Sphingomyelin synthases 1 and 2 exhibit phosphatidylcholine phospholipase C activity

Abstract

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo. Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo. Phosphatidylcholine (PC) is an essential phospholipid for cell formation, growth, and death (1van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: Where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (3973) Google Scholar). The steady-state levels of PC should be controlled by its biosynthesis (Kennedy pathway) and catabolic pathways, including PC-phospholipase C (PC-PLC) (2Wolf R.A. Gross R.W. Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium.J. Biol. Chem. 1985; 260: 7295-7303Abstract Full Text PDF PubMed Google Scholar, 3Sheikhnejad R.G. Srivastava P.N. Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma.J. Biol. Chem. 1986; 261: 7544-7549Abstract Full Text PDF PubMed Google Scholar). PLCs are a group of enzymes that produce a diacylglycerol (DAG) and a phosphorylated molecule, such as phosphorylcholine (P-choline) and phosphorylethanolamine (P-ethanolamine) (4Cheng M. Bhujwalla Z.M. Glunde K. Targeting phospholipid metabolism in cancer.Front. Oncol. 2016; 6: 266Crossref PubMed Scopus (76) Google Scholar). It is conceivable that PC-PLC activity is important in maintaining steady-state levels of PC, thus influencing cell membrane integrity and function. Although bacterial PC-PLC was cloned previously (5Vazquez-Boland J.A. Kocks C. Dramsi S. Ohayon H. Geoffroy C. Mengaud J. Cossart P. Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread.Infect. Immun. 1992; 60: 219-230Crossref PubMed Google Scholar), the gene, which is responsible for mammalian PC-PLC, is still unknown so far. Therefore, PC-PLC studies in both mammalian cells and in vivo have to rely on PC-PLC inhibitors, such as tricyclodecan-9-yl-potassium xanthate (D609) (6Amtmann E. The antiviral, antitumoural xanthate D609 is a competitive inhibitor of phosphatidylcholine-specific phospholipase C.Drugs Exp. Clin. Res. 1996; 22: 287-294PubMed Google Scholar). Mammalian sphingomyelin synthase (SMS) family contains three members (7Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. Identification of a family of animal sphingomyelin synthases.EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (448) Google Scholar, 8Tafesse F.G. Ternes P. Holthuis J.C. The multigenic sphingomyelin synthase family.J. Biol. Chem. 2006; 281: 29421-29425Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). SMS1 and SMS2 can transfer P-choline from PC onto ceramide to form SM. SMS-related protein (SMSr) has no SMS activity; however, in vitro, it can transfer P-ethanolamine from phosphatidylethanolamine (PE) onto ceramide to form ceramide P-ethanolamine (CPE), an SM-like molecule (9Vacaru A.M. Tafesse F.G. Ternes P. Kondylis V. Hermansson M. Brouwers J.F. Somerharju P. Rabouille C. Holthuis J.C. Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER.J. Cell Biol. 2009; 185: 1013-1027Crossref PubMed Scopus (116) Google Scholar). While SMS activity uses ceramide as the acceptor for the released phosphocholine head group, PC-PLC activity uses water as the acceptor (8Tafesse F.G. Ternes P. Holthuis J.C. The multigenic sphingomyelin synthase family.J. Biol. Chem. 2006; 281: 29421-29425Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10Sigal Y.J. McDermott M.I. Morris A.J. Integral membrane lipid phosphatases/phosphotransferases: Common structure and diverse functions.Biochem. J. 2005; 387: 281-293Crossref PubMed Scopus (136) Google Scholar). It is well known that D609, a PC-PLC inhibitor, can also inhibit SMS (11Adibhatla R.M. Hatcher J.F. Gusain A. Tricyclodecan-9-yl-xanthogenate (D609) mechanism of actions: A mini-review of literature.Neurochem. Res. 2012; 37: 671-679Crossref PubMed Scopus (75) Google Scholar, 12Luberto C. Hannun Y.A. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?.J. Biol. Chem. 1998; 273: 14550-14559Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). From SMS catalytic activity, we noticed that SMS1- or SMS2-mediated SM formation can be separated into two steps: (1) PC hydrolysis step, where PC is hydrolyzed into P-choline and DAG and (2) SM formation step, where P-choline is added onto ceramide. Besides six membrane-spanning domains, SMS1 and SMS2 also contain four highly conserved sequence motifs, designated D1, D2, D3, and D4 (7Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. Identification of a family of animal sphingomyelin synthases.EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (448) Google Scholar). Motifs D3 and D4 contain conserved three amino acids, His-His-Asp, which can form a catalytic triad mediating the nucleophilic attack on the phosphodiester bond of PC (7Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. Identification of a family of animal sphingomyelin synthases.EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (448) Google Scholar, 13Yeang C. Varshney S. Wang R. Zhang Y. Ye D. Jiang X.C. The domain responsible for sphingomyelin synthase (SMS) activity.Biochim. Biophys. Acta. 2008; 1781: 610-617Crossref PubMed Scopus (50) Google Scholar), that is, the first step of SMS activity. Thus, potentially, SMS1 and SMS2 can be functional PC-PLCs. In this study, we showed that SMS1 and SMS2 have dual activities, that is, SM biosynthesis and PC-PLC activities, which regulate steady-state levels of PC and DAG as well as SM. To examine whether SMS1 and SMS2 have PC-PLC activity, we overexpressed human SMS1-flag, SMS2-flag, and SMSr-flag in Cos7 cells through expression vector transfection, respectively. To avoid the noise from endogenous SMS activity, we immunoprecipitated each flagged SMS using a flag antibody and then utilizing the precipitate to perform SMS activity measurement, using PC and nitrobenz-diazol (NBD)-ceramide as two substrates. We found that both SMS1-flag and SMS2-flag but not SMSr-flag and empty vector had SM synthase activity (Fig. 1A). Next, we utilized the same precipitates to measure PC-PLC activity, using 14C-PC, as a substrate (without ceramide). We found that SMS1-flag and SMS2-flag but not SMSr-flag and empty vector produced 14C-DAG (Fig. 1B). Thus, both SMS1-flag and SMS2-flag have PC-PLC activity, that is, producing DAG through hydrolysis of PC in the absence of ceramide. To more precisely show that SMS1 and SMS2 have PC-PLC activity, we expressed recombinant SMS (rSMS) rSMS1–, rSMS2–, and rSMSr–Strep-tag in Baculovirus/Spodoptera frugiperda (SF9) cell system and then purified it, using streptavidin affinity column chromatography. As shown in Figure 2A, the purity of rSMS1–Strep-tag and rSMS2–Strep-tag was about 96%, while rSMSr–Strep-tag was about 90%. We then utilized the purified rSMS1, rSMS2, and rSMSr to measure SM synthase and PC-PLC activities. We found that rSMS1 and rSMS2 but not rSMSr had SMS activity when NBD-ceramide and PC were used as two substrates (Fig. 2B). Importantly, rSMS1 and rSMS2 but not rSMSr had PC-PLC activity when NBD-PC or 14C-PC was used, as a substrate, for generating NBD-DAG or 14C-DAG (Fig. 2, C and D). Also, we found that rSMS2 had higher PC-PLC activity than that of rSMS1 when the same amount of recombinant protein was used (Fig. 2, C and D). Moreover, we directly measured P-choline, another product of PC-PLC, and found that both rSMS1 and rSMS2 produced P-choline, and the latter had a higher activity (Fig. 2E). To evaluate the specificity, next, we sought to measure PE-PLC activity of rSMS1, rSMS2, and rSMSr, using NBD-PE as a substrate. We found that both rSMS1 and rSMS2 had extremely weak or no PE-PLC activity, whereas rSMSr had the high activity (Fig. 2F) that was consistent with our previous observation (14Chiang Y.P. Li Z. Chen Y. Cao Y. Jiang X.C. Sphingomyelin synthase related protein is a mammalian phosphatidylethanolamine phospholipase C.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866159017Crossref PubMed Scopus (1) Google Scholar). We did Michaelis–Menten kinetics of SMS1- and SMS2-mediated PC-PLC reaction. The Michaelis–Menten constant (Km) was measured. We found that, without ceramide, the Km for rSMS1-mediated PC-PLC was 61 μΜ and for rSMS2-mediated PC-PLC was 57 μM. However, both Km (162 μM and 104 μM, respectively) became larger when ceramide was added (Fig. 3, A and B), indicating that both rSMS1- and rSMS2-mediated PC-PLCs have SMS activities. To prove that SMS1 and SMS2 have PC-PLC activity in vivo, we depleted all three members of SMS gene family in mice. We established liver-specific Sms1/global Sms2 double-KO (dKO) mice (15Li Z. Chiang Y.P. He M. Zhang K. Zheng J. Wu W. Cai J. Chen Y. Chen G. Chen Y. Dong J. Worgall T.S. Jiang X.C. Effect of liver total sphingomyelin synthase deficiency on plasma lipid metabolism.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866158898Crossref PubMed Scopus (3) Google Scholar) and crossed them with our global Smsr KO mice (16Ding T. Li Y. C. A. J. Dong J. H. T. M. Li Z. Jiang X.C. members in the sphingomyelin synthase gene family have ceramide synthase Res. Full Text Full Text PDF PubMed Scopus Google Scholar) to liver-specific Sms1/global Smsr mice. The and the mice have no total SMS activity in (Fig. We also utilized the PC-PLC to measure PC-PLC activity in the liver of or mice. We found that the mice had significantly liver PC-PLC activity (Fig. the liver is of the to plasma lipid it is conceivable that the deficiency in plasma PC, and ceramide We measured plasma in mice. We found that the and the mice had a significantly of SM (Fig. and a significantly higher of PC (Fig. than mice. However, were no the and in both (Fig. C and that SMSr is an SMS a We also noticed that DAG levels were significantly reduced in the and the mice with mice, whereas no the two KO (Fig. that than the liver could have on DAG in the with mice, the and the mice had significantly higher ceramide levels (Fig. but was no the and the mice, that synthase activity in vitro could not an role in vivo. We sought to measure liver As we found that, with mice, the mice and the mice had of SM (Fig. and a of PC levels (Fig. the of liver DAG levels is mice, the of SMS-related PC-PLC and PE-PLC (Fig. 2, and both and have more ceramide the and or is not (Fig. To prove that SMS1 and SMS2 have PC-PLC activity in vivo, we and into mice, respectively. the we the liver from the mice. As and but not and had SMS activity as a (Fig. We also used the PC-PLC to measure PC-PLC activity in the liver of or or mice. We found that the or significantly PC-PLC activity in the with (Fig. Moreover, both and but not and liver PC-PLC activity, when NBD-PC was used as a substrate (Fig. confirmed the of PC-PLC activity (Fig. We also noticed that can also NBD-DAG was a weak NBD-DAG in both and (Fig. C and D). Next, we sought to measure liver lipid levels in mice. As liver was in all not or but not SM levels (Fig. however, on ceramide is (Fig. DAG levels were in all (Fig. Although we did not of total PC levels (Fig. we found that expression of SMS1 or SMS2 in the mice could the high levels of liver PC to levels by Figure with Figure In the present study, we that SMS1 and SMS2 exhibit PC-PLC activities. we that deficiency steady-state levels of PC and of the of the present is that SMS1 and SMS2 have PC-PLC activity. PC-PLC is an important of family to the hydrolysis of the and in PC, producing DAG and P-choline K. van M. J. E. D. and of a of PC-PLC J. Chem. PubMed Scopus Google Scholar). the a of has to the of PC-PLC in growth, and of mammalian cells C. D. S. P. S. E. V. S. phospholipase C in Res. 2008; PubMed Scopus Google Scholar, J. Wang W. Zhang S. J. phospholipase C and were in to Cell PubMed Scopus Google Scholar, Y. S. Zhang S. J. phospholipase and in the of and in 2006; PubMed Scopus Google Scholar, K. H. S. of by of phosphatidylcholine-specific phospholipase C in PubMed Scopus Google Scholar). a in our of this important is to the fact that the mammalian PC-PLC has not been bacterial PC-PLC was cloned (5Vazquez-Boland J.A. Kocks C. Dramsi S. Ohayon H. Geoffroy C. Mengaud J. Cossart P. Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread.Infect. Immun. 1992; 60: 219-230Crossref PubMed Google Scholar). Thus, so PC-PLC studies in mammalian cells and in vivo have to rely on of PC-PLC, such D609 (6Amtmann E. The antiviral, antitumoural xanthate D609 is a competitive inhibitor of phosphatidylcholine-specific phospholipase C.Drugs Exp. Clin. Res. 1996; 22: 287-294PubMed Google Scholar) and so on K. van M. J. E. D. and of a of PC-PLC J. Chem. PubMed Scopus Google Scholar). A was more than SMS account for the putative C. Hannun Y.A. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?.J. Biol. Chem. 1998; 273: 14550-14559Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). the fact that D609 can inhibit both PC-PLC and SMS C. Hannun Y.A. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?.J. Biol. Chem. 1998; 273: 14550-14559Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), we that SMS1 and SMS2 have both PC-PLC and SMS activities. so we have to show that both SMS1 and SMS2 have PC-PLC activity. as of the we found that in a immunoprecipitated SMS1 and SMS2 had PC-PLC activity (Fig. 1B). Importantly, we found that purified rSMS1 and rSMS2 had PC-PLC but not PE-PLC activity (Fig. 2, and Moreover, we used to SMS1 and SMS2 in the liver of mice and found that and liver PC-PLC activity (Fig. C and D). A weak NBD-DAG in both and was by enzymes than SMS family (Fig. C and D). P-choline, as another we found that rSMS1 and rSMS2 had PC-PLC activity (Fig. with Km (Fig. 3, A and The Km became larger when ceramide was we found that PC-PLC activity was significantly reduced in the liver (Fig. with the In we noticed that the liver still levels of P-choline, that enzymes than SMS1 and SMS2 could be responsible for its or significantly PC-PLC activity in the liver (Fig. of the present is that SMS1 and SMS2 are in steady-state levels of DAG and We measured PC, and ceramide in and plasma and The and the mice have a of SM levels in the plasma and indicating that SMS1 and SMS2 but not SMSr are SMS and (7Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. Identification of a family of animal sphingomyelin synthases.EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (448) Google Scholar, S. M. T. H. T. of a human sphingomyelin and cell in sphingomyelin Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the and the mice have a of PC with mice, in the plasma and that SMS1 and SMS2 but not SMSr have PC-PLC activity and In we found that plasma DAG levels are significantly reduced in the mice (Fig. with and the the and the mice is The could be related with of DAG PubMed Scopus Google Scholar). DAG levels in the liver were reduced in the of and (Fig. indicating that all SMS family members to DAG levels in the liver of PC-PLC and PE-PLC (Fig. 2, and Although DAG is known as an of The mechanism of protein C Full Text PDF PubMed Scopus Google Scholar), DAG from have on DAG from hydrolysis of can whereas DAG produced from A. T. C. and by C and respectively, have and diacylglycerol not protein C in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of DAG we found that the mice have significantly higher ceramide levels than mice in the plasma (Fig. that both SMS1 and SMS2 deficiency using ceramide as a substrate to produce SM (7Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. Identification of a family of animal sphingomyelin synthases.EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (448) Google Scholar, S. M. T. H. T. of a human sphingomyelin and cell in sphingomyelin Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). However, SMSr deficiency has no on plasma ceramide indicating SMSr is not a functional synthase that ceramide (9Vacaru A.M. Tafesse F.G. Ternes P. Kondylis V. Hermansson M. Brouwers J.F. Somerharju P. Rabouille C. Holthuis J.C. Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER.J. Cell Biol. 2009; 185: 1013-1027Crossref PubMed Scopus (116) Google Scholar). of into the mice to the of PLCs from a in the not was that are not functional synthase in vivo, we found that all have this activity when NBD-ceramide and two were used in (16Ding T. Li Y. C. A. J. Dong J. H. T. M. Li Z. Jiang X.C. members in the sphingomyelin synthase gene family have ceramide synthase Res. Full Text Full Text PDF PubMed Scopus Google Scholar). DAG in the liver was in all three which also that all SMS family members in liver DAG levels (Fig. and have shown that SMSr can DAG the hydrolysis of including PC, and in the absence of ceramide C. Sphingomyelin synthase-related protein diacylglycerol the hydrolysis of glycerophospholipids in the absence of Biol. Chem. 2021; Full Text Full Text PDF PubMed Scopus (3) Google Scholar). present with and C. Sphingomyelin synthase-related protein diacylglycerol the hydrolysis of glycerophospholipids in the absence of Biol. Chem. 2021; Full Text Full Text PDF PubMed Scopus (3) Google Scholar), as well as our (14Chiang Y.P. Li Z. Chen Y. Cao Y. Jiang X.C. Sphingomyelin synthase related protein is a mammalian phosphatidylethanolamine phospholipase C.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866159017Crossref PubMed Scopus (1) Google Scholar), that all three SMS family members are a group of studies that have SMS1 and SMS2 have PC-PLC activity, with SMS activity (7Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. Identification of a family of animal sphingomyelin synthases.EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (448) Google Scholar, 8Tafesse F.G. Ternes P. Holthuis J.C. The multigenic sphingomyelin synthase family.J. Biol. Chem. 2006; 281: 29421-29425Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), whereas SMSr has PE-PLC activity (14Chiang Y.P. Li Z. Chen Y. Cao Y. Jiang X.C. Sphingomyelin synthase related protein is a mammalian phosphatidylethanolamine phospholipase C.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866159017Crossref PubMed Scopus (1) Google Scholar), consistent with its in vitro synthase activity (9Vacaru A.M. Tafesse F.G. Ternes P. Kondylis V. Hermansson M. Brouwers J.F. Somerharju P. Rabouille C. Holthuis J.C. Sphingomyelin synthase-related protein SMSr controls ceramide homeostasis in the ER.J. Cell Biol. 2009; 185: 1013-1027Crossref PubMed Scopus (116) Google Scholar). Although the in PC and DAG levels in KO mice are in with PC-PLC activity of SMS1 and SMS2 by enzymatic in vitro, be to to are by PC-PLC activity in vivo. We that SMS1 and SMS2 can DAG and P-choline through the hydrolysis of PC in the absence of ceramide. that SMS1 and SMS2 are two for the that can be by D609 (11Adibhatla R.M. Hatcher J.F. Gusain A. Tricyclodecan-9-yl-xanthogenate (D609) mechanism of actions: A mini-review of literature.Neurochem. Res. 2012; 37: 671-679Crossref PubMed Scopus (75) Google Scholar, 12Luberto C. Hannun Y.A. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?.J. Biol. Chem. 1998; 273: 14550-14559Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). The and of PC-PLC activity it is not possible to global mice by all three global KO mice we crossed liver-specific Sms1/global Sms2 mice (15Li Z. Chiang Y.P. He M. Zhang K. Zheng J. Wu W. Cai J. Chen Y. Chen G. Chen Y. Dong J. Worgall T.S. Jiang X.C. Effect of liver total sphingomyelin synthase deficiency on plasma lipid metabolism.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866158898Crossref PubMed Scopus (3) Google Scholar) with global Smsr KO mice (16Ding T. Li Y. C. A. J. Dong J. H. T. M. Li Z. Jiang X.C. members in the sphingomyelin synthase gene family have ceramide synthase Res. Full Text Full Text PDF PubMed Scopus Google Scholar), liver-specific Sms1 Smsr mice. are on a were with the of the of and The were in with Cos7 cells were in the with and in Cos7 cells were established by using with cells were in The cells were The SMS1-flag or SMS2-flag or cells were in the and a inhibitor were to for and of the were with a using as the from liver the from cells were with of of protein for for the were then with of of affinity for were by for for activity The for human and SMSr respectively, cloned into an expression vector from with a amino sequence and then a sequence added to the of of The SMS were expressed using a frugiperda (SF9) cell The were purified to a J. P. T. Dong J. M. Ye D. Chen Y. K. Cao Y. Jiang X.C. not sphingomyelin synthase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the was as an affinity for the of on streptavidin affinity the could be by hydrolysis for However, we used the for both SMS and activity The was as (16Ding T. Li Y. C. A. J. Dong J. H. T. M. Li Z. Jiang X.C. members in the sphingomyelin synthase gene family have ceramide synthase Res. Full Text Full Text PDF PubMed Scopus Google Scholar). the without and In P-choline or P-ethanolamine has two that is, ceramide and the acceptor is ceramide, the SMS activity. the acceptor is the purified by or was with the and in total of as well as substrates with for the without and The were water for and then by of with The were then and the lipid was in a of and to a or substrates and or were separated using a the the were the whereas were on which were using the PC was with rSMS1, rSMS2, or rSMSr or liver in μM choline water for Km measurement, PC were rSMS1 or rSMS2 with or without ceramide, in the water for The P-choline was measured by the as S. M. C. and role of phosphatidylcholine-specific phospholipase C in human and J. 2006; PubMed Scopus Google Scholar). PC, and SM were measured by a PC a DAG and an SM the respectively. was measured by the of on the were prepared by mice were with respectively. was used as the the and plasma were for and lipid was two were expressed as the two were by the and were by by the were are the contains The that they have no of with the of this was by a and of and Z. and Z. Y. and Y. Cao J. The is the of the and not the of the of

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