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• W2004142844 abstract "The acid sphingomyelinase (ASM) gene, which has been implicated in ceramide-mediated cell signaling and atherogenesis, gives rise to both lysosomal SMase (L-SMase), which is reportedly cation-independent, and secretory SMase (S-SMase), which is fully or partially dependent on Zn2+ for enzymatic activity. Herein we present evidence for a model to explain how a single mRNA gives rise to two forms of SMase with different cellular trafficking and apparent differences in Zn2+ dependence. First, we show that both S-SMase and L-SMase, which contain several highly conserved zinc-binding motifs, are directly activated by zinc. In addition, SMase assayed from a lysosome-rich fraction of Chinese hamster ovary cells was found to be partially zinc-dependent, suggesting that intact lysosomes from these cells contain subsaturating levels of Zn2+. Analysis of Asn-linked oligosaccharides and of N-terminal amino acid sequence indicated that S-SMase arises by trafficking through the Golgi secretory pathway, not by cellular release of L-SMase during trafficking to lysosomes or after delivery to lysosomes. Most importantly, when Zn2+-dependent S-SMase was incubated with SMase-negative cells, the enzyme was internalized, trafficked to lysosomes, and became zinc-independent. We conclude that L-SMase is exposed to cellular Zn2+ during trafficking to lysosomes, in lysosomes, and/or during cell homogenization. In contrast, the pathway targeting S-SMase to secretion appears to be relatively sequestered from cellular pools of Zn2+; thus S-SMase requires exogeneous Zn2+ for full activity. This model provides important information for understanding the enzymology and regulation of L- and S-SMase and for exploring possible roles of ASM gene products in cell signaling and atherogenesis. The acid sphingomyelinase (ASM) gene, which has been implicated in ceramide-mediated cell signaling and atherogenesis, gives rise to both lysosomal SMase (L-SMase), which is reportedly cation-independent, and secretory SMase (S-SMase), which is fully or partially dependent on Zn2+ for enzymatic activity. Herein we present evidence for a model to explain how a single mRNA gives rise to two forms of SMase with different cellular trafficking and apparent differences in Zn2+ dependence. First, we show that both S-SMase and L-SMase, which contain several highly conserved zinc-binding motifs, are directly activated by zinc. In addition, SMase assayed from a lysosome-rich fraction of Chinese hamster ovary cells was found to be partially zinc-dependent, suggesting that intact lysosomes from these cells contain subsaturating levels of Zn2+. Analysis of Asn-linked oligosaccharides and of N-terminal amino acid sequence indicated that S-SMase arises by trafficking through the Golgi secretory pathway, not by cellular release of L-SMase during trafficking to lysosomes or after delivery to lysosomes. Most importantly, when Zn2+-dependent S-SMase was incubated with SMase-negative cells, the enzyme was internalized, trafficked to lysosomes, and became zinc-independent. We conclude that L-SMase is exposed to cellular Zn2+ during trafficking to lysosomes, in lysosomes, and/or during cell homogenization. In contrast, the pathway targeting S-SMase to secretion appears to be relatively sequestered from cellular pools of Zn2+; thus S-SMase requires exogeneous Zn2+ for full activity. This model provides important information for understanding the enzymology and regulation of L- and S-SMase and for exploring possible roles of ASM gene products in cell signaling and atherogenesis. SMases 1The abbreviations used are: SMase, sphingomyelinase; S-SMase, secreted sphingomyelinase; ASM, acid sphingomyelinase; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; endo H, β-endo-N-acetylglucosaminidase H; HI-FBS, heat-inactivated fetal bovine serum; LDL, low density lipoprotein; L-SMase, lysosomal sphingomyelinase; PBS, phosphate-buffered saline; PSG, penicillin, streptomycin, and glutamine; PAGE, polyacrylamide gel electrophoresis; SM, sphingomyelin; TIMP-1, tissue inhibitor of metalloproteinase-1; BSA, bovine serum albumin. (SM phosphodiesterase, EC 3.1.4.12) have been implicated in a wide variety of physiologic and pathophysiologic processes, including lysosomal hydrolysis of endocytosed SM (1Levade T. Salvayre R. Blazy-Douste L. J. Clin. Chem. Biochem. 1986; 24: 205-220PubMed Google Scholar, 2Brady R.O. Stanbury J.B. Wyngarden J.B. Fredrickson D.S. Goldstein J.L. Brown M.S. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1983: 831-841Google Scholar), ceramide-mediated cell signaling (3Kolesnick R.N. Prog. Lipid Res. 1991; 30: 1-38Crossref PubMed Scopus (258) Google Scholar, 4Hannun Y.A. Bell R.M. Science. 1989; 243: 500-507Crossref PubMed Scopus (1111) Google Scholar), membrane vesiculation (5Skiba P.J. Zha X. Maxfield F.R. Schissel S.L. Tabas I. J. Biol. Chem. 1996; 271: 13392-13400Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 6Zha X. Leopold P.L. Skiba P.J. Tabas I. Maxfield F.R. J. Cell Biol. 1998; 140: 39-47Crossref PubMed Scopus (174) Google Scholar), alterations in intracellular cholesterol trafficking (5Skiba P.J. Zha X. Maxfield F.R. Schissel S.L. Tabas I. J. Biol. Chem. 1996; 271: 13392-13400Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 7Slotte J.P. Bierman E.L. Biochem. J. 1988; 250: 653-658Crossref PubMed Scopus (274) Google Scholar, 8Porn M.I. Slotte J.P. Biochem. J. 1995; 308: 269-274Crossref PubMed Scopus (47) Google Scholar, 9Okwu A.K. Xu X. Shiratori Y. Tabas I. J. Lipid Res. 1994; 35: 644-655Abstract Full Text PDF PubMed Google Scholar), and atherogenesis (10Xu X. Tabas I. J. Biol. Chem. 1991; 266: 24849-24858Abstract Full Text PDF PubMed Google Scholar, 11Tabas I. Li Y. Brocia R.W. Wu S.W. Swenson T.L. Williams K.J. J. Biol. Chem. 1993; 268: 20419-20432Abstract Full Text PDF PubMed Google Scholar, 12Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (273) Google Scholar, 13Williams K.J. Tabas I. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar). One type of mammalian SMase is a magnesium-dependent, membrane-bound neutral SMase, and Tomiuk et al. (14Tomiuk S. Hofmann K. Nix M. Zumbansen M. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3638-3643Crossref PubMed Scopus (259) Google Scholar) have recently reported the cloning of an enzyme that has several properties in common with this SMase. Two other types of mammalian SMases are lysosomal SMase (L-SMase) and secretory SMase (S-SMase), both of which arise from the “acid SMase” or “ASM” gene (15Schuchman E.H. Suchi M. Takahashi T. Sandhoff K. Desnick R.J. J. Biol. Chem. 1991; 266: 8531-8539Abstract Full Text PDF PubMed Google Scholar, 16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Both enzymes are soluble hydrolases that function optimally at acid pH in a standard in vitro micellar assay (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 17Spence M.W. Adv. Lipid Res. 1993; 26: 3-23PubMed Google Scholar), although we have shown that S-SMase can hydrolyze physiologic SM-containing substrates at neutral pH (Ref. 18Schissel S.L. Jiang X. Tweedie-Hardman J. Jeong T. Camejo E.H. Najib J. Rapp J.H. Williams K.J. Tabas I. J. Biol. Chem. 1998; 273: 2738-2746Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar and see below). Both L- and S-SMase are absent from the cells of patients with types A and B Niemann-Pick disease, which is due to mutations in the ASM gene, and from the cells of ASM knock-out mice (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). S-SMase may have significant physiologic roles, since extracellular SM hydrolysis may be involved in some or all of the non-lysosomal processes listed above. For example, several lines of evidence have implicated extracellular SM hydrolysis in atherogenesis. First, treatment of LDL with SMase in vitro leads to LDL aggregation (10Xu X. Tabas I. J. Biol. Chem. 1991; 266: 24849-24858Abstract Full Text PDF PubMed Google Scholar, 11Tabas I. Li Y. Brocia R.W. Wu S.W. Swenson T.L. Williams K.J. J. Biol. Chem. 1993; 268: 20419-20432Abstract Full Text PDF PubMed Google Scholar), which is a prominent event during atherogenesis (19Hoff H.F. Morton R.E. Ann. N. Y. Acad. Sci. 1985; 454: 183-194Crossref PubMed Scopus (63) Google Scholar, 20Nievelstein P.F.E.M. Fogelman A.M. Mottino G. Frank J.S. Arterioscler. Thromb. 1991; 11: 1795-1805Crossref PubMed Google Scholar, 21Guyton J.R. Klemp K.F. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 4-11Crossref PubMed Scopus (207) Google Scholar) and one that leads to massive macrophage foam cell formation (10Xu X. Tabas I. J. Biol. Chem. 1991; 266: 24849-24858Abstract Full Text PDF PubMed Google Scholar, 11Tabas I. Li Y. Brocia R.W. Wu S.W. Swenson T.L. Williams K.J. J. Biol. Chem. 1993; 268: 20419-20432Abstract Full Text PDF PubMed Google Scholar, 22Hoff H.F. O'Neill J. Pepin J.M. Cole T.B. Eur. Heart J. 1990; 11: 105-115Crossref PubMed Google Scholar, 23Khoo J.C. Miller E. McLoughlin P. Steinberg D. Arteriosclerosis. 1988; 8: 348-358Crossref PubMed Google Scholar, 24Suits A.G. Chait A. Aviram M. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2713-2717Crossref PubMed Scopus (163) Google Scholar). Second, aggregated LDL from human and animal atherosclerotic lesions shows evidence of hydrolysis by extracellular SMase, and LDL retained in rabbit aortic strips ex vivo is hydrolyzed by an extracellular, cation-dependent SMase (12Schissel S.L. Tweedie-Hardman J. Rapp J.H. Graham G. Williams K.J. Tabas I. J. Clin. Invest. 1996; 98: 1455-1464Crossref PubMed Scopus (273) Google Scholar). Third, S-SMase, a leading candidate for this arterial wall enzyme, is secreted by macrophages (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) and endothelial cells (25Marathe S. Schissel S.L. Yellin M.J. Beatini N. Mintzer R. Williams K.J. Tabas I. J. Biol. Chem. 1998; 273: 4081-4088Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), cell types found in atherosclerotic lesions. Fourth, S-SMase is able to hydrolyze the SM in atherogenic lipoproteins at neutral pH (18Schissel S.L. Jiang X. Tweedie-Hardman J. Jeong T. Camejo E.H. Najib J. Rapp J.H. Williams K.J. Tabas I. J. Biol. Chem. 1998; 273: 2738-2746Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Other possible roles for S-SMase may be in ceramide-mediated cell signaling (26Cifone M.G. De Maria R. Roncaioli P. Rippo M.R. Azuma M. Lanier L.L. Santoni A. Testi R. J. Exp. Med. 1994; 180: 1547-1552Crossref PubMed Scopus (598) Google Scholar, 27Cifone M.G. Roncaioli P. De Maria R. Camarda G. Santoni A. Ruberti G. Testi R. EMBO J. 1995; 14: 5859-5868Crossref PubMed Scopus (277) Google Scholar, 28Wiegmann K. Schutze S. Machleidt T. Witte D. Kronke M. Cell. 1994; 78: 1005-1015Abstract Full Text PDF PubMed Scopus (678) Google Scholar, 29Santana P. Pena L.A. Haimovitz-Friedman A. Martin S. Green D. McLoughlin M. Cordon-Cardo C. Schuchman E.H. Fuks Z. Kolesnick R. Cell. 1996; 86: 189-199Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 30Haimovitz-Friedman A. Cordon-Cardod C. Bayoumyb S. Garzottob M. McLoughlin M. Gallily R. Edwards III, C.K. Schuchman E.H. Fuks Z. Kolesnick R. J. Exp. Med. 1997; 186: 1831-1841Crossref PubMed Scopus (384) Google Scholar), perhaps after re-uptake of the secreted enzyme into endosomal vesicles; in extracellular sphingomyelin catabolism after nerve injury and during demyelination (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 31Svensson M. Eriksson P. Persson J.K. Molander C. Arvidsson J. Aldskogius H. Brain Res. Bull. 1993; 30: 499-506Crossref PubMed Scopus (88) Google Scholar, 32Bauer J. Sminia T. Wouterlood F.G. Dijkstra C.D. J. Neurosci. Res. 1994; 38: 365-375Crossref PubMed Scopus (172) Google Scholar, 33Hartung H.P. Jung S. Stoll G. Zielasek J. Schmidt B. Archelos J.J. Toyka K.V. J. Neuroimmunol. 1992; 40: 197-210Abstract Full Text PDF PubMed Scopus (226) Google Scholar); and in defense against viruses, many of which are enriched in SM (34van Genderen I.L. Brandimarti R. Torrisi M.R. Campadelli G. van Meer G. Virology. 1994; 200: 831-836Crossref PubMed Scopus (95) Google Scholar, 35Aloia R.C. Jensen F.C. Curtain C.C. Mobley P.W. Gordon L.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 900-904Crossref PubMed Scopus (189) Google Scholar) and can be inactivated by treatment with SMase in vitro. 2S. L. Schissel and I. Tabas, unpublished data. L- and S-SMase are very similar proteins. Previous work from our laboratories has shown that cells transfected with an ASM cDNA overexpress both L-SMase and S-SMase (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), indicating that S-SMase does not arise by alternative processing of the ASM gene. In addition, antibodies made against L-SMase recognize S-SMase, demonstrating that the common mRNA is translated in the same reading frame, and the molecular weights of the enzymes on Western blot are similar (see Ref. 16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar and below). Nevertheless, S-SMase requires exogenously added Zn2+ for activation in in vitro assays, whereas L-SMase isolated from cell or tissue homogenates does not (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). In fact, the lack of stimulation of L-SMase by any cations and its lack of inhibition by EDTA has led to a long-standing body of literature labeling L-SMase as a “cation-independent” enzyme (1Levade T. Salvayre R. Blazy-Douste L. J. Clin. Chem. Biochem. 1986; 24: 205-220PubMed Google Scholar). Despite the widespread interest in mammalian SMases in general and in products of the ASM gene in particular, little is known about cellular itineraries of L-SMase and S-SMase or about the mechanism for their apparent difference in zinc dependence. For example, does S-SMase arise by release or exocytosis of L-SMase from lysosomes or by a separate trafficking pathway, and how could two enzymes that are so similar differ in their requirement for zinc? In this report, we show that S-SMase is secreted through a non-lysosomal secretory pathway, and we present evidence that both forms of the enzyme are zinc-activated. According to our model, L-SMase is exposed to cellular Zn2+during trafficking to lysosomes, in lysosomes, and/or during cell homogenization. Most likely, the Zn2+ dependence of L-SMase has been overlooked because it is already saturated with Zn2+ upon isolation from cell homogenates and thus does not respond to exogenous Zn2+ at the time of assay. Furthermore, as is the case with known zinc metalloenzymes (cf. Ref. 36Little C. Otnass A. Biochim. Biophys. Acta. 1975; 391: 326-333Crossref PubMed Scopus (56) Google Scholar), the Zn2+ cannot be stripped from L-SMase by simple exposure to EDTA. In contrast, the pathway targeting S-SMase to secretion appears to be relatively sequestered from cellular pools of Zn2+. Thus, this enzyme requires Zn2+during subsequent in vitro assay. The information in this report should prove useful for future studies that explore the enzymology, regulation, and functions of these important SMases. The Falcon tissue culture plasticware used in these studies was purchased from Fisher. Tissue culture media and other tissue culture reagents were obtained from Life Technologies, Inc. Fetal bovine serum was obtained from HyClone Laboratories (Logan, UT) and was heat-inactivated for 1 h at 65 °C (HI-FBS). [9,10-3H]Palmitic acid (56 Ci/mmol) was purchased from NEN Life Science Products, and [N-palmitoyl-9–10-3H]sphingomyelin was synthesized as described previously (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 37Sripada P.K. Maulik P.R. Hamilton J.A. Shipley G.G. J. Lipid Res. 1987; 28: 710-718Abstract Full Text PDF PubMed Google Scholar, 38Ahmad T.Y. Sparrow J.T. Morrisett J.D. J. Lipid Res. 1985; 26: 1160-1165Abstract Full Text PDF PubMed Google Scholar).N,N-Dimethylformamide, 1,3-dicyclohexylcarbodiimide,N-hydroxysuccinimide, andN,N-diisopropylethylamine were purchased from Aldrich. Precast 4–20% gradient polyacrylamide gels were purchased from NOVEX (San Diego, CA). Nitrocellulose was from Schleicher & Schuell. Rabbit anti-FLAG-tagged S-SMase was kindly provided by Dr. Henri Lichenstein (Amgen, Boulder, CO). FLAG-tagged S-SMase was purified by anti-FLAG immunoaffinity chromatography from the conditioned medium of CHO cells transfected with a human ASM-FLAG cDNA. FLAG-tagged L-SMase was purified from a 16,000 × g pellet (see below) of FLAG-ASM-transfected CHO cells using anti-FLAG immunoaffinity chromatography, Superose-12 gel filtration chromatography, and a second round of anti-FLAG immunoaffinity chromatography. Peroxidase-conjugated goat anti-rabbit IgG was purchased from Pierce. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was a kind gift from Dr. Yasunori Okada (Kanazawa University, Kanazawa, Japan). The thiol-based metalloproteinase inhibitors, HS-CH2-R-CH(CH2-CH(CH3)2)-C)-Phe-Ala-NH2and HO-NH-CO-CH2-CH(CH2CH(CH3)2)-C)-naphthyl-Ala-NH-CH2-CH2-NH2, were purchased from Peptides International, Inc. (Louisville, KY). β-Endo-N-acetylglucosaminidase H (endo H) and peptide-N-glycanase F were purchased from Boehringer Mannheim. Bovine liver 215-kDa mannose 6-phosphate receptor linked to Affi-Gel 15 was made as described by Varki and Kornfeld (39Varki A. Kornfeld S. J. Biol. Chem. 1983; 258: 2808-2818Abstract Full Text PDF PubMed Google Scholar) and was kindly supplied by Walter Gregory and Dr. Stuart Kornfeld (Washington University, St. Louis). Sphingosylphosphorylcholine, 1,10-phenanthroline, and all other chemicals and reagents were from Sigma, and all organic solvents were from Fisher. Monolayer cultures of J774.A1 cells (from the American Type Culture Collection, see Ref. 40Khoo J.C. Miller E. McLoughlin P. Tabas I. Rosoff W.J. Biochim. Biophys. Acta. 1989; 1012: 215-217Crossref PubMed Scopus (14) Google Scholar) were grown and maintained in spinner culture with DMEM/HI-FBS/PSG as described previously (9Okwu A.K. Xu X. Shiratori Y. Tabas I. J. Lipid Res. 1994; 35: 644-655Abstract Full Text PDF PubMed Google Scholar, 40Khoo J.C. Miller E. McLoughlin P. Tabas I. Rosoff W.J. Biochim. Biophys. Acta. 1989; 1012: 215-217Crossref PubMed Scopus (14) Google Scholar). Human skin fibroblasts obtained from a patient with type A Niemann-Pick disease (R496L mutation (41Levran O. Desnick R.J. Schuchman E.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3748-3752Crossref PubMed Scopus (78) Google Scholar)) were grown in DMEM/HI-FBS/PSG. CHO-K1 cells were grown in Hams' F-12 containing 10% HI-FBS and PSG. CHO cells stably transfected with ASM cDNA 3X. He, S. R. P. Miranda, A. Dagan, S. Gatt, and E. H. Schuchman, submitted for publication. were maintained in DMEM/HI-FBS/PSG (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Cells were plated in 35-mm (6-well) or 100-mm dishes in media containing HI-FBS for 48 h. The cells were then washed 3 times with PBS and incubated for 24 h in fresh serum-free media (1 and 6 ml per 35-mm and 100-mm dishes, respectively) containing 0.2% BSA. This 24-h conditioned medium was collected for SMase assays. Following the incubations described above and in the figure legends, cells were placed on ice, and the serum-free conditioned media were removed. The cells were washed two times with ice-cold 0.25 m sucrose and scraped into 0.3 and 3.0 ml of this sucrose solution per 35- and 100-mm dishes, respectively. Unless indicated otherwise, the scraped cells were disrupted by sonication on ice using three 5-s bursts (Branson 450 Sonifier), and the cellular homogenates were assayed for total protein by the method of Lowry et al. (42Merrill A.H. Jones D.D. Biochim. Biophys. Acta. 1990; 1044: 1-12Crossref PubMed Scopus (396) Google Scholar) and for SMase activity as described below. The conditioned media were spun at 800 × g for 5 min to pellet any contaminating cells and concentrated 6-fold using a Centriprep 30 (Amicon; Beverly, MA) concentrator (molecular weight cut-off = 30,000). For the experiment in Fig. 5, CHO-K1 cells were incubated in 100-mm dishes in serum-free media and washed as described above. Cells were then scraped in 5 ml of 0.25 m sucrose and broken open under 500 p.s.i. of nitrogen pressure for 1.5 min using a nitrogen cell disruption bomb (Parr Instruments, Moline, IL). Following disruption, a portion of the cells was subjected to brief sonication as described above; this portion of cells is referred to as the cell homogenate. The remainder of disrupted cells was spun at 1300 × g for 5 min to pellet any remaining intact cells and nuclei. This post-nuclear supernatant was collected, and the volume was increased to 15 ml with 0.25 m sucrose and then spun at 16,000 ×g for 30 min. The pellet from this centrifugation was resuspended in 1 ml of 0.25 m sucrose and sonicated as above, and this material, as well as the cell homogenate, was assayed for SMase activity. As described previously (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), the standard 200-μl assay mixture consisted of up to 40 μl of sample (conditioned media or homogenized cells; see above) and a volume of assay buffer (0.1 m sodium acetate, pH 5.0) to bring the volume to 160 μl. The reaction was initiated by the addition of 40 μl of substrate (50 pmol of [3H]sphingomyelin) in 0.25m sucrose containing 3% Triton X-100 (final concentration of Triton X-100 in the 200-μl assay mix = 0.6%). When added, the final concentrations of EDTA and Zn2+ were 5 and 0.1 mm, respectively, unless indicated otherwise. The assay mixtures were incubated at 37 °C for no longer than 3 h and then extracted by the method of Bligh and Dyer (43Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar); the lower, organic phase was harvested, evaporated under N2, and fractionated by TLC using chloroform/methanol (95:5). The ceramide spots were scraped and directly counted to quantify [3H]ceramide. Typically, our assay reactions contained approximately 20 μg of cellular homogenate protein and a volume of conditioned media derived from a quantity of cells equivalent to approximately 50 μg of cellular protein. Protein samples were boiled in buffer containing 1% SDS and 10 mm dithiothreitol for 10 min, loaded onto 4–20% gradient polyacrylamide gels, and electrophoresed for 50 min at 35 mA in buffer containing 0.1% SDS (SDS-PAGE). Following electrophoresis, some gels were fixed in methanol/glacial acetic acid/water (5:2:3, v/v) and then silver-stained using reagents from Bio-Rad. Other gels were electrotransferred (100 V for 1.5 h) to nitrocellulose for immunoblotting. For immunoblotting, the nitrocellulose membranes were incubated with 5% Carnation nonfat dry milk in buffer A (24 mm Tris, pH 7.4, containing 0.5m NaCl) for 3 h at room temperature. The membranes were then incubated with rabbit anti-FLAG-tagged S-SMase polyclonal antiserum (1:2000) in buffer B (buffer A containing 0.1% Tween 20, 3% nonfat dry milk, and 0.1% bovine serum albumin) for 1 h at room temperature. After washing four times with buffer A containing 0.1% Tween 20, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000) for 1 h in buffer B at room temperature. The membranes were subsequently washed twice with 0.3% Tween 20 in buffer A and twice with 0.1% Tween 20 in buffer A. Finally, the blots were soaked in the enhanced chemiluminescence reagent (NEN Life Science Products) for 2 min and exposed to x-ray film for 1 min. We followed the procedure described by Hurwitz et al. (44Hurwitz R. Ferlinz K. Vielhaber G. Moczall H. Sandhoff K. J. Biol. Chem. 1994; 269: 5440-5445Abstract Full Text PDF PubMed Google Scholar). CHO-K1 cells were incubated overnight with serum-free medium (CHO-S-SFM II from Life Technologies, Inc.). Fifty μg of 30-fold-concentrated conditioned medium and 50 μg of cell homogenate were diluted 1:1 (v/v) with 50 mmsodium acetate buffer, pH 5.0, containing 2% SDS and 20 mmβ-mercaptoethanol (44Hurwitz R. Ferlinz K. Vielhaber G. Moczall H. Sandhoff K. J. Biol. Chem. 1994; 269: 5440-5445Abstract Full Text PDF PubMed Google Scholar). One set of aliquots of the diluted conditioned medium and cell homogenate was treated for 16 h at 37 °C with 4 milliunits of endo H. Another set of aliquots was diluted another 15-fold with 50 mm sodium phosphate buffer, pH 7.2, containing 1% Nonidet P-40 and treated for 16 h at 37 °C with 100 milliunits of peptide-N-glycanase F. The endo H digest and a trichloroacetic acid pellet of the peptide-N-glycanase F digest (44Hurwitz R. Ferlinz K. Vielhaber G. Moczall H. Sandhoff K. J. Biol. Chem. 1994; 269: 5440-5445Abstract Full Text PDF PubMed Google Scholar) were boiled in SDS/dithiothreitol buffer and then electrophoresed and immunoblotted as described above. We used a modification of the method of Hortin and Gibson (45Hortin G.L. Gibson B.L. Prep. Biochem. 1989; 19: 49-59PubMed Google Scholar). Packed 1-ml columns of chelating Sepharose 6B (iminodiacetic acid coupled to agarose gel via a hydrophilic spacer; from Amersham Pharmacia Biotech) were washed with 10 mm sodium acetate buffer, pH 6.0, containing 10 mm EDTA, to leave the column uncharged, or containing 60 mm ZnCl2, to charge the column with Zn2+. The columns were then equilibrated with 50 mm Hepes buffer, pH 7.4, containing 50 mm NaCl. One-ml samples of a 1:1 (v/v) mixture of this equilibration buffer and unconcentrated conditioned medium from ASM-transfected CHO cells (above) were loaded onto the columns and incubated for 15 min at room temperature. The columns were then washed with 7.5 ml of 50 mm Hepes, pH 7.4, containing either 100 mm NaCl or 1 m NaCl, which was collected as 10 0.75-ml fractions. The columns were eluted with 3.75 ml of 50 mm Hepes, pH 7.4, containing 50 mm EDTA plus 1 mm1,10-phenanthroline, which was collected as 5 0.75-ml fractions. Aliquots of each of the fractions were spotted on nitrocellulose using a slot-blot apparatus and then immunoblotted using goat anti-human L-SMase polyclonal antiserum as described above. Unless otherwise indicated, results are given as means ± S.D. (n = 3); absent error bars in the figures signify S.D. values smaller than the graphic symbols. We sought to address how L- and S-SMase acquire their apparent differences in zinc dependence. One explanation would be that the secreted form requires a Zn2+-dependent cofactor. Because many lysosomal enzymes undergo proteolytic activation (46Hasilik A. Experientia ( Basel ). 1992; 48: 130-151Crossref PubMed Scopus (141) Google Scholar), an obvious candidate for a Zn2+-dependent cofactor would be a zinc metalloproteinase. Five sets of results, however, ruled out this possibility. First, Zn2+-activated S-SMase can be subsequently inactivated by Zn2+ chelation (see below); reversibility of Zn2+-induced activation is not consistent with proteolytic activation. Second, inhibitors of zinc metalloproteinases, such as tissue inhibitor of metalloproteinase-1 (TIMP-1) (47Nagase H. Suzuki K. Itoh Y. Kan C.C. Gehring M.R. Huang W. Brew K. Adv. Exp. Med. Biol. 1996; 389: 23-31Crossref PubMed Scopus (34) Google Scholar) and two different thiol-based peptide inhibitors, HS-CH2-R-CH(CH2-CH(CH3)2)-C)-Phe-Ala-NH2and HO-NH-CO-CH2-CH(CH2CH(CH3)2)-C)-naphthyl-Ala-NH-CH2-CH2-NH2(48Panchenko M.V. Stetler-Stevenson W.G. Trubetskoy O.V. Gacheru S.N. Kagan H.M. J. Biol. Chem. 1996; 271: 7113-7119Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), did not affect the ability of Zn2+ to activate S-SMase (data not shown). Third, mammalian zinc metalloproteinases require Ca2+ as well as Zn2+ for activity (49Reynolds J.J. Oral Diseases. 1996; 2: 70-76Crossref PubMed Scopus (182) Google Scholar), whereas Ca2+ is not a requirement for the activation of S-SMase (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas I. J. Biol. Chem. 1996; 271: 18431-18436Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Fourth, comparison of Zn2+-activated S-SMase from CHO cells with that of the intracellular (lysosomal) enzyme by immunoblot analysis showed that the activated secreted form had a somewhat higher, not lower, apparent molecular weight (see control data in Fig. 3, below); in addition, S-SMase not activated with Zn2+ had the same apparent molecular weight as Zn2+-activated S-SMase (data not shown). Fifth, we found that highly purified S-SMase, obtained by either anti-FLAG immunoaffinity purification of a FLAG-tagged S-SMase or by concanavalin A chromatography followed by anti-SMase immunoaffinity purification of S-SMase from ASM-transfected CHO cells (16Schissel S.L. Schuchman E.H. Williams K.J. Tabas" @default.
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• W2004142844 title "The Cellular Trafficking and Zinc Dependence of Secretory and Lysosomal Sphingomyelinase, Two Products of the Acid Sphingomyelinase Gene" @default.
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• W2004142844 doi "https://doi.org/10.1074/jbc.273.29.18250" @default.
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