Novel Tumor Necrosis Factor-responsive Mammalian Neutral Sphingomyelinase-3 Is a C-tail-anchored Protein
2006; Elsevier BV; Volume: 281; Issue: 19 Linguagem: Inglês
10.1074/jbc.m511306200
ISSN1083-351X
AutoresOleg Krut, Katja Wiegmann, Hamid Kashkar, Benjamin Yazdanpanah, Martin Krönke,
Tópico(s)Phagocytosis and Immune Regulation
ResumoTwo genes encoding neutral sphingomyelinases-1 and -2 (sphingomyelin phosphodiesterases-2 and -3) have been recently identified that hydrolyze sphingomyelin to phosphorylcholine and ceramide. Data bank searches using a peptide sequence derived from a previously purified bovine neutral sphingomyelinase (nSMase) allowed us to identify a cDNA encoding a novel human sphingomyelinase, nSMase3, that shows only a little homology to nSMase1 and -2. nSMase3 was biochemically characterized by overexpression in a yeast strain, JK9-3dΔIsc1p, lacking endogenous SMase activity. Similar to nSMase2, nSMase3 is Mg2+-dependent and shows optimal activity at pH 7, which is enhanced in the presence of phosphatidylserine and inhibited by scyphostatin. nSMase3 is ubiquitously expressed as a 4.6-kb mRNA species. nSMase3 lacks an N-terminal signal peptide, yet contains a 23-amino-acid transmembrane domain close to the C terminus, which is indicative for the family of C-tail-anchored integral membrane proteins. Cellular localization studies with hemagglutinin-tagged nSMase3 demonstrated colocalization with markers of the endoplasmic reticulum as well as with Golgi markers. Tumor necrosis factor stimulates rapid activation of nSMase3 in MCF7 cells with peak activity at 1.5 min, which was impaired by expression of dominant negative FAN. Two genes encoding neutral sphingomyelinases-1 and -2 (sphingomyelin phosphodiesterases-2 and -3) have been recently identified that hydrolyze sphingomyelin to phosphorylcholine and ceramide. Data bank searches using a peptide sequence derived from a previously purified bovine neutral sphingomyelinase (nSMase) allowed us to identify a cDNA encoding a novel human sphingomyelinase, nSMase3, that shows only a little homology to nSMase1 and -2. nSMase3 was biochemically characterized by overexpression in a yeast strain, JK9-3dΔIsc1p, lacking endogenous SMase activity. Similar to nSMase2, nSMase3 is Mg2+-dependent and shows optimal activity at pH 7, which is enhanced in the presence of phosphatidylserine and inhibited by scyphostatin. nSMase3 is ubiquitously expressed as a 4.6-kb mRNA species. nSMase3 lacks an N-terminal signal peptide, yet contains a 23-amino-acid transmembrane domain close to the C terminus, which is indicative for the family of C-tail-anchored integral membrane proteins. Cellular localization studies with hemagglutinin-tagged nSMase3 demonstrated colocalization with markers of the endoplasmic reticulum as well as with Golgi markers. Tumor necrosis factor stimulates rapid activation of nSMase3 in MCF7 cells with peak activity at 1.5 min, which was impaired by expression of dominant negative FAN. Sphingomyelinases (SMases 3The abbreviations used are: SMase, sphingomyelinase; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ER, endoplasmic reticulum; HA, hemagglutinin; lysoPAF, lyso platelet activation factor; ORF, open reading frame; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin; nSMase, neutral sphingomyelinase; SMPD, sphingomyelin phosphodiesterase; TLC, thin layer chromatography; TM, transmembrane; TNF, tumor necrosis factor; TRITC, tetramethylrhodamine isothiocyanate; FAN, factor associated with neutral sphingomyelinase activation. 3The abbreviations used are: SMase, sphingomyelinase; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ER, endoplasmic reticulum; HA, hemagglutinin; lysoPAF, lyso platelet activation factor; ORF, open reading frame; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin; nSMase, neutral sphingomyelinase; SMPD, sphingomyelin phosphodiesterase; TLC, thin layer chromatography; TM, transmembrane; TNF, tumor necrosis factor; TRITC, tetramethylrhodamine isothiocyanate; FAN, factor associated with neutral sphingomyelinase activation.; EC3.1.4.12) are sphingomyelin (SM) phosphodiesterases (SMPD) that catalyze hydrolysis of membrane SM to form ceramide. Ceramide has been suggested to play important roles in cell cycle arrest, apoptosis, inflammation, and the eukaryotic stress response (1Gulbins E. Kolesnick R. Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (348) Google Scholar, 2Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 3Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 4Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). Although ceramide can be generated by de novo synthesis through ceramide synthase, for the majority of cellular responses, it is generated from SM by the action of neutral or acid SMases. Different SMases have been described in eukaryotes and prokaryotes and are distinguished by their subcellular localization, pH optima, and requirement for metal ions. Three mammalian SMases have been molecularly cloned. The best characterized of these enzymes is the acid SMase (SMPD1) that is predominantly located in lysosomes but also secreted in response to different ligand-receptor interactions at the cell surface (5Schuchman E.H. Suchi M. Takahashi T. Sandhoff K. Desnick R.J. J. Biol. Chem. 1991; 266: 8531-8539Abstract Full Text PDF PubMed Google Scholar). SMPD1 deficiency results in lipid storage disorders that manifest in Niemann-Pick disease (6Ferlinz K. Hurwitz R. Sandhoff K. Biochem. Biophys. Res. Commun. 1991; 179: 1187-1191Crossref PubMed Scopus (39) Google Scholar). The regulated breakdown of SM to ceramide by activation of SMPD1 has been implicated in numerous cell responses from cell survival to apoptosis (1Gulbins E. Kolesnick R. Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (348) Google Scholar, 2Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 3Kolesnick R.N. Krönke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 4Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar).Two Mg2+-dependent neutral SMases, SMPD2 and -3, have been recently molecularly cloned based on their sequence homology to bacterial nSMases (7Tomiuk S. Hofmann K. Nix M. Zumbansen M. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3638-3643Crossref PubMed Scopus (257) Google Scholar, 8Hofmann K. Tomiuk S. Wolff G. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5895-5900Crossref PubMed Scopus (258) Google Scholar). The availability of two cDNAs encoding neutral sphingomyelin phosphodiesterase-2 and -3 (SMPD2 and -3) has allowed their biochemical and functional characterization. The ubiquitously expressed nSMase1 is Mg2+-dependent, localizes to the endoplasmic reticulum (ER), and is reversibly inhibited by oxidized glutathione and reactive oxygen species. Recently, it has been shown that nSMase1 has phospholipase C activity toward specific lysophospholipids (9Sawai H. Domae N. Nagan N. Hannun Y.A. J. Biol. Chem. 1999; 274: 38131-38139Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Using an antisense strategy, Tonetti et al. (10Tonnetti L. Veri M.C. Bonvini E. D'Adamio L. J. Exp. Med. 1999; 189: 1581-1589Crossref PubMed Scopus (86) Google Scholar) have shown that nSMAse1 could be involved in ceramide-mediated apoptosis triggered by T cell receptor ligation. However, gene-targeted mice deficient for nSMase1 do not show any overt functional phenotype and no accumulation or changed metabolism of SM or other lipids (11Zumbansen M. Stoffel W. Mol. Cell. Biol. 2002; 22: 3633-3638Crossref PubMed Scopus (49) Google Scholar). Thus, although some insights into the properties of nSMase1 have been provided, its functional significance remains elusive.NSMase2 is expressed predominantly in brain colocalizing with a Golgi marker in neuronal cells (8Hofmann K. Tomiuk S. Wolff G. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5895-5900Crossref PubMed Scopus (258) Google Scholar). NSMase2 has a neutral pH optimum, depends on Mg2+ ions, and is activated by unsaturated fatty acids and phosphatidylserine (PS). Biochemical characterization of nSMase2 overexpressed in yeast cells lacking inositol phosphosphingolipid phospholipase (Isc1p) reveals that nSMase2 is a structural gene for nSMase that acts as a bona fide nSMase in cells (12Sawai H. Okamoto Y. Luberto C. Mao C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: 39793-39798Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 13Marchesini N. Luberto C. Hannun Y.A. J. Biol. Chem. 2003; 278: 13775-13783Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Stable overexpression of nSMase2 in MCF7 cells results in a markedly decreased growth rate at the late exponential phase, suggesting that nSMase2 is involved in the regulation of cell growth (13Marchesini N. Luberto C. Hannun Y.A. J. Biol. Chem. 2003; 278: 13775-13783Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Deletions in the gene encoding nSMase2 result in developmental and postnatal abnormalities, yet the precise function of these enzymes remain elusive. Stoffel et al. (14Stoffel A. Chaurushiya M. Singh B. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9079-9084Crossref PubMed Scopus (48) Google Scholar) have recently demonstrated that gene-targeted mice deficient for nSMase2 develop a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency, suggesting that nSMase2 plays a pivotal role in the control of late embryonic and postnatal development. These authors have also observed immature architecture and delayed ossification of long bones. Strikingly, positional cloning of the recessive mutation fragilitas ossium (fro) in mice identified a deletion in the gene encoding nSMase2 (Smpd3) that led to complete loss of enzymatic activity (15Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Negre-Salvayre A. Goldberg M. Guenet J.L. Poirier C. Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (138) Google Scholar). The mouse mutation fragilitas ossium leads to a syndrome of severe osteogenesis and dentinogenesis imperfecta with no collagen defect. At birth, homozygous fro/fro mice are smaller than normal with deformities and multiple fractures of ribs and long bones (15Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Negre-Salvayre A. Goldberg M. Guenet J.L. Poirier C. Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (138) Google Scholar). Notably, the precise relationship between the impairment of nSMase2, its cascade of intracellular effects, and the production of defective extracellular mineralized tissue is not yet fully understood.We have recently purified to homogeneity and characterized a Mg2+ ion-dependent, 97-kDa nSMase from bovine brain (16Bernardo K. Krut O. Wiegmann K. Kreder D. Micheli M. Schäfer R. Sickman A. Schmidt W.E. Schröder J.M. Meyer H.E. Sandhoff K. Krönke M. J. Biol. Chem. 2000; 275: 7641-7647Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Amino acid sequencing of tryptic peptides showed no apparent homology to nSMase1 or -2 nor to any known bacterial SMase. By data bank searches, we identified a human protein with sequence homologies to the bovine nSMase. Here we investigated the hypothesis that the human homolog designated nSMase3 (SMPD4) functions as a nSMase in cells. In this study, we have established that nSMase3 is a structural gene for neutral sphingomyelinase and have determined its biochemical properties. Furthermore, we have shown that nSMase3 is responsive to TNF.EXPERIMENTAL PROCEDURESCell Culture and Materials—MCF7, HeLa, and HEK293 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 μgml-1 streptomycin and 100 units ml-1 penicillin (Biochrom) in a humidified 5% CO2 incubator.Yeast Strain and Culture Media—The Saccharomyces cerevisiae strain JK9-3dΔIsc1p deficient for the Isc1p gene (12Sawai H. Okamoto Y. Luberto C. Mao C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: 39793-39798Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) was kindly provided by Yusuf Hannun (Biochemistry and Molecular Biology, Medical University, Charleston, SC). Yeast extracts and peptone were from Difco (Heidelberg, Germany). Synthetic minimal medium (SD), SD/Gal, and uracil dropout supplement were purchased from Clontech (Heidelberg, Germany). Scyphostatin was a kind gift of Dr. T. Ogita and F. Nara, Sankyo Co. Ltd., Tokyo, Japan.Sequence Identification and Analysis and cDNA Cloning—Homology search using the bovine peptide KGLPYLEQLFR against protein sequences derived from a draft of the human genome project was performed using stand-alone BLAST program (17Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59166) Google Scholar). The protein sequence was analyzed for conserved patterns or motifs by search against the Prosite or Pfam data bases, respectively. The ClustalW algorithm was employed for protein sequence alignment. Plasmids containing the full-length coding sequence of human nSMase3 (DKFZp434A1711Q) and nSMase2 (IRALp962J187Q) were obtained from German Resource Center for Genome Research (RZPD) Deutsches Ressourcenzentrum für Genomforschung and reamplified by PCR for subcloning.Expression of nSMase3 in Yeast—The pYES2 yeast expression vector containing a galactose-inducible promoter was purchased from Invitrogen (Karlsruhe, Germany). cDNA of the human nSMase3 was generated by PCR using Pfu proofreading polymerase (Promega, Mannheim, Germany). The following PCR primers were used to introduce EcoRI- and SalI-endonuclease restriction sites: forward, 5′-GGAATTCTGATGACGACTTTCGGCG-3′; reverse, 5′-GTCGACGTCAGGGCTGGTGCAGCTT-3′ to facilitate subcloning. YES2 and the PCR product were digested by the restriction enzymes, purified, and ligated. Human nSMase2 was cloned in a similar way using XhoI and BamHI restriction sites (forward, 5′-GGGATCCATGGTTTTGTACACGACC-3′; reverse 5′-CCTCGAGCTATGCCTCCTCCTCCC-3′) and served as a control. The sequence of the resulting plasmids were confirmed by sequencing. Plasmids were transfected into yeast JK9-3dΔIsc1p cells as described by Marchesini et al. (13Marchesini N. Luberto C. Hannun Y.A. J. Biol. Chem. 2003; 278: 13775-13783Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and the expression of nSMase3 was induced by incubating the cells in synthetic complete uracil medium containing 2% galactose overnight.Preparation of Lysates of Yeast Cells—Yeast cells were suspended in buffer containing 25 mm Tris (pH 7.4), 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 4 μg/ml each of chymostatin, leupeptin, anti-pain, and pepstatin A. Cells were disrupted with glass beads as described previously (12Sawai H. Okamoto Y. Luberto C. Mao C. Bielawska A. Domae N. Hannun Y.A. J. Biol. Chem. 2000; 275: 39793-39798Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The glass beads and cell debris were removed by centrifugation at 2,000 × g for 10 min, and the supernatant was centrifuged at 100,000 × g to obtain the microsomal and cytosolic fractions. For the studies on cation and lipid effects and substrate requirements of nSMase2, membranes were delipidated by incubation in lysis buffer in the presence of 1% Triton X-100 and incubated at 4 °C for 1 h. The suspension was centrifuged at 100,000 × g for 90 min, and the supernatant was used for enzymatic determinations. Protein concentration was determined using a Bio-Rad protein assay reagent.Overexpression of nSMase3 in Mammalian Cells—The open reading frame (ORF) of human nSMase3 (and nSMase2) was amplified by PCR using nSMase primers containing XhoI/EcoRI restriction sites. To obtain a stably expressing MCF7 cell line, full-length nSMase3 subcloned into the pRK vector was transfected into MCF7 cells using the Ca2+-phosphate precipitation method (18Sambrook J. Russel D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, New York2001Google Scholar). For the selection of stable transfectants, 800 μg/ml G418 (Invitrogen) was added to the medium. Independent colonies were picked and cultured in separate wells, and three clones from single cells expressing the highest nSMase activity in vitro were used as nSMase3 overexpressors. To determine the subcellular localization of nSMase3, three pcDNA3 expression vectors encoding HA-tagged nSMase were generated. The HA tag (YPYDVPDYA) was either added by PCR at the 5′ or at the 3′ end of the nSMase3 (pnSMase3HA5 or pSMase3HA3). Alternatively, the HA tag was inserted internally, that is, at nucleotide position 1233 (amino acid 411) of the nSMase3 ORF (pnSMase3HAi) by the PCR overlap extension procedure (19Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6797) Google Scholar). Briefly, the HA sequence (5′-TACCCATACGATGTTCCAGATTACGCT-3′) was introduced into the nSMase ORF by the use of mutagenic forward and reverse oligonucleotides that prime downstream (1233-1253 ORF) and upstream (1213-1233) of the insertion site, respectively. Primers contained an overlapping complementary 5′ extension (HA sequence) to generate two DNA fragments having overlapping ends. These fragments were combined in a subsequent "fusion" PCR reaction in which the overlapping ends annealed, allowing the 3′ overlap of each strand to serve as a primer for the 3′ extension of the complementary strand. The resulting fusion product was amplified further by PCR and cloned into the multicloning site (EcoRI/XhoI) of the pcDNA3 expression vector. HA-tagged nSMAse3 was detected by affinity-purified polyclonal rabbit anti-HA antibody (eBioscience) and visualized by Alexa-488-conjugated goat anti-rabbit secondary antibody (Invitrogen).Assay for Phosphodiesterase Activity of Neutral SMase toward SM and Other Phospholipids—The micellar SMase assay using exogenous radiolabeled SM was described by Wiegmann et al. (20Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (673) Google Scholar). To measure phosphodiesterase activity of neutral SMase, pellets were dissolved in a buffer containing 20 mm Hepes (pH 7.4), 10 mm MgCl2, 2mm EDTA, 5 mm dithiothreitol, 0.1 mm Na3VO4, 0.1 mm Na2MoO4,30mm p-nitrophenylphosphate, 10 mm β-glycerophosphate, 750 μm ATP, 1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μm pepstatin, and 0.5% Chaps. After 15 min at 4 °C, cells were homogenized by repeated squeezing of the cells through an 18-gauge needle. Nuclei and cell debris were removed by a low speed centrifugation (800 × g). The protein concentration in supernatants containing the cytosolic and membrane fractions was measured using a protein assay (Pierce, Hamburg, Germany). 50 μg of protein were incubated for 2 h at 37 °C in a buffer containing 20 mm Hepes, 1 mm MgCl2 (pH 7.4) and 2.25 μ l of [N-methyl-14C]SM. For specificity controls, the substrates used were PE, PS, PC, or LysoPAF. All radiolabeled substrates were obtained from Amersham Biosciences, Braunschweig, Germany. Phosphorylcholine was then extracted with 800 μl of chloroform:methanol (2:1 v/v) and 250 μl of H2O. Radioactive phosphorylcholine produced from [14C]SM was quantified by counting 200 μl of the aqueous phase by scintillation counting.To measure phosphodiesterase activity toward LysoPAF, the reaction mixture was incubated with 0.59 pmol of [3H]lysoPAF diluted 100-fold with non-radioactive lysoPAF (Biomol). After 60 min of incubation at 37 °C, lysoPAF was extracted by the Bligh and Dyer (21Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42174) Google Scholar) method and separated by thin layer chromatography (TLC) in a solvent containing chloroform/methanol/2 n NH4OH (60:35:5, v/v/v). The bands corresponding to the lysoPAF standard were scraped from the TLC plate for liquid scintillation counting.Sphingomyelin Measurement—Cells were seeded at 0.3 × 106 cells/10-cm dish in 8 ml of complete growth medium. The next day, the cells were labeled with [methyl-3H]choline chloride (1 μCi/ml final concentration in 10 ml of growth medium/plate) for 48 h. The cells were then chased with 10 ml of complete medium. At the indicated time points, the medium from each plate was collected, and the cells were washed once with 2 ml of ice-cold PBS. The cells were scraped off on ice in 2 ml of PBS, and each plate was washed with an additional 2 ml of PBS. Cells and washes were pooled with the medium and centrifuged for 5 min at 2,000 × g (4 °C). Lipids were extracted by the Bligh and Dyer (21Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42174) Google Scholar) method, and aliquots of 250 μl were used for phosphorous and SM determination as previously described (20Wiegmann K. Schütze S. Machleidt T. Witte D. Krönke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (673) Google Scholar).Detection of Ceramide—The generation of the neutral lipid cleavage product of SMases, ceramide, was detected by the diacylglycerol-kinase assay combined with TLC analysis. Briefly, MCF7 cells were washed two times in ice-cold PBS. Pellets were resuspended in 1.825 ml of H2O, and the supernatants were transferred into glass tubes. Subsequently, 6 ml of CHCl3/MeOH/1 n HCl (100/100/1 v/v/v) were added. Tubes were sonicated for 5 min in a water bath sonicator and then centrifuged for 10 min at 6000 × g. The lower organic phase was dried down under nitrogen. The dry samples were resuspended in 2 ml of 0.1 n KOH and incubated for 1 h at 37 °C (alkaline hydrolysis). 6 ml of CHCl3/MeOH/1 n HCl (100/100/1 v/v/v), 1 ml of CHCl3, and 2.25 ml of H2O were added. After vortexing, the samples were centrifuged for 10 min at 6000 × g. The lower organic phase was dried down under nitrogen and resuspended in 100 μl of CHCl3 followed by transfer into Eppendorf tubes. From this point, the diacylglycerol-kinase assay kit protocol was followed according to the manufacturer (Amersham Biosciences). For thin layer chromatography, plates were pre-run in a solvent system composed of methanol/chloroform (1:1). The plates were removed from the tank and air-dried. The TLC chambers were pre-equilibrate for 1 h at room temperature with the solvent system cloroform:methanol:acidic acid (65:15:5,v/v/v). Dried samples were resuspend in 20 μl of chloroform:methanol (95:5, v/v). Samples were streaked onto a 10 × 10 cm silica gel thin layer plate. The plates were placed in a paper-lined TLC developing tank, and the solvent was allowed to migrate to the top of the plate. The plates were removed from the tank, air-dried, and exposed to Kodak XAR films at room temperature.Immunofluorescence—HeLa cells were grown on coverslips and transfected with 1 μg of pnSMase3-HAc using ExGen 500 in vitro transfection reagent (Fermentas, St. Leon-Rot, Germany) and incubated for 24 h. The cells were washed twice with cold PBS and then fixed with 3% paraformaldehyde for 20 min, permeabilized with 0.1% saponin in PBS for 10 min, and blocked with 3% bovine serum albumin, 0.005% sodium azide, 0,2% teleostean gelatin, and 0.1% saponin in PBS for 30 min. For immunostaining, cells were incubated for 1 h with rabbit anti-HA tag (eBioscience), rat anti-KDEL (carboxyl-terminal ER retention tetrapeptide) (kindly provided by Dr. J. C. Howard, Institute for Genetics, University of Cologne, Cologne, Germany), or mouse anti-p230 antibody (BD Transduction Laboratories), washed with 0.1% saponin in PBS, and then incubated for 30 min with Cy3-conjugated goat anti-rat Ig as a secondary reagent. The Golgi apparatus was stained by TRITC-conjugated wheat germ agglutinin. Nuclei were counterstained with Hoechst 33258 and examined using a confocal fluorescence microscope.RESULTSIdentification of a Human Homolog to Bovine nSMase—We have previously reported the purification to homogeneity and biochemical characterization of a Mg2+-dependent nSMase from bovine brain (16Bernardo K. Krut O. Wiegmann K. Kreder D. Micheli M. Schäfer R. Sickman A. Schmidt W.E. Schröder J.M. Meyer H.E. Sandhoff K. Krönke M. J. Biol. Chem. 2000; 275: 7641-7647Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Amino acid sequencing of tryptic peptides of bovine nSMase showed no homologies to any known nSMases. Allowing for mismatches due to possible species-specific primary sequences, extensive sequence data base searches revealed a homology to the human hypothetical protein FLJ20297 (synonyms KIAA1418 and LOC55627; GenBank™ accession number BAA92656). The predicted mRNA contains an ORF that encodes a protein of 866 amino acids resulting in a predicted molecular mass of 97.8 kDa, which matches the molecular mass of the nSMase previously purified from bovine brain. An alignment of the novel nSMase3 with nSMases1 and -2 is shown in Fig. 1A. Apparently, nSMase3 lacks an N-terminal signal peptide. In addition, a C-terminal hydrophobic region (amino acids 828-850) representing a putative transmembrane (TM) domain is followed by only 16 amino acid residues. The lack of an N-terminal leader sequence combined with a hydrophobic TM domain close to the C terminus defines the group of tail-anchored integral membrane proteins (22Borgese N. Colombo S. Pedrazzini E. J. Cell Biol. 2003; 161: 1013-1019Crossref PubMed Scopus (203) Google Scholar). The precise location of the entire functional N-terminal portion of nSMase3 cannot be unequivocally deduced, because according to the specific algorithm applied, one to three TM regions can be identified. We employed different prediction algorithms based on analysis hydrophobicity profiles (Toppred2) (23von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1399) Google Scholar), multiple sequence alignment analysis (TMAP, TMpred) (24Persson B. Argos P. J. Mol. Biol. 1994; 237: 182-192Crossref PubMed Scopus (422) Google Scholar), bias of amino acids in the TM domains (DAS) (25Cserzo M. Wallin E. Simon I. von Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar), or hidden Markov's models (TMHMM) (26Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (8882) Google Scholar). All of the algorithms predicted with a high probability the existence of at least one TM domain near to the C terminus. The total number of predicted TM regions differ from one (TMHMM) to two (DAS) or even four (Toppred2, TMpred). Thus, although integral membrane association of nSMase3 can be readily predicted, the exact number of TM regions is uncertain. Other than that, no statistically significant patterns or motifs were recognized. Strikingly, the alignment with mammalian nSMases1 and -2 showed no apparent homologies. The data base search revealed proteins closely related to nSMase3 in mouse, dog, chicken, zebrafish, Drosophila, and Caenorhabditis elegans (Fig. 1B).FIGURE 1Comparison of the sequence of nSMase3 with functional analogs and homologous proteins. A, the deduced amino acid sequence of nSMase3 was aligned with nSMase1 and nSMase2 (GenBank™ accession numbers NP_003071 and NP_061137, respectively). Positions of amino acids are numbered on the left. Gray shaded boxes indicate identical or similar amino acids, and highly conserved amino acids are shaded in black. The solid-lined frame indicates the putative TM domain, and the dashed frame indicates the match with bovine peptide sequences. B, proteins with significant amino acid sequence homology were identified by a BLAST search of the GenBank™ data base. If more than one sequence from the same species were identified, sequences with the highest degrees of similarity were preferred. The sequence of nSMase3 was aligned with deduced amino acid sequences of homologous proteins from dog (GenBank™ accession number XP_543560), mouse (BAB31737), chicken (XP_415236), zebrafish (XP_706729), Drosophila (NP_650124), and C. elegans (NP_492324) using the ClustalW algorithm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The gene for nSMase3 is encoded on chromosome 2, chromosomal locus 2q21.1. Although the predicted gene comprises ∼31 alternative exons and may result in 12 different splice variants, we found that nSMase3 mRNA was ubiquitously expressed as a single 4.6 kb mRNA species, with the exception of leukocytes where two signals were visible. The greatest abundance of nSMase3 mRNA was observed in striated muscle and heart muscle (Fig. 2). It should be noted that the current version of the GenBank™ data base defines two shorter isoforms of FLJ20297 mRNA as a reference sequence. This reflects alternate splicing events that were hypothesized to occur on theoretical grounds and should result in mRNAs with 3,600 and 3,300 nucleotides, respectively. The 4.6 kb size of nSMase3 mRNA, however, suggests abundant expression of full-length cDNA corresponding to the cDNA clone with GenBank™ accession number AB037839.FIGURE 2Expression of nSMase3 mRNA in human tissues. Poly(A)+ mRNA from different human tissues (Clontech, human 12-lane multiple tissue Northern blot) was analyzed by Northern blotting using as a hybridization probe a randomly primed 32P-labeled full-length nSMase3 cDNA.View Large Image Fig
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