Publications

• Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase.

  Hofmann K., Tomiuk S., Wolff G., Stoffel W.

  The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular dat ... >> More

  The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular data on the neutral sphingomyelinases (nSMases). We recently identified a mammalian nSMase, an integral membrane protein with remote similarity to bacterial sphingomyelinases. However, its ubiquitous expression pattern is in contrast to previous findings that sphingomyelinase activity is found mainly in brain tissues. By using an improved database search method, combined with phylogenetic analysis, we identified a second mammalian nSMase (nSMase2) with predominant expression in the brain. The sphingomyelinase activity of nSMase2 has a neutral pH optimum, depends on Mg(2+) ions, and is activated by unsaturated fatty acids and phosphatidylserine. Immunofluorescence reveals a neuron-specific punctate perinuclear staining, which colocalizes with a Golgi marker in a number of cell lines. The likely identity of nSMase2 with cca1, a rat protein involved in contact inhibition of 3Y1 fibroblasts, suggests a role for this enzyme in cell cycle arrest. Both mammalian nSMases are members of a superfamily of Mg(2+)-dependent phosphohydrolases, which also contains nucleases, inositol phosphatases, and bacterial toxins. << Less

  Proc. Natl. Acad. Sci. U.S.A. 97:5895-5900(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle.

  Moylan J.S., Smith J.D., Wolf Horrell E.M., McLean J.B., Deevska G.M., Bonnell M.R., Nikolova-Karakashian M.N., Reid M.B.

  <h4>Aims</h4>Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase ... >> More

  <h4>Aims</h4>Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production.<h4>Results</h4>We demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown.<h4>Innovation</h4>These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production.<h4>Conclusion</h4>Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity. << Less

  Redox Biol. 2:910-920(2014) [PubMed] [EuropePMC]

• Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases.

  Miura Y., Gotoh E., Nara F., Nishijima M., Hanada K.

  Sphingosylphosphocholine (SPC), the N-deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC-phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 a ... >> More

  Sphingosylphosphocholine (SPC), the N-deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC-phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 and nSMase2, human neutral sphingomyelinases (SMases), are capable of hydrolyzing SPC efficiently under detergent-free conditions. Bacterial and plasmodial neutral SMases also showed SPC-PLC activity. The substrate specificity of neutral SMases that hydrolyze SM, SPC, and monoradyl glycerophosphocholine, but not diradyl glycerophosphocholine, suggested that a hydrogen-bond donor at the C-2 or sn-2 position in the substrate is required for recognition by the enzymes. << Less

  FEBS Lett. 557:288-292(2004) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Human acid sphingomyelinase. Isolation, nucleotide sequence and expression of the full-length and alternatively spliced cDNAs.

  Schuchman E.H., Suchi M., Takahashi T., Sandhoff K., Desnick R.J.

  Two types of partial cDNAs encoding human acid sphingomyelinase (EC 3.1.4.12; ASM) were recently isolated from fibroblast and placental cDNA libraries (Quintern, L. E., Schuchman, E.H., Levran, O., Suchi. M., Ferlinz, K., Reinke, H., Sandhoff, K., and Desnick, R. J. (1989) EMBO J. 8, 2469-2473). T ... >> More

  Two types of partial cDNAs encoding human acid sphingomyelinase (EC 3.1.4.12; ASM) were recently isolated from fibroblast and placental cDNA libraries (Quintern, L. E., Schuchman, E.H., Levran, O., Suchi. M., Ferlinz, K., Reinke, H., Sandhoff, K., and Desnick, R. J. (1989) EMBO J. 8, 2469-2473). The cDNA inserts had identical sequences with the exception of an internal region; type 1 cDNAs (representing approximately 90% of the ASM cDNAs isolated) had 172 in-frame base pairs (bp), which were replaced in the type 2 cDNAs by a 40-bp in-frame sequence. Northern hybridization and RNase protection studies indicated that both type 1 and 2 transcripts were approximately 2.5 kilobases; therefore, efforts were directed to isolate full-length type 1 and 2 cDNAs by screening human placental, testis, hepatoma, and retinal cDNA libraries. In addition to type 1 and 2 cDNAs, a new type of ASM cDNA (type 3), which did not contain the type 1- or 2-specific regions, was isolated and sequenced. The full-length type 1 and the reconstructed full-length type 2 and 3 cDNAs were transiently expressed in COS-1 cells. Only the full-length type 1 transcript encoded catalytically active human ASM, demonstrating its functional integrity. The 2347-bp full-length type 1 placental cDNA (pASM-1FL) had an 87-bp 5'-untranslated region, an 1890-bp open reading frame encoding 629 amino acids, and a 370-bp 3'-untranslated sequence. The predicted location of the signal peptide cleavage site was after alanine 46. Two base differences were identified in codons 322 and 506 and shown to be polymorphisms with the common alleles having frequencies of 0.6 and 0.7, respectively. To determine the genomic organization of the type 1, 2, and 3 sequences, a 1665-bp genomic region containing both the unique type 1 (172 bp) and type 2 (40 bp) sequences was amplified by the polymerase chain reaction and sequenced. The 172-bp sequence was exonic, flanked by 5'- and 3'-intronic sequences of 1052 and 229 bp, respectively. The 40-bp type 2 sequence was intronic, occurring at the 5' end of the 1052-bp intron due to the use of a cryptic 5' donor splice site, which deleted the entire 172-bp exon and both flanking intronic sequences. The type 3 cDNA resulted from an alternative splicing event, which excised the 172-bp exon. These studies demonstrate the occurrence of alternatively splicing of the ASM transcript, but the existence of only one functional mRNA. << Less

  J. Biol. Chem. 266:8531-8539(1991) [PubMed] [EuropePMC]

• Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family.

  Duan R.-D., Bergman T., Xu N., Wu J., Cheng Y., Duan J., Nelander S., Palmberg C., Nilsson A.

  Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, an ... >> More

  Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alk-SMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30-36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn2+. Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity. << Less

  J. Biol. Chem. 278:38528-38536(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells.

  Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M., Hannun Y.A.

  Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). ... >> More

  Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). In this study, the role of endogenous nSMase2 in regulating growth arrest was investigated. The results show that endogenous nSMase2 mRNA was up-regulated approximately 5-fold when MCF7 cells became growth-arrested at confluence, and total neutral SMase activity was increased by 119 +/-41% with respect to control. Cell cycle analysis showed that up-regulation of endogenous nSMase2 correlated with G(0)/G(1) cell cycle arrest and an increase in total ceramide levels (2.4-fold). Analysis of ceramide species showed that confluence caused selective increases in very long chain ceramide C(24:1) (370 +/- 54%) and C(24:0) (266 +/-81%) during arrest. The role of endogenous nSMase2 in growth regulation and ceramide metabolism was investigated using short interfering RNA (siRNA)-mediated loss-of-function analysis. Down-regulation of nSMase2 with specific siRNA increased the cell population of cells in S phase of the cell cycle by 59 +/-14% and selectively reverted the effects of growth arrest on the increase in levels of very long chain ceramides. Mechanistically, confluence arrest also induced hypophosphorylation of the retinoblastoma protein (6-fold) and induction of p21(WAF1) (3-fold). Down-regulation of nSMase2 with siRNA largely prevented the dephosphorylation of the retinoblastoma protein and the induction of p21(WAF1), providing a link between the action of nSMase2 and key regulators of cell cycle progression. Moreover, studies on nSMase2 localization in MCF7 cells showed that nSMase2 distributed throughout the cells in subconfluent, proliferating cultures. In contrast, nSMase2 became nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that nSMase2 functions as a growth suppressor in MCF7 cells, linking confluence to the G(0)/G(1) cell cycle check point. << Less

  J. Biol. Chem. 279:25101-25111(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria.

  Johansen K.A., Gill R.E., Vasil M.L.

  Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis). Thin-layer chromatography ... >> More

  Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis). Thin-layer chromatography (TLC) using radiolabeled phosphatidylcholine and sphingomyelin as substrates indicated that cell extracts of M. tuberculosis contain both phospholipase C (PLC) and phospholipase D (PLD) activities. In contrast, only PLD activity was detected in cell extracts of M. smegmatis. Neither activity was detected in cell-free culture supernatants from these organisms. We and others recently identified two open reading frames in M. tuberculosis with the potential to encode proteins which are highly homologous to the nonhemolytic (PlcN) and hemolytic (PlcH) phospholipase C enzymes of Pseudomonas aeruginosa. In contrast to the plc genes in P. aeruginosa, which are considerably distal to each other (min 34 and 64 on the chromosome), the mycobacterial genes, designated mpcA and mpcB, are tandemly arranged in the same relative orientation and separated by only 191 bp. Both the mpcA and the mpcB genes were individually cloned in M. smegmatis, and PLC activity was expressed from each gene in this organism. Hybridization experiments using the mpcA and the mpcB genes as probes under conditions of moderate stringency identified sequences homologous to these genes in M. bovis, M. bovis BCG, and M. marinum but not in several other Mycobacterium species, including M. smegmatis, M. avium, and M. intracellulare. TLC analysis using radiolabeled substrates indicated that M. bovis and M. marinum cell extracts contain PLC and PLD activities, but only PLD activity was detected in M. bovis BCG cell extracts. Sphingomyelinase activity was also detected in whole-cell extracts of M. tuberculosis, M. marinum, M. bovis, and M. bovis BCG, but this activity was not detected in extracts of M. smegmatis. Sphingomyelinase activity was detected in cell extracts from M. smegmatis harboring either recombinant mpcA or mpcB. These data indicate that PLC and sphingomyelinase activities are associated with the most virulent mycobacterial species, while PLD activity was detected in both virulent and saprophytic strains. << Less

  Infect. Immun. 64:3259-3266(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.