Molecular Cloning, Characterization, and Expression of a Novel Human Neutral Sphingomyelinase

Neutral sphingomyelinase (N-SMase) has emerged as an important cell membrane-associated enzyme that participates in several signal transduction and cell regulatory phenomena. Using expression cloning, we have identified a 3.7-kilobase pair cDNA transcript for N-SMase whose open reading frame predicts a 397-amino acid polypeptide. Transfection of COS-7 cells with cDNA for N-SMase resulted in a marked increase in N-SMase activity. Recombinant N-SMase (r-N-SMase) had the following physical-chemical properties. Mg2+ activated and Cu2+ and glutathione inhibited the activity of r-N-SMase. In contrast, dithiothreitol did not alter the activity of the enzyme. Of several phospholipids examined, sphingomyelin was the preferred substrate for r-N-SMase. The apparent molecular mass of r-N-SMase derived from COS-7 cells was ∼90 kDa, similar to the native neutral sphingomyelinase prepared from human urine. However, upon expression inEscherichia coli, the apparent molecular mass of the recombinant enzyme was ∼45 kDa. We speculate that this apparent difference in recombinant enzymes derived from COS-7 and E. coli cells may be due to extensive post-transcriptional changes. r-N-SMase has numerous post-transcriptional modification sites such as phosphorylation sites via protein kinase C, casein kinase II, tyrosine kinase, and cAMP- and cGMP-dependent protein kinases as well as sites for glycosylation and myristoylation. Amino acid sequence alignment studies revealed that r-N-SMase has some similarity to acid sphingomyelinase and significant homology to the death domains of tumor necrosis factor-α receptor-1 and Fas/Apo-I. We believe that the molecular cloning and characterization of N-SMase cDNA will accelerate the process to define its role as a key regulator in apoptosis, lipid and lipoprotein metabolism, and other cell regulatory pathways. Neutral sphingomyelinase (N-SMase) has emerged as an important cell membrane-associated enzyme that participates in several signal transduction and cell regulatory phenomena. Using expression cloning, we have identified a 3.7-kilobase pair cDNA transcript for N-SMase whose open reading frame predicts a 397-amino acid polypeptide. Transfection of COS-7 cells with cDNA for N-SMase resulted in a marked increase in N-SMase activity. Recombinant N-SMase (r-N-SMase) had the following physical-chemical properties. Mg2+ activated and Cu2+ and glutathione inhibited the activity of r-N-SMase. In contrast, dithiothreitol did not alter the activity of the enzyme. Of several phospholipids examined, sphingomyelin was the preferred substrate for r-N-SMase. The apparent molecular mass of r-N-SMase derived from COS-7 cells was ∼90 kDa, similar to the native neutral sphingomyelinase prepared from human urine. However, upon expression inEscherichia coli, the apparent molecular mass of the recombinant enzyme was ∼45 kDa. We speculate that this apparent difference in recombinant enzymes derived from COS-7 and E. coli cells may be due to extensive post-transcriptional changes. r-N-SMase has numerous post-transcriptional modification sites such as phosphorylation sites via protein kinase C, casein kinase II, tyrosine kinase, and cAMP- and cGMP-dependent protein kinases as well as sites for glycosylation and myristoylation. Amino acid sequence alignment studies revealed that r-N-SMase has some similarity to acid sphingomyelinase and significant homology to the death domains of tumor necrosis factor-α receptor-1 and Fas/Apo-I. We believe that the molecular cloning and characterization of N-SMase cDNA will accelerate the process to define its role as a key regulator in apoptosis, lipid and lipoprotein metabolism, and other cell regulatory pathways. tumor necrosis factor-α TNF-α receptor neutral sphingomyelinase recombinant N-SMase low density lipoprotein polymerase chain reaction glutathioneS-transferase base pair(s) kilobase pair(s) Type C sphingomyelinases (sphingomyelin phosphodiesterase, EC3.1.4.12) are a group of phospholipases that catalyze the hydrolytic cleavage of sphingomyelin to ceramide and phosphocholine (1Chatterjee S. Adv. Lipid Res. 1993; 26: 25-48PubMed Google Scholar). Neutral sphingomyelinase from human urine and cultured human kidney proximal tubular cell membranes has an apparent molecular mass of 92 kDa and neutral pH optima and is heat-unstable. This enzyme is associated with the cell membrane in tissues and cultured cells (1Chatterjee S. Adv. Lipid Res. 1993; 26: 25-48PubMed Google Scholar, 2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google Scholar, 3Spence M. Adv. Lipid Res. 1993; 26: 3-23PubMed Google Scholar, 4Lui B. Obeid L.M. Hannun Y.A. Cell. Dev. Biol. 1997; 8: 311-322Google Scholar). In cultured mammalian cells, the addition of diverse agonists,i.e. vitamin D3, tumor necrosis factor-α (TNF-α),1 interferon-γ, and nerve growth factor, results in the activation of N-SMase and the consequent production of ceramide. Ceramide and its higher homologs have been shown to serve as lipid second messengers that lead to diverse cell regulatory phenomena, such as differentiation, proliferation, and programmed cell death or apoptosis (1Chatterjee S. Adv. Lipid Res. 1993; 26: 25-48PubMed Google Scholar, 4Lui B. Obeid L.M. Hannun Y.A. Cell. Dev. Biol. 1997; 8: 311-322Google Scholar, 5Kolesnick R. Golde D.W. Cell. 1994; 77: 325-328Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 6Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar). Activation of N-SMase by TNF-α in human skin fibroblasts results in not only the hydrolytic cleavage of sphingomyelin, residing on the cell surface, but also the mobilization of cholesterol to the interior of the cell. Such cholesterol is esterified by the action of fatty-acyl-coenzyme A acyltransferase to form cholesteryl esters (1Chatterjee S. Adv. Lipid Res. 1993; 26: 25-48PubMed Google Scholar,7Chatterjee S. J. Biol. Chem. 1994; 269: 879-882Abstract Full Text PDF PubMed Google Scholar). In a human hepatocyte cell line, TNF-α-induced N-SMase activation and ceramide production led to the maturation of sterol regulatory element-binding protein-1 and a subsequent increase in LDL receptor mRNA expression (8Lawler Jr., J.F. Yin M. Diehl A.M. Roberts E. Chatterjee S. J. Biol. Chem. 1998; 273: 5053-5059Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Interestingly, this phenomenon is not accompanied by apoptosis and occurs in a sterol-independent fashion. Thus, LDL receptors may be up-regulated via this cascade of reactions involving N-SMase independent of the presence of sterols in the culture medium. These studies suggest that N-SMase may be involved in the regulation of lipid and lipoprotein metabolism and sterol influx (7Chatterjee S. J. Biol. Chem. 1994; 269: 879-882Abstract Full Text PDF PubMed Google Scholar, 8Lawler Jr., J.F. Yin M. Diehl A.M. Roberts E. Chatterjee S. J. Biol. Chem. 1998; 273: 5053-5059Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Rabbit skeletal muscle has been shown to contain at least two kinds of N-SMase. These are the classical 92-kDa Mg2+-dependent N-SMase and a Mg2+-independent 53-kDa protein. The localization of N-SMase in skeletal muscle transferase tubule membrane is in agreement with the production of the sphingomyelin-derived second messenger, sphingosine, in such tubules. Since sphingosine has been shown to modulate calcium release from sarcoplasmic reticulum membranes, these studies imply that N-SMase/sphingosine signaling systems may be a physiologically relevant mechanism of regulation of Ca2+ levels in skeletal muscle and may well be involved in muscle contraction (9Ghosh N. Sabbadini R. Chatterjee S. Mol. Cell. Biochem. 1998; 189: 161-168Crossref PubMed Google Scholar). In additional studies, we have shown that Sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase and the release of ceramide. 2J. T. Jan, S. Chatterjee, and D. F. Griffin, submitted for publication. Collectively, these studies imply that N-SMase may play a central role in diverse cell regulatory phenomena. Nevertheless, the evidence accumulated so far in support of this view has been largely indirect and has recently been the subject of rigorous debate (11Hofman K. Dixit V.M. Trends Biochem. Sci. 1999; 24: 227-228Abstract Full Text Full Text PDF PubMed Google Scholar), particularly in regard to the controversial role of ceramide in apoptosis (12Kolesick R. Hannun Y.A. Trends Biochem. Sci. 1999; 24: 224-226Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). We rationalized that to unambiguously demonstrate the functional role of N-SMase in the various cell regulatory phenomena discussed above, it was essential to first clone this protein. Therefore, we employed a monospecific polyclonal antibody against human N-SMase and a human kidney cDNA library to pursue expression cloning. In this work, we describe the molecular cloning and characterization of recombinant N-SMase (r-N-SMase) expressed in Escherichia coli as well as in cultured COS-7 cells. In work to be published elsewhere, we will show that overexpression of N-SMase results in spontaneous apoptosis in human aortic smooth muscle cells independent of the presence of agonists. 3S. Chatterjee, K. Gakenheimer, H. Han, S. Dey, G. Hutchins, I. Dobromilskaya, and A. Snowden, submitted for publication. α-32PdATP (specific activity of 0.25 Bq/mmol), α-32PdCTP (specific activity of 9.25 Bq/mmol), andN-methyl14Csphingomyelin (specific activity of 1.85 Bq/mmol were purchased from Amersham Pharmacia Biotech. E. coli strain 1090r was purchased from Life Technologies, Inc. COS-7 cells were purchased from American Type Culture Collection (Manassas, VA). These cells were grown in Eagle's minimal essential medium containing 10% dialyzed fetal bovine serum (Hyclone Laboratories, Logan, Utah), penicillin, streptomycin, and nonessential amino acids. The multiple tissue Northern blot and human kidney library were purchased from CLONTECH (Palo Alto, CA). Restriction endonucleases were purchased from Amersham Pharmacia Biotech. The transient expression vector pSV-SPOT-1, LipofectAMINETM, and cell culture medium were purchased from Life Technologies, Inc. Bacterial cells and COS-7 cells transfected with N-SMase cDNA were homogenized in Tris/glycine buffer (pH 7.4) containing 0.1% cutscum. The samples were mixed vigorously and sonicated for 10 s. Next, the samples were transferred to a 4 °C incubator and shaken for ∼2 h. Every hour, the samples were sonicated again on ice and further shaken. Subsequently, the samples were centrifuged at 10,000 ×g for 10 min. The supernatants were collected, and the protein content was measured using bovine serum albumin as a standard and subjected to N-SMase assay using 14Csphingomyelin as a substrate (2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google Scholar). COS-7 cells transfected with pSV-SPOT-1 and pHH1 were subjected to detergent extraction as described above. The supernatants were subjected to sodium lauryl sarcosine-polyacrylamide (7.5%) gel electrophoresis at 4 °C as described previously (13Taki T. Chatterjee S. Anal. Biochem. 1995; 224: 490-493Crossref PubMed Scopus (7) Google Scholar). The gel was calibrated with prestained proteins of known molecular mass (Bio-Rad). Subsequently, the gel was sliced, and pieces were transferred to a glass test tube and subjected to N-SMase assay at pH 7.4 using 14Csphingomyelin as a substrate. The human kidney library was screened using the anti-N-SMase antibody available in our laboratory according to the manufacturer's protocol. Briefly, λgt11 phage was plated at 3 × 104 plaque-forming units/150-mm plate on a lawn ofE. coli strain y1090r. Incubation was carried out at 42 °C for 3.5 h to allow lytic phage growth. Then, a filter saturated with 10 mmisopropyl-β-d-thiogalactopyranoside was placed on top of the plate and incubated overnight at 37 °C. Next, the filter was blocked with a solution of 5% nonfat dry milk for 1 h at room temperature. Subsequently, the filter was incubated with antibody against N-SMase at 1:200 dilution at room temperature overnight, and signal was detected by the enhanced chemiluminescence technique (ECL, Amersham Pharmacia Biotech). Sixty-three clones were obtained by screening 1 × 106 λgt11 phage clones. The most intense clones of cells were subjected to secondary and tertiary screening. All positive clones were subjected to PCR to identify their insert size. a clone containing the insert was for further by and We the as described by the In this the in the was with phage and incubated for h at °C. Next, was and was at 37 °C. The cell were at and and and h. The cells were with and for further a expression was with and A insert the N-SMase open reading frame that is of sequence was with a containing of sequence at the at The and a vector and Pharmacia Biotech). and was into E. coli cells. A of was grown in medium at °C cell density was was to protein expression for h. were and the protein was using according to by the Pharmacia Biotech). N-SMase was from the protein by Such were subjected to activity and N-SMase cDNA into a transient expression was with endonucleases The insert containing N-SMase was and into the transient expression vector A pHH1 was COS-7 cells with pHH1 and we 3 × COS-7 in a plate in of Eagle's medium with 10% dialyzed fetal were incubated in a 10% 37 °C incubator were The cells were transfected with pHH1 using in medium. The medium was overnight the cells were at various and h by at × for 10 with and at °C. r-N-SMase derived from COS-7 cells was as described previously (2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google and was employed for characterization studies with regard to pH substrate for for and other previously described of N-SMase as well as with anti-N-SMase antibody and serum The were subjected to and the activity of N-SMase was measured in the We of sequence from as the to out as described above. A was obtained and of this was with of α-32PdATP and of α-32PdCTP using Technologies, The activity of this was × Next, the multiple tissue Northern blot was in of solution at 42 °C for h with Then, 10 of was Next, 10 of solution was and at °C. The were in and at room temperature for and in and for at °C. the blot was overnight to a at °C using two A containing a insert was as a in these for the molecular cloning of cDNA for N-SMase involved the of monospecific against human N-SMase (2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google Scholar). This from our in human cells TNF-α-induced activation of N-SMase was by such in cells and aortic smooth muscle cells, and apoptosis, was by anti-N-SMase and TNF-α-induced maturation of sterol regulatory element-binding protein-1 in human cells and TNF-α-induced cholesteryl in human skin fibroblasts were by of cells with such (7Chatterjee S. J. Biol. Chem. 1994; 269: 879-882Abstract Full Text PDF PubMed Google Scholar). We this antibody to the human kidney λgt11 cDNA Sixty-three positive clones were obtained by screening 1 × 106 clones. The most intense clones were subjected to secondary and tertiary screening. All positive clones were subjected to PCR to identify their insert size. a clone containing the insert was subjected to into to The insert was with using and by The sequence of cDNA revealed an open reading frame of base predicts a 397-amino acid polypeptide. The amino acid sequence of N-SMase is shown in are several modification sites in this protein. at tyrosine phosphorylation at and two cAMP- and cGMP-dependent protein kinase phosphorylation sites at and were This protein also has casein kinase phosphorylation sites at and sites at and were are 10 protein kinase C phosphorylation sites at and of N-SMase that are a the presence of a The at the tyrosine phosphorylation at and several other phosphorylation sites are on the Such sites may be subject to further glycosylation and The alignment of the death of N-SMase with the death domains of TNF-α receptor-1 and is shown in N-SMase homology to the death to the death N. S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). N-SMase and have of the at the C of the death we that a acid N-SMase had and similarity to acid sphingomyelinase M. T. K. J. Biol. Chem. Full Text PDF PubMed Google not Since is in our amino acid this may suggest multiple of N-SMase. this we expressed N-SMase in E. coli and COS-7 cells. that the cDNA N-SMase with an apparent molecular mass of kDa, we of the with glutathione We transfected this into E. coli to and to protein. The expression of the protein was by for h. The protein was using N-SMase was from the protein by it as a with a molecular mass of kDa and the is The N-SMase by previously has an apparent molecular mass of kDa and a of (2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google Scholar). This difference may be due to multiple post-transcriptional of the mammalian N-SMase with the N-SMase The N-SMase was by antibody against human N-SMase r-N-SMase expressed in E. an activity of of The amino acid sequence of N-SMase the of further the cDNA we it into a mammalian cell transient expression vector and transfected it into COS-7 cells. 4 A that cells transfected with N-SMase cDNA a increase in N-SMase activity with cells transfected with vector h of N-SMase activity following sodium lauryl gel of r-N-SMase activity to an protein 4 This was further by assay of revealed that the apparent molecular mass of r-N-SMase is ∼90 kDa, and this was similar to the native N-SMase derived from human urine not shown in r-N-SMase derived from cells the enzyme had a pH of r-N-SMase did not have enzyme activity in the or studies revealed of several phospholipids sphingomyelin to be the most preferred substrate not on revealed that r-N-SMase was with Mg2+ In contrast, Cu2+ inhibited the activity of the enzyme. studies revealed that r-N-SMase was to with but the activity was inhibited by glutathione characterization studies using antibody against N-SMase and serum as a revealed the the were subjected to an anti-N-SMase antibody increase in the mass of N-SMase protein was revealed the other of N-SMase activity in the supernatants obtained following with anti-N-SMase antibody revealed a Collectively, these that anti-N-SMase antibody r-N-SMase. In contrast, serum did not the Mg2+-dependent N-SMase and most of the N-SMase activity was associated with the The at the of the N-SMase was to N-SMase mRNA in various human N-SMase was expressed in the human tissues the transcript and from tissue to the of N-SMase in human and from to in an However, Northern blot revealed that the transcript of N-SMase expressed in of the human tissues is The transcript may be derived from or from A Northern blot shown at the of was to serve as a positive Using a human N-SMase monospecific polyclonal we have screened a human kidney cDNA library and have identified a cDNA The open reading frame of the cDNA predicts a 397-amino acid polypeptide. expressed in E. coli, it a protein of ∼45 kDa upon gel expressed in COS-7 cells, the cDNA to r-N-SMase a higher activity to cells and had an apparent molecular mass of ∼90 kDa, multiple post-transcriptional The presence of numerous phosphorylation sites via the action of protein kinase C, casein kinase II, cAMP- and cGMP-dependent protein and tyrosine kinase is of the of this enzyme to of protein kinase C and and casein kinases B. Obeid L.M. Hannun Y.A. Cell. Dev. Biol. 1997; 8: 311-322Google Scholar, A. T. G. J. 1997; PubMed Scopus Google Scholar, E. J. Res. Google Scholar). The amino acid sequence of N-SMase the of This may its to dithiothreitol and S. Chem. 1999; PubMed Scopus Google Scholar, B. Hannun J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of enzyme activity by glutathione is with studies a regulatory mechanism for this enzyme B. Hannun J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). characterization of r-N-SMase revealed similar to of the human N-SMase described by previously (1Chatterjee S. Adv. Lipid Res. 1993; 26: 25-48PubMed Google Scholar, 2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google Scholar, S. Chem. 1999; PubMed Scopus Google Scholar). These were a pH of for activity of activity at °C for of and detergent for activation and by The amino acid sequence and suggest this enzyme to be a N-SMase. Collectively, the physical-chemical of r-N-SMase derived from COS-7 cells, molecular are to of the native enzyme derived from human urine (2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google Scholar). sequence alignment of N-SMase revealed some similarity to acid M. T. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar), it was to with not a that has been shown to acid activity (2Chatterjee S. Ghosh N. J. Biol. Chem. 1989; 264: 12554-12561Abstract Full Text PDF PubMed Google Scholar). r-N-SMase was by antibody against but not by serum the activity of the enzyme in the not be have been previously using and the This may be on the that the polyclonal antibody to the in it of or such as and the pH to 4 to the enzyme from the did not the of as the enzyme was to such Nevertheless, we a increase in the of the by anti-N-SMase antibody and a in the activity of N-SMase in the In the the of a antibody against N-SMase of the to the enzyme may the activity of the enzyme. The of r-N-SMase not were also similar to of human such as a increase in cholesteryl and of the maturation of sterol regulatory element-binding protein-1 and LDL receptor mRNA expression in a of human (8Lawler Jr., J.F. Yin M. Diehl A.M. Roberts E. Chatterjee S. J. Biol. Chem. 1998; 273: 5053-5059Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). overexpression of r-N-SMase in human aortic smooth muscle cells resulted in apoptosis and apoptosis in these cells, 3S. Chatterjee, K. Gakenheimer, H. Han, S. Dey, G. Hutchins, I. Dobromilskaya, and A. Snowden, submitted for publication. and antibody against N-SMase this Han, T. and S. Chatterjee, submitted for publication. The of two receptors and that significant homology has been the These two proteins contain that are also in the nerve growth of proteins such as and The for and are and and apoptosis in cells S. 1993; Scopus Google Scholar, 1995; Full Text PDF PubMed Scopus Google Scholar). The death domains of and have been to with several and proteins that have homology to the death and that a to activation and apoptosis N. S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). studies have shown that overexpression of or lead to apoptosis in the of due to of the death I. D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, it was not to spontaneous apoptosis in cells that was This is further by the alignment of the death of N-SMase with the death domains of and homology N-SMase and have of the at the C of the death studies will be to the of these cDNA of and at and These from the but were in and and of the in Such due to their at derived from and and medium and skeletal muscle the is N-SMase activity and N-SMase mRNA levels in a human tissues had the enzyme activity for N-SMase. muscle and kidney had and N-SMase as with the N-SMase mRNA levels in and kidney are This may be due to regulation at multiple sites this was in in the the cloning of a mammalian neutral sphingomyelinase S. K. M. M. Sci. S. A. 1998; PubMed Scopus Google using sequence homology with a The role of this N-SMase in signaling was also we several and functional the two neutral is amino acid sequence homology the two our N-SMase has multiple sites for protein post-transcriptional and its physical-chemical are overexpression of our N-SMase in human aortic smooth muscle cells apoptosis the presence of an such as However, LDL had an on apoptosis in cells N-SMase. In the overexpression of N-SMase in and cells did not apoptosis independent of the presence of TNF-α S. K. M. M. Sci. S. A. 1998; PubMed Scopus Google Scholar). may be several for the in these two may be to in the molecular cloning of these is that spontaneous apoptosis in cells N-SMase in our be due to the presence of death domains in other S. K. M. M. Sci. S. A. 1998; PubMed Scopus Google Scholar). However, the of the N-SMase and its role in diverse cell signaling we are not to molecular of N-SMase We in the additional will be that will sphingomyelin but may have diverse relevant to human and such proteins may of the of N-SMase are as from a 1995; D. Scopus Google Scholar). We believe that the molecular cloning of N-SMase cDNA will accelerate the process to define its role as a key regulator in apoptosis and other cell regulatory pathways. We for of this