Neutral ceramidase encoded by the Asah2 gene is essential for the intestinal degradation of sphingolipids

Complex sphingolipids are abundant as eukaryotic cell membrane components, whereas their metabolites, in particular ceramide, sphingosine, and sphingosine 1-phosphate, are involved in diverse cell signaling processes. In mammals, degradation of ceramide by ceramidase yields sphingosine, which is phosphorylated by the action of sphingosine kinase to generate sphingosine 1-phosphate. Therefore, ceramidases are key enzymes in the regulation of the cellular levels of ceramide, sphingosine, and sphingosine 1-phosphate. To explore the physiological functions of a neutral ceramidase with diverse cellular locations, we disrupted the Asah2 gene in mice. Asah2 null mice have a normal life span and do not show obvious abnormalities or major alterations in total ceramide levels in tissues. The Asah2-encoded neutral ceramidase is highly expressed in the small intestine along the brush border, suggesting that the neutral ceramidase may be involved in a pathway for the digestion of dietary sphingolipids. Indeed, Asah2 null mice were deficient in the intestinal degradation of ceramide. Thus, the results indicate that the Asah2-encoded neutral ceramidase is a key enzyme for the catabolism of dietary sphingolipids and regulates the levels of bioactive sphingolipid metabolites in the intestinal tract.     

The reverse activity of human acid ceramidase

An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [14C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was approximately 5.5, as compared with approximately 4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The Km and Vmax values toward sphingosine were 23.75 microM and 208.3 pmol/microg/h, respectively, whereas for lauric acid they were 73.76 microM and 232.5 pmol/microg/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced approximately 30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.     

Overexpression and mass spectrometry analysis of mature human acid ceramidase

Human acid ceramidase catalyzes the last step of lysosomal sphingolipid degradation, the hydrolysis of ceramide to sphingosine and free fatty acid. Inherited deficiency of acid ceramidase activity leads to Farber disease (Farber lipogranulomatosis). In this study, we describe the overexpression and processing of recombinant human acid ceramidase in Sf21 insect cells, its purification and characterization. Infection of Sf21 cells with a recombinant baculovirus encoding acid ceramidase precursor led to a mixture of human acid ceramidase precursor and mature enzyme secreted into the medium. Acidification of the cell culture supernatant to pH 4.2-4.3 triggered the processing of the precursor and resulted in a homogeneous sample of mature human acid ceramidase. The enzyme was purified by chromatography on Concanavalin A Sepharose and Octyl Sepharose yielding 1 mg purified protein per liter of supernatant. The recombinant enzyme was deglycosylated with peptide N-glycosidase F and the main component of the released oligosaccharides was identified as GlcNAc(2)(Fuc)Man(3) by electrospray mass spectrometry. Apparently, five of the six potential N-glycosylation sites were used. Tryptic digestion of the functional recombinant enzyme and matrix-assisted laser desorption/ionization time-of-flight- and electrospray ionization-mass spectrometry analysis of the resulting peptides indicated disulfide bridges between C10-C319, C122-C271 and C367-C371.     

Rat intestinal ceramidase: purification, properties, and physiological relevance

Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of approximately 116 kDa. The enzyme acts on both [14)]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249-262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.     

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2–3.3. This enzyme prefers to hydrolyze the acidic GSLs, II³SO₃LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4–4.5. Since oyster ceramidase greatly prefers ceramides with C₆ to C₈ fatty acids, C₆-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C₁₆ and C₁₈ fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.     

Involvement of neutral ceramidase in ceramide metabolism at the plasma membrane and in extracellular milieu

Neutral ceramidase is a type II integral membrane protein, which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor. We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell surface ceramide, which increased in amount after the treatment of cells with bacterial sphingomyelinase, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate. On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The treatment of cells with bovine serum albumin significantly reduced the cellular level of sphingosine, but not sphingosine 1-phosphate, generated by overexpression of the enzyme. The cellular level of sphingosine 1-phosphate increased with overexpression of the cytosolic sphingosine kinase. These results suggest that sphingosine 1-phosphate is mainly produced inside of the cell after the incorporation of sphingosine generated on the plasma membranes. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as sphingosine 1-phosphate were generated in the cell-free conditioned medium of ceramidase transfectants, compared with mock transfectants. No increase in these metabolites was observed if serum or bacterial sphingomyelinase was omitted from the conditioned medium, suggesting that the major source of ceramide is the serum-derived sphingomyelin. A sphingosine 1-phosphate receptor, S1P(1), was internalized much faster by the treatment of S1P(1)-overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Collectively, these results clearly indicate that the enzyme is involved in the metabolism of ceramide at the plasma membrane and in the extracellular milieu, which could regulate sphingosine 1-phosphate-mediated signaling through the generation of sphingosine.     

Purification and characterization of a second type of neutral ceramidase from rat brain: a second more hydrophobic form of rat brain ceramidase

Ceramidases (CDase) are enzymes that catalyze the hydrolysis of N-acyl linkage of ceramide (Cer) to generate sphingosine and free fatty acids. In this study we report the purification and characterization of a novel second type of neutral ceramidase from rat brain (RBCDase II). Triton X-100 protein extract from rat brain membrane was purified sequentially using Q-Sepharose, HiLoad16/60 Superdex 200pg, heparin-Sepharose, phenyl-Sepharose HP, and Mono Q columns. After Mono Q, the specific activity of the enzyme increased by ~15,000-fold over that of the rat brain homogenate. This enzyme has pH optima of 7.5, and it has a larger apparent molecular weight (110kDa) than the previously purified (90kDa) and characterized neutral rat brain CDase (RBCDase I). De-glycosylation experiments show that the differences in molecular mass of RBCDase I and II on SDS-PAGE are not due to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca(2+) and is inhibited by pyrimidine mono nucleotides such as TMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on a CDase activity. The enzyme was also inhibited by both oxidized and reduced GSH. The effects of metal ions were examined, and we found that the enzyme is very sensitive to Hg(2+) and Fe(3+), while it is not affected by Mn(2+). EDTA was somewhat inhibitory at a 20mM concentration.     

Tracking Sphingosine Metabolism and Transport in Sphingolipidoses: NPC1 Deficiency as a Test Case

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann-Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [(3) H] in carbon-3 of the sphingosine backbone and targeted them to LE/LY in low-density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [(3) H]-sphingomyelin-derived [(3) H]-ceramide and depletion of [(3) H]-sphingosine upon acid ceramidase depletion, and (2) accumulation of [(3) H]-sphingosine-derived [(3) H]-ceramide and attenuation of [(3) H]-sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [(3) H]-sphingosine derived from [(3) H]-sphingomyelin/LDL or [(3) H]-ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [(3) H]-sphingolipids or a fluorescent sphingosine derivative in NPC1 knock-out cells. Instead, NPC1-deficient cells displayed an increased affinity for sphingosine independently of protein-mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.     

CDase is a pan-ceramidase in Drosophila

Ceramidases catalyze the conversion of ceramide to sphingosine. They are acylaminohydrolases that catalyze the deacylation of the amide-linked saturated fatty acid from ceramide to generate sphingosine. They also catalyze the reverse reaction of ceramide biosynthesis using sphingosine and fatty acid. In mammals, different proteins catalyze these reactions while individually exhibiting optimal activity over a narrow pH range and have been accordingly called acid, neutral, and alkaline ceramidases. Several genes encode for variants of alkaline ceramidase in mammals. Brainwashing (Bwa) is the only putative alkaline ceramidase homologue present in Drosophila. In this study we have demonstrated that BWA does not exhibit ceramidase activity and that bwa null mutants display no loss of ceramidase activity. Instead, the neutral ceramidase gene CDase encodes the protein that is responsible for all measurable ceramidase activity in Drosophila. Our studies show strong genetic interaction of Bwa with CDase and the Drosophila ceramide kinase gene (DCERK). We show that, although BWA is unlikely to be a ceramidase, it is a regulator of sphingolipid flux in Drosophila. Bwa exhibits strong genetic interaction with other genes coding for ceramide-metabolizing enzymes. This interaction might partly explain its original identification as a ceramidase.     

Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.     

Ceramidase enhances phospholipase C-induced hemolysis by Pseudomonas aeruginosa

We previously reported the purification, molecular cloning, and characterization of a neutral ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616-36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cell-free culture supernatant. Ceramide and sphingosine were also effective in promoting the production of ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the ceramidase. A ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcH-induced hemolysis by the ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.     

Purification and characterization of a neutral ceramidase from mouse liver. A single protein catalyzes the reversible reaction in which ceramide is both hydrolyzed and synthesized

We report here a novel ceramidase that was purified more than 150, 000-fold from the membrane fraction of mouse liver. The enzyme was a monomeric polypeptide having a molecular mass of 94 kDa and was highly glycosylated with N-glycans. The amino acid sequence of a fragment obtained from the purified enzyme was homologous to those deduced from the genes encoding an alkaline ceramidase of Pseudomonas aeruginosa and a hypotheical protein of the slime mold Dictyostelium discoideum. However, no significant sequence similarities were found in other known functional proteins including acid ceramidases of humans and mice. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, sulfatide, GM1a, or sphingomyelin. The enzyme exhibited the highest activity around pH 7.5 and was thus identified as a type of neutral ceramidase. The apparent K(m) and V(max) values for C12-4-nitrobenzo-2-oxa-1, 3-diazole-ceramide and C16-(14)C-ceramide were 22.3 microM and 29.1 micromol/min/mg and 72.4 microM and 3.6 micromol/min/mg, respectively. This study also clearly demonstrated that the purified 94-kDa ceramidase catalyzed the condensation of fatty acid to sphingosine to generate ceramide, but did not catalyze acyl-CoA-dependent acyl-transfer reaction.     

Simultaneous quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene-2,3-dicarboxyaldehyde derivatization after hydrolysis with ceramidase

Ceramide and sphingosine are sphingolipids with important functional and structural roles in cells. In this paper we report a new enzyme-based method to simultaneously quantify the levels of ceramide and sphingosine in biological samples. This method utilizes purified human recombinant acid ceramidase to completely hydrolyze ceramide to sphingosine, followed by derivatization of the latter with naphthalene-2,3-dialdehyde (NDA) and quantification by reverse-phase high-performance liquid chromatography. The limits of detection for sphingosine-NDA and ceramidase-derived sphingosine-NDA were 9.6 and 12.3 fmol, respectively, and the limits of quantification were 34.2 and 45.7 fmol, respectively. The recovery of sphingosine and ceramide standards quantified by this assay were between 95.6 and 104.6%. The relative standard deviations for the intra- and interday sphingosine assay were 2.1 and 4.5%, respectively, and those for the ceramide assay were 3.3 and 4.1%, respectively. To validate this procedure, we quantified ceramide and sphingosine in mouse plasma, white blood cells, and hemoglobin, the first reported time that the amounts of these lipids have been documented in individual blood components. We also used this technique to evaluate the ability of a novel ceramide analog, AD2646, to inhibit the hydrolytic activity of acid ceramidase. The results demonstrate that this new procedure can provide sensitive, reproducible, and simultaneous ceramide and sphingosine quantification. The technique also may be used for determining the activity and inhibition of ceramidases and may be adapted for quantifying sphingomyelin and sphingosine-1-phosphate levels. In the future it could be an important tool for investigators studying the role of ceramide/sphingosine metabolism in signal transduction, cell growth and differentiation, and cancer pathogenesis and treatment.     

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.     

Sphingomyelinase activity in mother's milk is essential for juvenile development: a case from lactating tsetse flies

Sphingosine is a structural component of sphingolipids. The metabolism of phosphoethanolamine ceramide (sphingomyelin) by sphingomyelinase (SMase), followed by the breakdown of ceramide by ceramidase (CDase) yields sphingosine. Female tsetse fly is viviparous and generates a single progeny within her uterus during each gonotrophic cycle. The mother provides her offspring with nutrients required for development solely via intrauterine lactation. Quantitative PCR showed that acid smase1 (asmase1) increases in mother's milk gland during lactation. aSMase1 was detected in the milk gland and larval gut, indicating this protein is generated during lactation and consumed by the larva. The higher levels of SMase activity in larval gut contents indicate that this enzyme is activated by the low gut pH. In addition, cdase is expressed at high levels in the larval gut. Breakdown of the resulting ceramide is likely accomplished by the larval gut-secreted CDase, which allows absorption of sphingosine. We used the tsetse system to understand the critical role(s) of SMase and CDase during pregnancy and lactation and their downstream effects on adult progeny fitness. Reduction of asmase1 by short interfering RNA negatively impacted pregnancy and progeny performance, resulting in a 4-5-day extension in pregnancy, 10%-15% reduction in pupal mass, lower pupal hatch rates, impaired heat tolerance, reduced symbiont levels, and reduced fecundity of adult progeny. This study suggests that the SMase activity associated with tsetse lactation and larval digestion is similar in function to that of mammalian lactation and represents a critical process for juvenile development, with important effects on the health of progeny during their adulthood.     

Acid ceramidase and human disease

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is the lipid hydrolase responsible for the degradation of ceramide into sphingosine and free fatty acids within lysosomes. The enzymatic activity was first identified over four decades ago, and is deficient in the inherited lipid storage disorder, Farber Lipogranulomatosis (Farber disease). Importantly, AC not only hydrolyzes ceramide into sphingosine, but also can synthesize ceramide from sphingosine and free fatty acids in vitro and in situ. This "reverse" enzymatic activity occurs at a distinct pH from the hydrolysis ("forward") reaction (6.0 vs. 4.5, respectively), suggesting that the enzyme may have diverse functions within cells dependent on its subcellular location and the local pH. Most information concerning the role of AC in human disease stems from work on Farber disease. This lipid storage disease is caused by mutations in the gene encoding AC, leading to a profound reduction in enzymatic activity. Recent studies have also shown that AC activity is aberrantly expressed in several human cancers, and that the enzyme may be a useful cancer drug target. For example, AC inhibitors have been used to slow the growth of cancer cells, alone or in combination with other established, anti-oncogenic treatments. Aberrant AC activity also has been described in Alzheimer's disease, and overexpression of AC may prevent insulin resistant (Type II) diabetes induced by free fatty acids. Current information concerning the biology of this enzyme and its role in human disease is reviewed within.     

Acid ceramidase and human disease

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23; AC) is the lipid hydrolase responsible for the degradation of ceramide into sphingosine and free fatty acids within lysosomes. The enzymatic activity was first identified over four decades ago, and is deficient in the inherited lipid storage disorder, Farber Lipogranulomatosis (Farber disease). Importantly, AC not only hydrolyzes ceramide into sphingosine, but also can synthesize ceramide from sphingosine and free fatty acids in vitro and in situ. This “reverse” enzymatic activity occurs at a distinct pH from the hydrolysis (“forward”) reaction (6.0 vs. 4.5, respectively), suggesting that the enzyme may have diverse functions within cells dependent on its subcellular location and the local pH. Most information concerning the role of AC in human disease stems from work on Farber disease. This lipid storage disease is caused by mutations in the gene encoding AC, leading to a profound reduction in enzymatic activity. Recent studies have also shown that AC activity is aberrantly expressed in several human cancers, and that the enzyme may be a useful cancer drug target. For example, AC inhibitors have been used to slow the growth of cancer cells, alone or in combination with other established, anti-oncogenic treatments. Aberrant AC activity also has been described in Alzheimer's disease, and overexpression of AC may prevent insulin resistant (Type II) diabetes induced by free fatty acids. Current information concerning the biology of this enzyme and its role in human disease is reviewed within.     

Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase

We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.     

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.     

Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures

The neutral ceramidase is a key enzyme in the regulation of cellular ceramide levels. Previously we have reported that stimulation of rat renal mesangial cells with nitric oxide (NO) donors leads to an inhibition of neutral ceramidase activity which is due to increased degradation of the enzyme. This and the concomitant activation of the sphingomyelinase results in an amplification of ceramide levels. Here, we show that the NO-triggered degradation of neutral ceramidase involves activation of the ubiquitin/proteasome complex. The specific proteasome inhibitor lactacystin completely reverses the NO-induced degradation of ceramidase protein and neutral ceramidase activity. As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin. Furthermore, ubiquitinated neutral ceramidase accumulates after NO stimulation. In summary, our data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral ceramidase activity and thereby regulates the availability of ceramide.