up by cells–sphingoid base salvage for ceramide and complex sphingolipid synthesis–has not been clearly delineated. Here, we use a genome-wide clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 KO screen in HeLa cells to identify the genes that populate this pathway.DiscussionS1P is an extracellular-signaling sphingolipid whose metabolism regulates its signaling activity. It has two major intracellular metabolic fates (1Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics., 2Sphingolipids and their metabolism in physiology and disease., 3Sandhoff R. Schulze H. Sandhoff K. Ganglioside Metabolism in Health and Disease.): 1) degradation by S1P lyase and removal of sphingoid base substrate from the sphingolipid pathway or 2) salvage and recycling of its sphingoid base for sphingolipid synthesis. Recent studies have illuminated the pathway for the cellular uptake and S1P lyase–mediated degradation of extracellular S1P in hepatocytes (12Kharel Y. Huang T. Salamon A. Harris T.E. Santos W.L. Lynch K.R. Mechanism of sphingosine 1-phosphate clearance from blood.). However, the pathway used for intracellular salvage of the sphingoid base from extracellular S1P and its recycling has not been clearly defined. We have used a genome-wide CRISPR/Cas9 KO screen to identify the genes responsible for this S1P cellular uptake and sphingoid base salvage for recycling into the sphingolipid synthesis pathway.Among the genes we identified for uptake sphingoid base salvage and recycling were two distinct lipid phosphatases, PLPP3 and SGPP1. Also known as LPP3, PAP-2b, or PPAP2B, PLPP3 is an integral-membrane protein with an extracellular-facing active site that catalyzes the dephosphorylation of a variety of lipid phosphates, including S1P (35Hooks S.B. Ragan S.P. Lynch K.R. Identification of a novel human phosphatidic acid phosphatase type 2 isoform., 36Jamal Z. Martin A. Gomez-Munoz A. Brindley D.N. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol., 37Kai M. Wada I. Imai S. Sakane F. Kanoh H. Cloning and characterization of two human isozymes of Mg2+-independent phosphatidic acid phosphatase., 38Roberts R. Sciorra V.A. Morris A.J. Human type 2 phosphatidic acid phosphohydrolases. Substrate specificity of the type 2a, 2b, and 2c enzymes and cell surface activity of the 2a isoform.). We found that PLPP3 was exclusively expressed at the plasma membrane in WT HeLa cells, which was in agreement with previous studies (39Ishikawa T. Kai M. Wada I. Kanoh H. Cell surface activities of the human type 2b phosphatidic acid phosphatase., 40Kai M. Wada I. Imai S. Sakane F. Kanoh H. Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase., 41Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells.). At the hepatocyte surface, PLPP3 has been shown to dephosphorylate blood S1P, allowing sphingosine to enter hepatocytes for rephosphorylation by Sphk2 and subsequent degradation by S1P lyase (12Kharel Y. Huang T. Salamon A. Harris T.E. Santos W.L. Lynch K.R. Mechanism of sphingosine 1-phosphate clearance from blood.). Similarly, in human myeloid-derived HAP1 cells, the three-member PLPP family, including PLPP3, was demonstrated to be important for the efficient handling of exogenous S1P, although some PLPP-independent uptake was also detected (42Goto H. Miyamoto M. Kihara A. Direct uptake of sphingosine-1-phosphate independent of phospholipid phosphatases.). In human lung endothelial cells, PLPP1 was found to stimulate uptake of the sphingoid base of S1P, which was then subjected to rephosphorylation by SPHK1 (43Zhao Y. Kalari S.K. Usatyuk P.V. Gorshkova I. He D. Watkins T. et al.Intracellular generation of sphingosine 1-phosphate in human lung endothelial cells: role of lipid phosphate phosphatase-1 and sphingosine kinase 1.). Interestingly, adipocyte PLPP3 deficiency was found to regulate sphingolipid synthesis, resulting in reduced ceramide and sphingomyelin accumulation during adipose-tissue expansion (44Federico L. Yang L. Brandon J. Panchatcharam M. Ren H. Mueller P. et al.Lipid phosphate phosphatase 3 regulates adipocyte sphingolipid synthesis, but not developmental adipogenesis or diet-induced obesity in mice.). The other two PLPP family members, although expressed in HeLa cells (Human Protein Atlas proteinatlas.org), were not detected in the screen indicating that PLPP3 is dominant in initiating S1P uptake by dephosphorylation of exogenous S1P.SGPP1 is a lipid phosphatase that specifically catalyzes dephosphorylation of S1P (45Sphingosine-1-phosphate phosphatases.). We found that SGPP1 localized to the ER in HeLa cells, which is consistent with previous findings that SGPP1 and its homologs in yeast (46Mao C. Saba J.D. Obeid L.M. The dihydrosphingosine-1-phosphate phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation and heat stress responses.), mouse (47Le Stunff H. Peterson C. Thornton R. Milstien S. Mandala S.M. Spiegel S. Characterization of murine sphingosine-1-phosphate phosphohydrolase.), and human (48Johnson K.R. Johnson K.Y. Becker K.P. Bielawski J. Mao C. Obeid L.M. Role of human sphingosine-1-phosphate phosphatase 1 in the regulation of intra- and extracellular sphingosine-1-phosphate levels and cell viability.) are ER integral-membrane proteins. Overexpression of SGPP1 has been shown to stimulate the incorporation of sphingosine into ceramide for the production of glycosphingolipids (49Le Stunff H. Giussani P. Maceyka M. Lepine S. Milstien S. Spiegel S. Recycling of sphingosine is regulated by the concerted actions of sphingosine-1-phosphate phosphohydrolase 1 and sphingosine kinase 2.). SGPP2, while structurally and functionally similar to SGPP1 (50Ogawa C. Kihara A. Gokoh M. Igarashi Y. Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase, hSPP2.), is not well expressed in HeLa cells possibly explaining its absence among the hits in the screen (Human Protein Atlas proteinatlas.org).The different locations of PLPP3 and SGPP1 imply that extracellular S1P is dephosphorylated in two disparate subcellular compartments during a dephosphorylation-phosphorylation-dephosphorylation cycle prior to the incorporation of its sphingoid base into the sphingolipid synthesis pathway. In this scheme, the sphingosine product of PLPP3 generated at the plasma membrane would be rephosphorylated in the cytoplasm by the sphingosine kinases. The newly formed intracellular S1P would then be transferred to the ER surface, by a yet to-be-explained process, where it would be dephosphorylated by SGPP1 to generate sphingoid base substrate for ceramide production (Fig. 7). Other top hits in the screen included core biosynthetic enzymes responsible for Gb3 production (CERS2, UGCG, B4GALT5, A4GALT), proteins that support glycosphingolipid synthesis in the Golgi (LAPTM4A, TM9SF2, SLC35A2, TMEM165, GOLPH3), and a transcription factor (AHR) that regulates glycosphingolipid synthesis gene expression (Fig. 7).

Fig. 7Proposed pathway for S1P cellular uptake and incorporation into the sphingolipid synthesis pathway. Plasma membrane–resident PLPP3 dephosphorylates exogenous S1P to sphingosine, which enters cells by a flip-flop mechanism and is phosphorylated to S1P by SPHKs 1 and 2. Intracellular S1P serves as a branch-point substrate for ER-resident SGPP1 and SGPL1. SGPP1 dephosphorylates S1P to sphingosine, which is utilized for ceramide synthesis (CERS2) and sequential modifications for glycosphingolipid production. SGPL1 irreversibly degrades S1P to hexadecenal and phosphoethanolamine, allowing exit of the substrate from the sphingolipid synthesis pathway. These enzymes work in concert to drive an S1P dephosphorylation-phosphorylation-dephosphorylation/degradation cycle. In the absence of sphingosine kinases, sphingosine may bypass the cycle and directly serve as a CERS2 substrate (dashed line), thereby being excluded from the SGPL1-mediated degradation pathway. UGCG, B4GALT5, and A4GALT are Golgi core glycosphingolipid synthesis enzymes for synthesis of Gb3. LAPTM4A, TM9SF2, SLC35A2, TMEM165, and GOLPH3 are positive regulators of Gb3 synthesis in the Golgi. AHR is a transcriptional activator of genes involved in glycosphingolipid synthesis. Deg, degradation; Dephos, dephosphorylation; Gb3, globotriaosylceramide; Phos, phosphorylation; PM, plasma membrane; Sph, sphingosine; S1P, sphingosine-1-phosphate

In yeast, previous studies have indicated that exogenous sphingoid bases undergo a cycle of phosphorylation and dephosphorylation to be efficiently incorporated into ceramides and sphingolipids (51Qie L. Nagiec M.M. Baltisberger J.A. Lester R.L. Dickson R.C. Identification of a Saccharomyces gene, LCB3, necessary for incorporation of exogenous long chain bases into sphingolipids., 52Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-phosphate phosphatase., 53Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. et al.Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response., 54Funato K. Lombardi R. Vallee B. Riezman H. Lcb4p is a key regulator of ceramide synthesis from exogenous long chain sphingoid base in Saccharomyces cerevisiae.). Although this cycle can be bypassed, sphingolipids are synthesized less efficiently if exogenous sphingoid bases cannot be phosphorylated (51Qie L. Nagiec M.M. Baltisberger J.A. Lester R.L. Dickson R.C. Identification of a Saccharomyces gene, LCB3, necessary for incorporation of exogenous long chain bases into sphingolipids., 52Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-phosphate phosphatase., 53Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. et al.Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response., 54Funato K. Lombardi R. Vallee B. Riezman H. Lcb4p is a key regulator of ceramide synthesis from exogenous long chain sphingoid base in Saccharomyces cerevisiae.).Our findings indicate that, in HeLa cells, the sphingosine kinases do not appear to be needed for Gb3 synthesis via the salvage of sphingoid base from extracellular S1P and its recycling into the sphingolipid synthesis pathway. First, our genome-wide CRISPR/Cas9 KO screen identified both phosphatases as positive regulators of sphingoid base recycling into the Gb3 synthesis pathway but did not pick up the sphingosine kinases as regulators of this process. Second, knocking down sphingosine-kinase expression in cells with the de novo sphingolipid synthesis pathway disabled led to elevated Gb3 cell-surface expression, apparently due to enhanced salvage of sphingoid bases for use in sphingolipid synthesis. The enhanced Gb3 cell-surface expression in cells deficient in sphingosine kinase may be due to the inability to produce a substrate for S1P lyase, effectively blocking the degradation pathway and thus salvaging sphingoid bases for incorporation into new sphingolipids (Fig. 7, dashed-line arrow). Indeed, we found a similar enhancement of Gb3 cell-surface expression when S1P lyase was knocked down in HeLa cells. Other studies have also confirmed that disruption of S1P lyase activity stimulates sphingoid base salvage and recycling (55Bektas M. Allende M.L. Lee B.G. Chen W. Amar M.J. Remaley A.T. et al.Sphingosine 1-phosphate lyase deficiency disrupts lipid homeostasis in liver., 56Hagen-Euteneuer N. Lutjohann D. Park H. Merrill Jr., A.H. van Echten-Deckert G. Sphingosine 1-phosphate (S1P) lyase deficiency increases sphingolipid formation via recycling at the expense of de novo biosynthesis in neurons.).S1P, when in circulation, is predominately bound to carrier proteins, HDL, and serum albumin (4Emerging biology of sphingosine-1-phosphate: its role in pathogenesis and therapy.). In our screen, S1P was added to HeLa cells without carrier proteins, a form that was taken up as well as carrier-bound S1P. However, under these conditions, we may not have identified cell surface receptors involved in the uptake of carrier-bound S1P for subsequent entry into the recycling pathway.The dephosphorylation-phosphorylation-dephosphorylation cycle for extracellular S1P provides several key functions. The dephosphorylation of extracellular S1P controls its extracellular levels by allowing lipid uptake into cells. The sphingoid base liberated by PLPP3 may pass though the plasma membrane by a flip-flop mechanism as proposed for hepatocytes (12Kharel Y. Huang T. Salamon A. Harris T.E. Santos W.L. Lynch K.R. Mechanism of sphingosine 1-phosphate clearance from blood.) (Fig. 7). The subsequent sphingosine kinase–mediated rephosphorylation of sphingoid bases entering cells prevents severe disturbances in endocytic trafficking and autophagic function (31Shen H. Giordano F. Wu Y. Chan J. Zhu C. Milosevic I. et al.Coupling between endocytosis and sphingosine kinase 1 recruitment., 57Young M.M. Takahashi Y. Fox T.E. Yun J.K. Kester M. Wang H.G. Sphingosine kinase 1 cooperates with autophagy to maintain endocytic membrane trafficking., 58Lima S. Milstien S. Spiegel S. Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking.). This rephosphorylation step also serves to produce a critical branch-point substrate (59Perez Rafael S. Vallee-Belisle A. Fabregas E. Plaxco K. Palleschi G. Ricci F. Employing the metabolic "branch point effect" to generate an all-or-none, digital-like response in enzymatic outputs and enzyme-based sensors., 60LaPorte D.C. Walsh K. Koshland Jr., D.E. The branch point effect. Ultrasensitivity and subsensitivity to metabolic control.), S1P, allowing either sphingoid base removal from the sphingolipid synthesis pathway via degradation by S1P lyase or its preservation in the pathway by the second S1P dephosphorylation step in the ER for sphingoid base salvage and subsequent recycling, a concerted process that is critical for the control of sphingolipid levels (49Le Stunff H. Giussani P. Maceyka M. Lepine S. Milstien S. Spiegel S. Recycling of sphingosine is regulated by the concerted actions of sphingosine-1-phosphate phosphohydrolase 1 and sphingosine kinase 2., 55Bektas M. Allende M.L. Lee B.G. Chen W. Amar M.J. Remaley A.T. et al.Sphingosine 1-phosphate lyase deficiency disrupts lipid homeostasis in liver.).Acknowledgments

We thank Linda Raab for editing and suggestions for improving the article. This work was supported by the Intramural Research Program of the National Institutes of Health , National Institute of Diabetes and Digestive and Kidney Diseases , USA.

Author contributions

M. K., L. H.-H., S. M., R. S., C. B., H. Z., and R. L. P. conceptualization; M. K., L. H.-H., S. M., R. S., C. B., and H. Z. investigation; M. K., L. H.-H., S. M., R. S., C. B., H. Z., and R. L. P. formal analysis; M. K., L. H.-H., and R. L. P. writing–original draft; M. K., L. H.-H., S. M., R. S., C. B., H. Z., and R. L. P. Writing—review and editing.

Funding and additional information

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