Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jul 1;365(Pt 1):69–77. doi: 10.1042/BJ20020120

Biochemical identification of a neutral sphingomyelinase 1 (NSM1)-like enzyme as the major NSM activity in the DT40 B-cell line: absence of a role in the apoptotic response to endoplasmic reticulum stress.

Amanda C Fensome 1, Michelle Josephs 1, Matilda Katan 1, Fernando Rodrigues-Lima 1
PMCID: PMC1222658  PMID: 12071841

Abstract

DT40 cells have approx. 10-fold higher Mg2+-dependent neutral sphingomyelinase (NSM) activity in comparison with other B-cell lines and contain very low acidic sphingomyelinase activity. Purification of this activity from DT40 cell membranes suggested the presence of one major NSM isoform. Although complete purification of this isoform could not be achieved, partially purified fractions were examined further with regard to the known characteristics of previously partially purified NSMs and the two cloned enzymes exhibiting in vitro NSM activity (NSM1 and NSM2). For a direct comparative study, highly purified brain preparations, purified NSM1 protein and Bacillus cereus enzyme were used. Analysis of the enzymic properties of the partially purified DT40 NSM, such as cation dependence, substrate specificity, redox regulation and stimulation by phosphatidylserine, together with the localization of this enzyme to the endoplasmic reticulum (ER), suggested that this NSM from DT40 cells corresponds to NSM1. Further studies aimed to correlate presence of the high levels of this NSM1-like activity in DT40 cells with the ability of these cells to accumulate ceramide and undergo apoptosis. When DT40 cells were stimulated to apoptose by a variety of agents, including the ER stress, an increase in endogenous ceramide levels was observed. However, these responses were not enhanced compared with another B-cell line (Nalm-6), characterized by low sphingomyelinase activity. In addition, DT40 cells were not more susceptible to ceramide accumulation and apoptosis when exposed to the ER stress compared with other apoptotic agents. Inhibition of de novo synthesis of ceramide partially inhibited its accumulation, indicating that the ceramide production in DT40 cells could be complex and, under some conditions, could involve both sphingomyelin hydrolysis and ceramide synthesis.

Full Text

The Full Text of this article is available as a PDF (196.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allan D. Lipid metabolic changes caused by short-chain ceramides and the connection with apoptosis. Biochem J. 2000 Feb 1;345(Pt 3):603–610. [PMC free article] [PubMed] [Google Scholar]
  2. Aronson N. N., Jr, Touster O. Isolation of rat liver plasma membrane fragments in isotonic sucrose. Methods Enzymol. 1974;31:90–102. doi: 10.1016/0076-6879(74)31009-9. [DOI] [PubMed] [Google Scholar]
  3. Bernardo K., Krut O., Wiegmann K., Kreder D., Micheli M., Schäfer R., Sickman A., Schmidt W. E., Schröder J. M., Meyer H. E. Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain. J Biol Chem. 2000 Mar 17;275(11):7641–7647. doi: 10.1074/jbc.275.11.7641. [DOI] [PubMed] [Google Scholar]
  4. Birbes H., El Bawab S., Hannun Y. A., Obeid L. M. Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis. FASEB J. 2001 Dec;15(14):2669–2679. doi: 10.1096/fj.01-0539com. [DOI] [PubMed] [Google Scholar]
  5. Dawson G., Kilkus J., Schieven G. L. Selective phosphotyrosine phosphatase inhibition and increased ceramide formation is associated with B-cell death by apoptosis. FEBS Lett. 2000 Aug 4;478(3):233–236. doi: 10.1016/s0014-5793(00)01853-6. [DOI] [PubMed] [Google Scholar]
  6. Dbaibo G. S., El-Assaad W., Krikorian A., Liu B., Diab K., Idriss N. Z., El-Sabban M., Driscoll T. A., Perry D. K., Hannun Y. A. Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death. FEBS Lett. 2001 Aug 10;503(1):7–12. doi: 10.1016/s0014-5793(01)02625-4. [DOI] [PubMed] [Google Scholar]
  7. Fensome A. C., Rodrigues-Lima F., Josephs M., Paterson H. F., Katan M. A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species. J Biol Chem. 2000 Jan 14;275(2):1128–1136. doi: 10.1074/jbc.275.2.1128. [DOI] [PubMed] [Google Scholar]
  8. Hannun Y. A., Luberto C., Argraves K. M. Enzymes of sphingolipid metabolism: from modular to integrative signaling. Biochemistry. 2001 Apr 24;40(16):4893–4903. doi: 10.1021/bi002836k. [DOI] [PubMed] [Google Scholar]
  9. Hannun Y. A., Luberto C. Ceramide in the eukaryotic stress response. Trends Cell Biol. 2000 Feb;10(2):73–80. doi: 10.1016/s0962-8924(99)01694-3. [DOI] [PubMed] [Google Scholar]
  10. Hofmann K., Tomiuk S., Wolff G., Stoffel W. Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5895–5900. doi: 10.1073/pnas.97.11.5895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jorgensen P. L. Isolation of (Na+ plus K+)-ATPase. Methods Enzymol. 1974;32:277–290. [PubMed] [Google Scholar]
  12. Kim Y. S., Perdomo J., Nordberg J. Glycoprortein biosynthesis in small intestinal mucosa. I. A study of glycosyltransferases in microsomal subfractions. J Biol Chem. 1971 Sep 10;246(17):5466–5476. [PubMed] [Google Scholar]
  13. Kroesen B. J., Pettus B., Luberto C., Busman M., Sietsma H., de Leij L., Hannun Y. A. Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria. J Biol Chem. 2001 Jan 17;276(17):13606–13614. doi: 10.1074/jbc.M009517200. [DOI] [PubMed] [Google Scholar]
  14. Krönke M. Involvement of sphingomyelinases in TNF signaling pathways. Chem Phys Lipids. 1999 Nov;102(1-2):157–166. doi: 10.1016/s0009-3084(99)00084-5. [DOI] [PubMed] [Google Scholar]
  15. Lahti J. M. Use of gene knockouts in cultured cells to study apoptosis. Methods. 1999 Apr;17(4):305–312. doi: 10.1006/meth.1999.0744. [DOI] [PubMed] [Google Scholar]
  16. Levade T., Jaffrézou J. P. Signalling sphingomyelinases: which, where, how and why? Biochim Biophys Acta. 1999 Apr 19;1438(1):1–17. doi: 10.1016/s1388-1981(99)00038-4. [DOI] [PubMed] [Google Scholar]
  17. Lewis V. A., Hynes G. M., Zheng D., Saibil H., Willison K. T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol. Nature. 1992 Jul 16;358(6383):249–252. doi: 10.1038/358249a0. [DOI] [PubMed] [Google Scholar]
  18. Liu B., Hassler D. F., Smith G. K., Weaver K., Hannun Y. A. Purification and characterization of a membrane bound neutral pH optimum magnesium-dependent and phosphatidylserine-stimulated sphingomyelinase from rat brain. J Biol Chem. 1998 Dec 18;273(51):34472–34479. doi: 10.1074/jbc.273.51.34472. [DOI] [PubMed] [Google Scholar]
  19. Maruo A., Oishi I., Sada K., Nomi M., Kurosaki T., Minami Y., Yamamura H. Protein tyrosine kinase Lyn mediates apoptosis induced by topoisomerase II inhibitors in DT40 cells. Int Immunol. 1999 Sep;11(9):1371–1380. doi: 10.1093/intimm/11.9.1371. [DOI] [PubMed] [Google Scholar]
  20. Mizutani Y., Tamiya-Koizumi K., Irie F., Hirabayashi Y., Miwa M., Yoshida S. Cloning and expression of rat neutral sphingomyelinase: enzymological characterization and identification of essential histidine residues. Biochim Biophys Acta. 2000 May 31;1485(2-3):236–246. doi: 10.1016/s1388-1981(00)00059-7. [DOI] [PubMed] [Google Scholar]
  21. Neuberger Y., Shogomori H., Levy Z., Fainzilber M., Futerman A. H. A lyso-platelet activating factor phospholipase C, originally suggested to be a neutral-sphingomyelinase, is located in the endoplasmic reticulum. FEBS Lett. 2000 Mar 3;469(1):44–46. doi: 10.1016/s0014-5793(00)01235-7. [DOI] [PubMed] [Google Scholar]
  22. Richarme G. Protein-disulfide isomerase activity of elongation factor EF-Tu. Biochem Biophys Res Commun. 1998 Nov 9;252(1):156–161. doi: 10.1006/bbrc.1998.9591. [DOI] [PubMed] [Google Scholar]
  23. Rodrigues-Lima F., Fensome A. C., Josephs M., Evans J., Veldman R. J., Katan M. Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis. J Biol Chem. 2000 Sep 8;275(36):28316–28325. doi: 10.1074/jbc.M003080200. [DOI] [PubMed] [Google Scholar]
  24. Sawai H., Domae N., Nagan N., Hannun Y. A. Function of the cloned putative neutral sphingomyelinase as lyso-platelet activating factor-phospholipase C. J Biol Chem. 1999 Dec 31;274(53):38131–38139. doi: 10.1074/jbc.274.53.38131. [DOI] [PubMed] [Google Scholar]
  25. Storrie B., Madden E. A. Isolation of subcellular organelles. Methods Enzymol. 1990;182:203–225. doi: 10.1016/0076-6879(90)82018-w. [DOI] [PubMed] [Google Scholar]
  26. Ségui B., Andrieu-Abadie N., Adam-Klages S., Meilhac O., Kreder D., Garcia V., Bruno A. P., Jaffrézou J. P., Salvayre R., Krönke M. CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway. J Biol Chem. 1999 Dec 24;274(52):37251–37258. doi: 10.1074/jbc.274.52.37251. [DOI] [PubMed] [Google Scholar]
  27. Takao N., Li Y., Yamamoto K. Protective roles for ATM in cellular response to oxidative stress. FEBS Lett. 2000 Apr 21;472(1):133–136. doi: 10.1016/s0014-5793(00)01422-8. [DOI] [PubMed] [Google Scholar]
  28. Takata M., Homma Y., Kurosaki T. Requirement of phospholipase C-gamma 2 activation in surface immunoglobulin M-induced B cell apoptosis. J Exp Med. 1995 Oct 1;182(4):907–914. doi: 10.1084/jem.182.4.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tepper A. D., Ruurs P., Borst J., van Blitterswijk W. J. Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis. Biochem Biophys Res Commun. 2001 Jan 26;280(3):634–639. doi: 10.1006/bbrc.2000.4166. [DOI] [PubMed] [Google Scholar]
  30. Tepper A. D., Ruurs P., Wiedmer T., Sims P. J., Borst J., van Blitterswijk W. J. Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology. J Cell Biol. 2000 Jul 10;150(1):155–164. doi: 10.1083/jcb.150.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tomiuk S., Hofmann K., Nix M., Zumbansen M., Stoffel W. Cloned mammalian neutral sphingomyelinase: functions in sphingolipid signaling? Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3638–3643. doi: 10.1073/pnas.95.7.3638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tomiuk S., Zumbansen M., Stoffel W. Characterization and subcellular localization of murine and human magnesium-dependent neutral sphingomyelinase. J Biol Chem. 2000 Feb 25;275(8):5710–5717. doi: 10.1074/jbc.275.8.5710. [DOI] [PubMed] [Google Scholar]
  33. Tonnetti L., Verí M. C., Bonvini E., D'Adamio L. A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction. J Exp Med. 1999 May 17;189(10):1581–1589. doi: 10.1084/jem.189.10.1581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Uckun F. M., Waddick K. G., Mahajan S., Jun X., Takata M., Bolen J., Kurosaki T. BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells. Science. 1996 Aug 23;273(5278):1096–1100. doi: 10.1126/science.273.5278.1096. [DOI] [PubMed] [Google Scholar]
  35. Wiesner D. A., Kilkus J. P., Gottschalk A. R., Quintáns J., Dawson G. Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-XL. J Biol Chem. 1997 Apr 11;272(15):9868–9876. doi: 10.1074/jbc.272.15.9868. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES