O-Glycosylation of Mucin-like Domain Retains the Neutral Ceramidase on the Plasma Membranes as a Type II Integral Membrane Protein
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m207932200
ISSN1083-351X
AutoresMotohiro Tani, Hiroshi Iida, Makoto Ito,
Tópico(s)Lysosomal Storage Disorders Research
ResumoCeramidase is a key enzyme involved in regulating cellular levels of ceramide, sphingosine, and possibly sphigosine 1-phosphate and thus could modulate sphingolipid signaling. Here we report that O-glycosylation of the mucin-like domain of neutral ceramidases was required for localization to the surface of plasma membranes. The deduced amino acid sequences of the mammalian enzymes contain a serine-threonine-rich domain (mucin box), which follows the signal/anchor sequence, whereas those of bacterial and invertebrate enzymes completely lack a mucin box, suggesting that the specific domain has been acquired during evolution. In HEK293 cells overexpressing ceramidase, the enzyme was not only secreted into the medium after cleavage of the NH2-terminal signal/anchor sequence but also localized at the plasma membrane as a type II integral membrane protein. Lectin blot analysis using peanut agglutinin revealed that the mucin box of the enzyme is highly glycosylated withO-glycans. Interestingly, a mutant lacking the mucin box or possible O-glycosylation sites in the mucin box was secreted into the medium but not localized at the surface of the cells. Furthermore, a mucin box-fused chimera green fluorescent protein (GFP), but not GFP itself, with the signal/anchor sequence was distributed on the surface of the cells. These results suggest thatO-glycosylation of the mucin box retains proteins on the plasma membranes. We also found that the 112-kDa membrane-bound enzyme from mouse kidney is O-glycosylated, whereas the 94-kDa soluble enzyme from liver is not. These results clearly indicate that post-translational modification of the enzyme withO-glycans is tissue-specific and helps the enzyme to localize at the surface of plasma membranes as a type II membrane protein. Ceramidase is a key enzyme involved in regulating cellular levels of ceramide, sphingosine, and possibly sphigosine 1-phosphate and thus could modulate sphingolipid signaling. Here we report that O-glycosylation of the mucin-like domain of neutral ceramidases was required for localization to the surface of plasma membranes. The deduced amino acid sequences of the mammalian enzymes contain a serine-threonine-rich domain (mucin box), which follows the signal/anchor sequence, whereas those of bacterial and invertebrate enzymes completely lack a mucin box, suggesting that the specific domain has been acquired during evolution. In HEK293 cells overexpressing ceramidase, the enzyme was not only secreted into the medium after cleavage of the NH2-terminal signal/anchor sequence but also localized at the plasma membrane as a type II integral membrane protein. Lectin blot analysis using peanut agglutinin revealed that the mucin box of the enzyme is highly glycosylated withO-glycans. Interestingly, a mutant lacking the mucin box or possible O-glycosylation sites in the mucin box was secreted into the medium but not localized at the surface of the cells. Furthermore, a mucin box-fused chimera green fluorescent protein (GFP), but not GFP itself, with the signal/anchor sequence was distributed on the surface of the cells. These results suggest thatO-glycosylation of the mucin box retains proteins on the plasma membranes. We also found that the 112-kDa membrane-bound enzyme from mouse kidney is O-glycosylated, whereas the 94-kDa soluble enzyme from liver is not. These results clearly indicate that post-translational modification of the enzyme withO-glycans is tissue-specific and helps the enzyme to localize at the surface of plasma membranes as a type II membrane protein. ceramide ceramidase fetal bovine serum green fluorescent protein horseradish peroxidase 4-nitrobenzo-2-oxa-1,3-diazole phosphate-buffered saline peanut agglutinin sphingomyelin sphingosine sphingosine 1-phosphate Chinese hamster ovary cells that express polyoma LT antigen Dulbecco's modified Eagle's medium endoplasmic reticulum Sphingolipids have emerged as a multifunctional lipid biomodulator within or among cells. Ceramide (N-acylsphingosine, Cer),1 a common lipid backbone of sphingomyelin (SM) and glycosphingoslipids, has been shown to mediate many cellular events, including cell growth arrest, differentiation, and apoptosis (1Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar, 2Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar), possibly regulating various cytoplasmic proteins such as protein kinases C-ζ (3Bourbon N.A. Yun J. Kester M. J. Biol. Chem. 2000; 275: 35617-35623Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), C-α, and C-δ (4Huwiler A. Fabbro D. Pfeilschifter J. Biochemistry. 1998; 37: 14556-14562Crossref PubMed Scopus (106) Google Scholar), and protein phosphatases 1 and 2A (5Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Sphingosine (Sph), the N-deacylated product of Cer, exerts mitogenic and apoptosis-inducing activities, depending on the cell type and cell cycle (6Zhang H. Buckley N.E. Gibson K. Spiegel S. J. Biol. Chem. 1990; 265: 76-81Abstract Full Text PDF PubMed Google Scholar, 7Ohta H. Sweeney E.A. Masamune A. Yatomi Y. Hakomori S. Igarashi Y. Cancer Res. 1995; 55: 691-697PubMed Google Scholar). Sph is known to be a potent inhibitor of protein kinase C (8Hannun Y.A. Bell R.M. Science. 1987; 235: 670-674Crossref PubMed Scopus (465) Google Scholar) and an activator of 3-phosphoinositide-dependent kinase 1, which is thought to be occasionally localized at the inner plasma membrane (9King C.C. Zenke F.T. Dawson P.E. Dutil E.M. Newton A.C. Hemmings B.A. Bokoch G.H. J. Biol. Chem. 2000; 275: 18108-18113Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Notably, Sph can be phosphorylated to yield Sph 1-phosphate (S1P), which regulates cell proliferation (10Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar), motility (11Kupperman E. An S. Osborne N. Waldron S. Stainier D.Y.R. Nature. 2000; 406: 192-194Crossref PubMed Scopus (341) Google Scholar), and morphology (12Van Brocklyn J.R. Tu Z. Edsall L.C. Schmidt R.R. Spiegel S. J. Biol. Chem. 1999; 274: 4626-4632Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). In contrast to Cer and Sph, S1P appears to act extracellularly by interacting with cell surface G protein-coupled receptors, the endothelial differentiation gene (EDG) family (13Lee M.J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (880) Google Scholar).Ceramidase (CDase, EC 3.5.1.23), an enzyme that catalyzes hydrolysis of the N-acyl linkage of Cer to produce Sph, has been classified into three types mainly based on catalytic pH optima,i.e. acidic, neutral, and alkaline. Neutral CDases, which have an optimum pH of 6.5–8.5, have been cloned from bacteria (14Okino N. Ichinose S. Omori A. Imayama S. Nakamura T. Ito M. J. Biol. Chem. 1999; 274: 36616-36622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), Drosophila (15Yoshimura Y. Okino N. Tani M. Ito M. J. Biochem. 2002; 132: 229-236Crossref PubMed Scopus (49) Google Scholar), mouse (16Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), rat (17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and human (18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Interestingly, phylogenetic analysis revealed that the three CDase isoforms having different pH optima may be derived from different ancestral genes (16Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Mammalian neutral CDase seems to regulate the balance of Cer/Sph/S1P in response to various stimuli, including cytokines (19Nikolova-Karakashian M. Morgan E.T. Alexander C. Liotta D.C. Merrill Jr., A.H. J. Biol. Chem. 1997; 272: 18718-18724Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 20Franzen R. Pautz A. Brautigam L. Geisslinger G. Pfeilschifter J. Huwiler A. J. Biol. Chem. 2001; 276: 35382-35389Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and growth factor (21Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and thus could modulate sphingolipid-mediated signaling. Furthermore, the fact that Sph is not produced by de novo synthesis (22Wang E. Norred W.P. Bacon C.W. Riley R.T. Merrill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar) implies a significant role for CDase in the generation of Sph and possibly S1P.We found that neutral CDases of bacteria (14Okino N. Ichinose S. Omori A. Imayama S. Nakamura T. Ito M. J. Biol. Chem. 1999; 274: 36616-36622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and Drosophila(15Yoshimura Y. Okino N. Tani M. Ito M. J. Biochem. 2002; 132: 229-236Crossref PubMed Scopus (49) Google Scholar) were released from cells as a soluble form, whereas those of mammalian origins were mainly recovered in membrane fractions (21Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Recently, it was found that the intracellular distribution of the mammalian enzyme was cell/tissue-specific. In rat kidney, neutral CDase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting dusts, whereas in liver the enzyme was distributed among endosome-like organelles in hepatocytes (17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Human neutral CDase was exclusively localized to mitochondria in HEK293 and MCF7 cells when overexpressed as a fusion construct with green fluorescent protein (GFP) at the NH2 terminus of the enzyme (18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Furthermore, both neutral and acid CDases were found to be released by murine endothelial cells (23Romiti E. Meacci E. Tani M. Nuti F. Farnararo M. Ito M. Bruni P. Biochem. Biophys. Res. Commun. 2000; 275: 746-751Crossref PubMed Scopus (47) Google Scholar). However, the molecular mechanism by which neutral CDases are localized to different organelles is not well understood.This report clearly shows that O-glycosylation of the mucin-like domain (mucin box) is required to retain neutral CDases on the plasma membranes as a type II integral membrane protein. It was found that the domain was occasionally lost by post-translational processing, resulting in a different localization of the enzyme. These findings facilitate the understanding of the mechanism for cell/tissue-specific localization of neutral CDases and provide some insight into sphingolipid metabolism at the cell surface or in the extracellular milieu.DISCUSSIONThis study demonstrated that the neutral CDase is sorted by a classic pathway via ER/Golgi compartments to the plasma membranes where it is expressed as a type II integral membrane protein or alternatively secreted out of cells after proteolytic processing of the NH2-terminal signal/anchor sequence. This report also clearly indicates that the fate of the protein (as a membrane protein or secretion protein) depends on the presence of a mucin box located in the NH2-terminal region of the enzyme. Because, in contrast to the wild-type CDase, the mucin box-deleted mutant CDase does not localize at the surface of cells and is almost entirely secreted. Furthermore, the fact that bacterial and invertebrate neutral CDases, which lack the mucin box, are secreted without cell surface expression may support this conclusion. In conclusion, the mucin box acquired by the process of evolution enables the mammalian CDase to localize on the cell surface as a type II integral membrane protein. It should be emphasized that the mucin box-mediated cell surface localization is not limited to the neutral CDase but occurs in general, because the mucin box-fused chimeric GFP, but not GFP itself, with signal/anchor sequence was found to be exclusively localized at the surfaces of HEK293 cells.The mechanism by which the mucin box retains CDase on the cell surface is unclear at present. However, the finding made here that replacement of possible O-glycosylation sites (Ser/Thr residues) with Ala greatly increased the secretion and reduced the expression of the CDase on plasma membranes may indicate specific functions ofO-glycans, although the structures of O-glycans remain to be elucidated.A diverse range of membrane proteins of type I or type II topology are occasionally released from the lipid bilayer by proteolysis catalyzed by a group of enzymes referred to as secretases (30Hooper N.H. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). The proteolytic processing of membrane-bound proteins by secretases is well characterized in amyloid precursor protein (31De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1540) Google Scholar), a type I integral membrane protein, and a Golgi-resident sialyltransferase (32Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (230) Google Scholar), a type II integral membrane protein. The latter is cleaved in its membrane-anchoring region by β-secretase and secreted out of the cell (32Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (230) Google Scholar). Thus, it is possible that the neutral CDase is detached from cells after processing of the NH2-terminal signal/anchor sequence by secretases. We indicate the possibility that the mucin box interferes with the action of secretases resulting in the generation of the type II membrane-bound CDase, because the mucin box exists very close to the possible cleavage site of the signal/anchor sequence. However, the molecular mechanism for proteolytic processing of the CDase, including the participation of secretases, remains to be clarified. Another possibility is that the mucin box is a potential signal for the sorting of proteins to plasma membranes without processing of the signal/anchor sequence, although the counterparts for the recognition of O-glycans have yet been characterized.There have been few reports on the role of theO-glycosylation of secretory proteins. It has been reported that O-glycosylation of the COOH-terminal tandem-repeated sequences regulates the secretion of rat pancreatic bile salt-dependent lipase. The O-glycosylation of the enzyme concealed the Pro-, Glu-, Ser-, and Thr-rich domain (PEST region), which is commonly present in rapidly degraded proteins, resulting in delivery to a secretion instead of a degradation pathway (33Bruneau N. Nganga A. Fisher E.A. Lombardo D. J. Biol. Chem. 1997; 272: 27353-27361Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In polarized cells, the O-glycosylation was also found to be required for apical sorting of sucrase isomaltase (34Jacob R. Alfalah M. Grunberg J. Obendorf M. Naim H.Y. J. Biol. Chem. 2000; 275: 6566-6572Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and the neurotrophin receptor (35Yeaman C. Le Gall A.H. Baldwin A.N. Monlauzeur L. Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (245) Google Scholar). It was also reported that a mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells (36Zheng X. Sadler J.E. J. Biol. Chem. 2002; 277: 6858-6863Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The neutral CDase was localized at the apical membranes of proximal tubules, distal tubules, and collecting ducts in rat kidney (17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and thus the possible functions of O-glycans in apical sorting should be examined in this CDase.In contrast to our observation, human neutral CDase has been reported to be expressed in mitochondria when overexpressed in HEK293 and MCF7 cells (18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). We found that the deduced NH2-terminal sequence of the human CDase lacks 19 amino acid residues in comparison with those of mouse and rat CDases (16Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This may result in the generation of an incomplete signal/anchor sequence for ER targeting, allowing the human CDase to target mitochondria instead of the lumen side of the ER.Recently, evidence has emerged that the outer leaflet of the plasma membrane is a site of SM metabolism; SM is abundant here particularly in lipid microdomain rafts (37Prinetti A. Chigorno V. Tettamanti G. Sonnino S. J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and sphingomyelinase as well as CDase is actively secreted from endothelial cells (23Romiti E. Meacci E. Tani M. Nuti F. Farnararo M. Ito M. Bruni P. Biochem. Biophys. Res. Commun. 2000; 275: 746-751Crossref PubMed Scopus (47) Google Scholar, 38Marathe 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 (214) Google Scholar). Furthermore, acid sphingomyelinase was reported to be secreted and helped to metabolize SM in oxidized lipoproteins (39Schissel 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 (274) Google Scholar), and CD95 induces the translocation of acid sphingomyelinase onto the cell surface, resulting in the generation of extracellularly orientated Cer (40Grassme H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). The neutral CDase is expressed at the plasma membrane as a type II membrane protein, the catalytic domain of which is located on the extracellular side, or is secreted out of the cell. Importantly, the generation of S1P in the extracellular milieu, which occurs due to the export of the Sph kinase, was reported (41Ancellin N. Colmont C. Su J. Li Q. Mittereder N. Chae S. Stefansson S. Liau G. Hla T. J. Biol. Chem. 2002; 277: 6667-6675Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Very recently, it was found that phorbol 12-myristate 13-acetate induced the protein kinase C-dependent translocation of Sph kinase to the plasma membrane, resulting in the extracellular release of S1P (42Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Taken together, all metabolic enzymes required for hydrolysis of SM to generate S1P could be present at the outer leaflet of the plasma membrane or in extra milieu, allowing for the formation of an alternative pathway from SM to S1P. In this context, it is important to note that the neutral CDase was actually detected in mouse serum, which may indicate an important role in vascular biology, i.e.regulating the extracellular content of Cer, Sph, and possibly S1P, all of which may function in autocrine/paracrine signaling. Sphingolipids have emerged as a multifunctional lipid biomodulator within or among cells. Ceramide (N-acylsphingosine, Cer),1 a common lipid backbone of sphingomyelin (SM) and glycosphingoslipids, has been shown to mediate many cellular events, including cell growth arrest, differentiation, and apoptosis (1Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1487) Google Scholar, 2Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar), possibly regulating various cytoplasmic proteins such as protein kinases C-ζ (3Bourbon N.A. Yun J. Kester M. J. Biol. Chem. 2000; 275: 35617-35623Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), C-α, and C-δ (4Huwiler A. Fabbro D. Pfeilschifter J. Biochemistry. 1998; 37: 14556-14562Crossref PubMed Scopus (106) Google Scholar), and protein phosphatases 1 and 2A (5Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Sphingosine (Sph), the N-deacylated product of Cer, exerts mitogenic and apoptosis-inducing activities, depending on the cell type and cell cycle (6Zhang H. Buckley N.E. Gibson K. Spiegel S. J. Biol. Chem. 1990; 265: 76-81Abstract Full Text PDF PubMed Google Scholar, 7Ohta H. Sweeney E.A. Masamune A. Yatomi Y. Hakomori S. Igarashi Y. Cancer Res. 1995; 55: 691-697PubMed Google Scholar). Sph is known to be a potent inhibitor of protein kinase C (8Hannun Y.A. Bell R.M. Science. 1987; 235: 670-674Crossref PubMed Scopus (465) Google Scholar) and an activator of 3-phosphoinositide-dependent kinase 1, which is thought to be occasionally localized at the inner plasma membrane (9King C.C. Zenke F.T. Dawson P.E. Dutil E.M. Newton A.C. Hemmings B.A. Bokoch G.H. J. Biol. Chem. 2000; 275: 18108-18113Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Notably, Sph can be phosphorylated to yield Sph 1-phosphate (S1P), which regulates cell proliferation (10Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar), motility (11Kupperman E. An S. Osborne N. Waldron S. Stainier D.Y.R. Nature. 2000; 406: 192-194Crossref PubMed Scopus (341) Google Scholar), and morphology (12Van Brocklyn J.R. Tu Z. Edsall L.C. Schmidt R.R. Spiegel S. J. Biol. Chem. 1999; 274: 4626-4632Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). In contrast to Cer and Sph, S1P appears to act extracellularly by interacting with cell surface G protein-coupled receptors, the endothelial differentiation gene (EDG) family (13Lee M.J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (880) Google Scholar). Ceramidase (CDase, EC 3.5.1.23), an enzyme that catalyzes hydrolysis of the N-acyl linkage of Cer to produce Sph, has been classified into three types mainly based on catalytic pH optima,i.e. acidic, neutral, and alkaline. Neutral CDases, which have an optimum pH of 6.5–8.5, have been cloned from bacteria (14Okino N. Ichinose S. Omori A. Imayama S. Nakamura T. Ito M. J. Biol. Chem. 1999; 274: 36616-36622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), Drosophila (15Yoshimura Y. Okino N. Tani M. Ito M. J. Biochem. 2002; 132: 229-236Crossref PubMed Scopus (49) Google Scholar), mouse (16Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), rat (17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and human (18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Interestingly, phylogenetic analysis revealed that the three CDase isoforms having different pH optima may be derived from different ancestral genes (16Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Mammalian neutral CDase seems to regulate the balance of Cer/Sph/S1P in response to various stimuli, including cytokines (19Nikolova-Karakashian M. Morgan E.T. Alexander C. Liotta D.C. Merrill Jr., A.H. J. Biol. Chem. 1997; 272: 18718-18724Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 20Franzen R. Pautz A. Brautigam L. Geisslinger G. Pfeilschifter J. Huwiler A. J. Biol. Chem. 2001; 276: 35382-35389Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and growth factor (21Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and thus could modulate sphingolipid-mediated signaling. Furthermore, the fact that Sph is not produced by de novo synthesis (22Wang E. Norred W.P. Bacon C.W. Riley R.T. Merrill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar) implies a significant role for CDase in the generation of Sph and possibly S1P. We found that neutral CDases of bacteria (14Okino N. Ichinose S. Omori A. Imayama S. Nakamura T. Ito M. J. Biol. Chem. 1999; 274: 36616-36622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and Drosophila(15Yoshimura Y. Okino N. Tani M. Ito M. J. Biochem. 2002; 132: 229-236Crossref PubMed Scopus (49) Google Scholar) were released from cells as a soluble form, whereas those of mammalian origins were mainly recovered in membrane fractions (21Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Recently, it was found that the intracellular distribution of the mammalian enzyme was cell/tissue-specific. In rat kidney, neutral CDase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting dusts, whereas in liver the enzyme was distributed among endosome-like organelles in hepatocytes (17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Human neutral CDase was exclusively localized to mitochondria in HEK293 and MCF7 cells when overexpressed as a fusion construct with green fluorescent protein (GFP) at the NH2 terminus of the enzyme (18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Furthermore, both neutral and acid CDases were found to be released by murine endothelial cells (23Romiti E. Meacci E. Tani M. Nuti F. Farnararo M. Ito M. Bruni P. Biochem. Biophys. Res. Commun. 2000; 275: 746-751Crossref PubMed Scopus (47) Google Scholar). However, the molecular mechanism by which neutral CDases are localized to different organelles is not well understood. This report clearly shows that O-glycosylation of the mucin-like domain (mucin box) is required to retain neutral CDases on the plasma membranes as a type II integral membrane protein. It was found that the domain was occasionally lost by post-translational processing, resulting in a different localization of the enzyme. These findings facilitate the understanding of the mechanism for cell/tissue-specific localization of neutral CDases and provide some insight into sphingolipid metabolism at the cell surface or in the extracellular milieu. DISCUSSIONThis study demonstrated that the neutral CDase is sorted by a classic pathway via ER/Golgi compartments to the plasma membranes where it is expressed as a type II integral membrane protein or alternatively secreted out of cells after proteolytic processing of the NH2-terminal signal/anchor sequence. This report also clearly indicates that the fate of the protein (as a membrane protein or secretion protein) depends on the presence of a mucin box located in the NH2-terminal region of the enzyme. Because, in contrast to the wild-type CDase, the mucin box-deleted mutant CDase does not localize at the surface of cells and is almost entirely secreted. Furthermore, the fact that bacterial and invertebrate neutral CDases, which lack the mucin box, are secreted without cell surface expression may support this conclusion. In conclusion, the mucin box acquired by the process of evolution enables the mammalian CDase to localize on the cell surface as a type II integral membrane protein. It should be emphasized that the mucin box-mediated cell surface localization is not limited to the neutral CDase but occurs in general, because the mucin box-fused chimeric GFP, but not GFP itself, with signal/anchor sequence was found to be exclusively localized at the surfaces of HEK293 cells.The mechanism by which the mucin box retains CDase on the cell surface is unclear at present. However, the finding made here that replacement of possible O-glycosylation sites (Ser/Thr residues) with Ala greatly increased the secretion and reduced the expression of the CDase on plasma membranes may indicate specific functions ofO-glycans, although the structures of O-glycans remain to be elucidated.A diverse range of membrane proteins of type I or type II topology are occasionally released from the lipid bilayer by proteolysis catalyzed by a group of enzymes referred to as secretases (30Hooper N.H. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). The proteolytic processing of membrane-bound proteins by secretases is well characterized in amyloid precursor protein (31De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1540) Google Scholar), a type I integral membrane protein, and a Golgi-resident sialyltransferase (32Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (230) Google Scholar), a type II integral membrane protein. The latter is cleaved in its membrane-anchoring region by β-secretase and secreted out of the cell (32Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (230) Google Scholar). Thus, it is possible that the neutral CDase is detached from cells after processing of the NH2-terminal signal/anchor sequence by secretases. We indicate the possibility that the mucin box interferes with the action of secretases resulting in the generation of the type II membrane-bound CDase, because the mucin box exists very close to the possible cleavage site of the signal/anchor sequence. However, the molecular mechanism for proteolytic processing of the CDase, including the participation of secretases, remains to be clarified. Another possibility is that the mucin box is a potential signal for the sorting of proteins to plasma membranes without processing of the signal/anchor sequence, although the counterparts for the recognition of O-glycans have yet been characterized.There have been few reports on the role of theO-glycosylation of secretory proteins. It has been reported that O-glycosylation of the COOH-terminal tandem-repeated sequences regulates the secretion of rat pancreatic bile salt-dependent lipase. The O-glycosylation of the enzyme concealed the Pro-, Glu-, Ser-, and Thr-rich domain (PEST region), which is commonly present in rapidly degraded proteins, resulting in delivery to a secretion instead of a degradation pathway (33Bruneau N. Nganga A. Fisher E.A. Lombardo D. J. Biol. Chem. 1997; 272: 27353-27361Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In polarized cells, the O-glycosylation was also found to be required for apical sorting of sucrase isomaltase (34Jacob R. Alfalah M. Grunberg J. Obendorf M. Naim H.Y. J. Biol. Chem. 2000; 275: 6566-6572Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and the neurotrophin receptor (35Yeaman C. Le Gall A.H. Baldwin A.N. Monlauzeur L. Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (245) Google Scholar). It was also reported that a mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells (36Zheng X. Sadler J.E. J. Biol. Chem. 2002; 277: 6858-6863Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The neutral CDase was localized at the apical membranes of proximal tubules, distal tubules, and collecting ducts in rat kidney (17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and thus the possible functions of O-glycans in apical sorting should be examined in this CDase.In contrast to our observation, human neutral CDase has been reported to be expressed in mitochondria when overexpressed in HEK293 and MCF7 cells (18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). We found that the deduced NH2-terminal sequence of the human CDase lacks 19 amino acid residues in comparison with those of mouse and rat CDases (16Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This may result in the generation of an incomplete signal/anchor sequence for ER targeting, allowing the human CDase to target mitochondria instead of the lumen side of the ER.Recently, evidence has emerged that the outer leaflet of the plasma membrane is a site of SM metabolism; SM is abundant here particularly in lipid microdomain rafts (37Prinetti A. Chigorno V. Tettamanti G. Sonnino S. J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and sphingomyelinase as well as CDase is actively secreted from endothelial cells (23Romiti E. Meacci E. Tani M. Nuti F. Farnararo M. Ito M. Bruni P. Biochem. Biophys. Res. Commun. 2000; 275: 746-751Crossref PubMed Scopus (47) Google Scholar, 38Marathe 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 (214) Google Scholar). Furthermore, acid sphingomyelinase was reported to be secreted and helped to metabolize SM in oxidized lipoproteins (39Schissel 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 (274) Google Scholar), and CD95 induces the translocation of acid sphingomyelinase onto the cell surface, resulting in the generation of extracellularly orientated Cer (40Grassme H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). The neutral CDase is expressed at the plasma membrane as a type II membrane protein, the catalytic domain of which is located on the extracellular side, or is secreted out of the cell. Importantly, the generation of S1P in the extracellular milieu, which occurs due to the export of the Sph kinase, was reported (41Ancellin N. Colmont C. Su J. Li Q. Mittereder N. Chae S. Stefansson S. Liau G. Hla T. J. Biol. Chem. 2002; 277: 6667-6675Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Very recently, it was found that phorbol 12-myristate 13-acetate induced the protein kinase C-dependent translocation of Sph kinase to the plasma membrane, resulting in the extracellular release of S1P (42Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Taken together, all metabolic enzymes required for hydrolysis of SM to generate S1P could be present at the outer leaflet of the plasma membrane or in extra milieu, allowing for the formation of an alternative pathway from SM to S1P. In this context, it is important to note that the neutral CDase was actually detected in mouse serum, which may indicate an important role in vascular biology, i.e.regulating the extracellular content of Cer, Sph, and possibly S1P, all of which may function in autocrine/paracrine signaling. This study demonstrated that the neutral CDase is sorted by a classic pathway via ER/Golgi compartments to the plasma membranes where it is expressed as a type II integral membrane protein or alternatively secreted out of cells after proteolytic processing of the NH2-terminal signal/anchor sequence. This report also clearly indicates that the fate of the protein (as a membrane protein or secretion protein) depends on the presence of a mucin box located in the NH2-terminal region of the enzyme. Because, in contrast to the wild-type CDase, the mucin box-deleted mutant CDase does not localize at the surface of cells and is almost entirely secreted. Furthermore, the fact that bacterial and invertebrate neutral CDases, which lack the mucin box, are secreted without cell surface expression may support this conclusion. In conclusion, the mucin box acquired by the process of evolution enables the mammalian CDase to localize on the cell surface as a type II integral membrane protein. It should be emphasized that the mucin box-mediated cell surface localization is not limited to the neutral CDase but occurs in general, because the mucin box-fused chimeric GFP, but not GFP itself, with signal/anchor sequence was found to be exclusively localized at the surfaces of HEK293 cells. The mechanism by which the mucin box retains CDase on the cell surface is unclear at present. However, the finding made here that replacement of possible O-glycosylation sites (Ser/Thr residues) with Ala greatly increased the secretion and reduced the expression of the CDase on plasma membranes may indicate specific functions ofO-glycans, although the structures of O-glycans remain to be elucidated. A diverse range of membrane proteins of type I or type II topology are occasionally released from the lipid bilayer by proteolysis catalyzed by a group of enzymes referred to as secretases (30Hooper N.H. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). The proteolytic processing of membrane-bound proteins by secretases is well characterized in amyloid precursor protein (31De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1540) Google Scholar), a type I integral membrane protein, and a Golgi-resident sialyltransferase (32Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (230) Google Scholar), a type II integral membrane protein. The latter is cleaved in its membrane-anchoring region by β-secretase and secreted out of the cell (32Kitazume S. Tachida Y. Oka R. Shirotani K. Saido T.C. Hashimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13554-13559Crossref PubMed Scopus (230) Google Scholar). Thus, it is possible that the neutral CDase is detached from cells after processing of the NH2-terminal signal/anchor sequence by secretases. We indicate the possibility that the mucin box interferes with the action of secretases resulting in the generation of the type II membrane-bound CDase, because the mucin box exists very close to the possible cleavage site of the signal/anchor sequence. However, the molecular mechanism for proteolytic processing of the CDase, including the participation of secretases, remains to be clarified. Another possibility is that the mucin box is a potential signal for the sorting of proteins to plasma membranes without processing of the signal/anchor sequence, although the counterparts for the recognition of O-glycans have yet been characterized. There have been few reports on the role of theO-glycosylation of secretory proteins. It has been reported that O-glycosylation of the COOH-terminal tandem-repeated sequences regulates the secretion of rat pancreatic bile salt-dependent lipase. The O-glycosylation of the enzyme concealed the Pro-, Glu-, Ser-, and Thr-rich domain (PEST region), which is commonly present in rapidly degraded proteins, resulting in delivery to a secretion instead of a degradation pathway (33Bruneau N. Nganga A. Fisher E.A. Lombardo D. J. Biol. Chem. 1997; 272: 27353-27361Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In polarized cells, the O-glycosylation was also found to be required for apical sorting of sucrase isomaltase (34Jacob R. Alfalah M. Grunberg J. Obendorf M. Naim H.Y. J. Biol. Chem. 2000; 275: 6566-6572Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and the neurotrophin receptor (35Yeaman C. Le Gall A.H. Baldwin A.N. Monlauzeur L. Le Bivic A. Rodriguez-Boulan E. J. Cell Biol. 1997; 139: 929-940Crossref PubMed Scopus (245) Google Scholar). It was also reported that a mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells (36Zheng X. Sadler J.E. J. Biol. Chem. 2002; 277: 6858-6863Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The neutral CDase was localized at the apical membranes of proximal tubules, distal tubules, and collecting ducts in rat kidney (17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and thus the possible functions of O-glycans in apical sorting should be examined in this CDase. In contrast to our observation, human neutral CDase has been reported to be expressed in mitochondria when overexpressed in HEK293 and MCF7 cells (18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). We found that the deduced NH2-terminal sequence of the human CDase lacks 19 amino acid residues in comparison with those of mouse and rat CDases (16Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Mitsutake S. Tani M. Okino N. Mori K. Ichinose S. Omori A. Iida H. Nakamura T. Ito M. J. Biol. Chem. 2001; 276: 26249-26259Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 18El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). This may result in the generation of an incomplete signal/anchor sequence for ER targeting, allowing the human CDase to target mitochondria instead of the lumen side of the ER. Recently, evidence has emerged that the outer leaflet of the plasma membrane is a site of SM metabolism; SM is abundant here particularly in lipid microdomain rafts (37Prinetti A. Chigorno V. Tettamanti G. Sonnino S. J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and sphingomyelinase as well as CDase is actively secreted from endothelial cells (23Romiti E. Meacci E. Tani M. Nuti F. Farnararo M. Ito M. Bruni P. Biochem. Biophys. Res. Commun. 2000; 275: 746-751Crossref PubMed Scopus (47) Google Scholar, 38Marathe 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 (214) Google Scholar). Furthermore, acid sphingomyelinase was reported to be secreted and helped to metabolize SM in oxidized lipoproteins (39Schissel 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 (274) Google Scholar), and CD95 induces the translocation of acid sphingomyelinase onto the cell surface, resulting in the generation of extracellularly orientated Cer (40Grassme H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). The neutral CDase is expressed at the plasma membrane as a type II membrane protein, the catalytic domain of which is located on the extracellular side, or is secreted out of the cell. Importantly, the generation of S1P in the extracellular milieu, which occurs due to the export of the Sph kinase, was reported (41Ancellin N. Colmont C. Su J. Li Q. Mittereder N. Chae S. Stefansson S. Liau G. Hla T. J. Biol. Chem. 2002; 277: 6667-6675Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Very recently, it was found that phorbol 12-myristate 13-acetate induced the protein kinase C-dependent translocation of Sph kinase to the plasma membrane, resulting in the extracellular release of S1P (42Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Taken together, all metabolic enzymes required for hydrolysis of SM to generate S1P could be present at the outer leaflet of the plasma membrane or in extra milieu, allowing for the formation of an alternative pathway from SM to S1P. In this context, it is important to note that the neutral CDase was actually detected in mouse serum, which may indicate an important role in vascular biology, i.e.regulating the extracellular content of Cer, Sph, and possibly S1P, all of which may function in autocrine/paracrine signaling. We are grateful to Dr. J. W. Dennis and Dr. K. Nara for the gift of CHOP cells. We also thank Dr. S. Tanaka for the gift of anti-Rab6 antibody. Thanks are also due to Dr. N. Okino and H. Monjusho in our laboratory for supplying the sequence data of the slime mold CDase.
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