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J. Biol. Chem., Vol. 278, Issue 32, 29948-29953, August 8, 2003
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¶ 
From the
Department of Human Genetics, Mount Sinai
School of Medicine, New York, New York 10029, the
Department of Bioscience and Biotechnology,
Kyushu University, Fukuoka 812-8581, Japan, the
||Department of Biochemistry, Hebrew
University-Hadassah School of Medicine, Jerusalem 91120, Israel, and the
**Kekule Institut für Organische Chemie und
Biochemie der Friedrich-Wilhelms Universität, D-53121 Bonn, Germany
Received for publication, March 31, 2003 , and in revised form, May 14, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5.5, as compared with 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 µM and 208.3 pmol/µg/h, respectively,
whereas for lauric acid they were 73.76 µM and 232.5
pmol/µg/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 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Acid ceramidase (N-acylsphingosine amidohydrolase (EC 3.5.1.23
[EC]
),
AC) is one of several enzymes responsible for ceramide degradation within
mammalian cells (8). Based on
its in vitro pH optimum of 4.5, the hydrolytic activity of this
enzyme is thought to occur within lysosomes and/or late endosomes. An
inherited deficiency of AC activity results in the lipid storage disorder,
Farber disease, characterized by progressive joint pain, lipid accumulation in
various tissues, and early death
(9). In 1995, human AC was
purified to apparent homogeneity from urine
(10). It was found to be a
heterodimeric enzyme containing two subunits, 
(13 kDa) and 
(40 kDa), both of which resulted from cleavage of a 55-kDa precursor
polypeptide. The full-length human and murine AC cDNA and genomic sequences
have been cloned and characterized
(11–13),
and several point mutations in the human AC gene have now been found in Farber
disease patients (11,
14). In addition, insertional
mutagenesis of the mouse AC gene led to an early embryonic lethal phenotype,
indicating that AC activity is essential for mammalian development
(15).
In the 1960s, Gatt first reported that partially purified AC preparations carried out ceramide synthesis using free fatty acids and Sph as substrates (i.e. the ceramidase-associated "reverse reaction") (8, 16). However, because these early studies did not use highly purified enzyme, the question of whether a single protein could catalyze the hydrolysis and reverse reactions remained unclear. Recent studies using several cloned neutral and alkaline ceramidases have confirmed these early observations and revealed that these enzymes, which are distinct from AC, also can catalyze both reactions in vitro (i.e. ceramide hydrolysis and synthesis) (17–22). However, based on the acidic pH optimum of AC and the fact that de novo ceramide synthesis is not thought to occur within lysosomes, it has remained unclear whether AC could catalyze the reverse reaction.
Recently, we established a Chinese hamster ovary (CHO) cell line overexpressing the full-length, human AC cDNA, and purified the recombinant enzyme to apparent homogeneity from the culture medium.2 In the course of these studies we determined that purified AC could indeed catalyze ceramide synthesis in vitro using lauric acid and Sph as substrates. To better understand the reverse activity of human AC and to determine whether this activity occurred in situ, as well as in vitro, we have now investigated the biochemical and mechanistic characteristics of the AC-associated reverse reaction using purified, recombinant AC and cultured cells obtained from Farber disease patients.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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AC Purification—The purification of recombinant, human AC
was carried out as previously described.2 Briefly, the enzyme was
purified from the culture media of overexpressing CHO cells by sequential
chromatography on concanavalin A-Sepharose, Blue Sepharose, and Superose 12.
The final preparation revealed only AC-specific polypeptides (i.e. AC
- and -subunits). Total protein was determined using the Bio-Rad
protein assay kit according to the manufacturer's instructions.
Cell Culture—CHO cells were cultured in Dulbecco's modified eagle's medium supplemented with L-glutamine (4 mM), penicillin (100 units/ml), streptomycin (100 µg/ml), and heat-inactivated fetal calf serum (10%) in a humid incubator containing 5% CO2 at 37 °C. Human Epstein-Barr virus-transformed lymphoid cell lines were derived from a normal individual or from a patient with Farber disease and routinely grown in a humidified 5% CO2 atmosphere at 37 °C in RPMI 1640 medium containing L-glutamine (4 mM), penicillin (100 units/ml), streptomycin (100 µg/ml), and heat-inactivated fetal calf serum (10%).
AC Assay—AC activity was measured using either BODIPY-conjugated C12 ceramide or 14C-labeled C12 ceramide as substrates, as previously described (24).
Reverse AC Assay—Unless otherwise noted, reverse AC activity was determined using the purified, CHO-derived AC as the enzyme source. A stock solution was prepared containing [14C]lauric acid (1 nmol/reaction), Sph (0.5 nmol/reaction), and Triton X-100 (0.1%, v/v), vortexed vigorously, and dried under a stream of 80% nitrogen. The dried mixture was then resuspended by sonication in 0.2 M citrate-phosphate buffer (pH 6.0) containing 300 mM NaCl. The standard 10-µl reaction mixture contained 5 µl of pure AC, [14C]lauric acid (1 nmol, 100 µM final concentration), Sph (0.5 nmol, 50 µM final concentration), 0.05% Triton X-100, and 150 mM NaCl in 0.1 M citrate-phosphate buffer, pH 6.0. To determine the effect of cations on the reverse activity, MES (pH 6.0) was used instead of citrate-phosphate buffer.
The reaction mixtures were incubated at 37 °C in a water bath for 1 h and then spotted onto a TLC plate and dried (using a hair dryer). [14C]Lauric acid and synthesized [14C]ceramide were separated by TLC using solvent system I consisting of chloroform/methanol/25% ammonium hydroxide (90:20:0.5, v/v). The TLC plate was exposed to a PhosphorImager screen that was subsequently scanned using a Strom 860 PhosphorImager system (Amersham Biosciences). The undigested, radioactive substrate (lauric acid) and product (ceramide) were identified by co-migration with standards, and the signal was quantified using the ImageQuaNT software (Amersham Biosciences).
To evaluate the specificity of individual fatty acids on the AC-associated reverse reaction, [3H]Sph was used as a substrate. Briefly, [3H]Sph and fatty acids of different chain lengths were mixed well with Triton X-100 as described above and dried. The dried mixture was then resuspended by sonication in 0.2 M citrate-phosphate buffer (pH 6.0) containing 300 mM NaCl. The final reaction mixtures contained 50 µM [3H]Sph (diluted with cold Sph), 100 µM of each fatty acid, 0.05% Triton-X-100, 150 mM NaCl, and 0.1 M citrate-phosphate buffer, pH 6.0. The reactions were incubated at 37 °C in a water bath for 1 h, spotted onto a TLC plate, and dried using a hair dryer. [3H]Sph and synthesized [3H]ceramide were separated by TLC using the solvent system described above. The plates were then sprayed with EN3HANCE and radiographed on Hyperfilm MP. For quantification of the lipids, the films were scanned and analyzed with Image software (National Institutes of Health).
Determination of AC-associated Reverse Activity in Lymphoid Cells— For in vitro studies, lymphoid cells were harvested, washed 3x with PBS, and centrifuged (1600 x g). Cell pellets were suspended and disrupted in 0.25 M sucrose by three cycles of freeze-thawing and sonication. The AC-associated reverse activity, as well as the ceramidase activity, were determined in the cell lysates using [14C]lauric acid and Sph, or BODIPY-conjugated C12 ceramide as substrates, respectively.
For in situ experiments, lymphoid cells (5 x 106 cells/ml in RPMI 1640 media supplemented with 10% fetal calf serum) were incubated with 20 µM NBD-C12-fatty acid and 10 µM Sph for 2 h. After incubation, the cells were harvested and washed once with PBS. Total lipids were extracted using chloroform/methanol (2/1, v/v) for 15 min with sonication and then centrifuged at 13,600 x g for 5 min. The supernatants were removed and dried using a Speed-Vac concentrator, applied to TLC plates, and developed using solvent system I. Signals were visualized and quantified using a Storm 860 PhosphorImager.
Fluorescence Microscopy—Normal human skin fibroblasts, grown on glass chamber slides at 37 °C, were fixed with methanol for 5 min at –20 °C and then dried for 10 min. After being washed twice with PBS, the fixed cells were incubated with PBS containing 10% normal goat serum and 0.1% Triton X-100 for 1 h and then incubated overnight at 4 °C in PBS containing 10% normal goat serum, 0.05% Triton X-100, and the anti-AC antibody (1:200 dilution of primary, rabbit serum) in combination with one of the following commercial antibodies: 1) anti-LAMP-2 (1.25 µg/ml), 2) anti-EEA1 (1.25 µg/ml), or 3) anti-BiP (1.25 µg/ml). The slides were then washed 3x with PBS for 5 min, incubated for 1 h with secondary antibody conjugated with Alexa Fluor 488 and 568 (1:500), and washed again 3x with PBS for 5 min. They were then observed with a fluorescent microscope (Eclipse E800, Nikon, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5.5, as compared with 4.5 for the
hydrolysis reaction.2 The effects of various detergents on the
reverse reaction were next examined. As shown in
Fig. 1, the reaction proceeded
most efficiently in the absence of detergents. Triton X-100 and Igepal CA-630,
both non-ionic detergents, inhibited the enzyme activity in a
concentration-dependent manner. Taurocholate, an anionic detergent, strongly
inhibited the enzyme activity at low concentrations, but the activity was
partially restored at concentrations around 0.2%. Based on these findings, in
the final assay mixture only a small amount of Triton X-100 (0.05%) was
included to solubilize the substrates.
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Effect of Cations—The addition of CaCl2,
MgCl2, and NaCl to the reaction mixtures increased AC reverse
activity moderately (Fig.
2A). In contrast, ZnCl2 was very inhibitory
toward this reaction. The addition of EDTA (10 mM) did not affect
the reverse activity.3
To further investigate the salt dependence of this reaction, the activity was
measured in the presence of increasing concentrations of NaCl. As shown in
Fig. 2B, NaCl
activation was concentration-dependent, with 2-fold activation seen at
150 mM. Therefore, in the final assay mixture the NaCl
concentration was maintained at 150 mM, close to the physiologic
salt concentration.
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Kinetics of the Reverse Activity—To study the kinetics of the reverse reaction, the activity was measured using various amounts of each substrate (Fig. 3). For these studies, the concentration of either lauric acid (Fig. 3A) or Sph (Fig. 3B) was fixed at 200 µM in the reaction mixture and that of the other was increased. As can be seen, the Lineweaver-Burk plots of these data were linear and followed normal Michaelis-Menten kinetics. The apparent Km and Vmax values of the reverse reaction for Sph were 23.75 µM and 208.3 pmol/µg/h and for lauric acid were 73.76 µM and 232.5 pmol/µg/h.
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Substrate Specificity of the Reverse Activity—Fig. 4A shows the substrate specificity of the reverse reaction toward various Sph stereoisomers. Among the stereoisomers tested, only the natural, D-erythro form was efficiently utilized as a substrate. D-erythro-dihydroSph and D-erythro-phytoSph were utilized to a limited extent, whereas D-threo, L-threo, and L-erythro were not utilized at all. We next evaluated which fatty acids could serve as substrates in this reaction. As seen in Fig. 4B, under the conditions studied fatty acids with chain lengths of 12 or 14 carbons were most efficiently used.
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Effects of Lipids—The effects of various phospholipids and sphingolipids on the reverse activity were next investigated and compared with their effects on the hydrolysis reaction (Fig. 5). Fig. 5A shows that the acidic phospholipids, phosphatidylserine (PS) and phosphatidic acid (PA), significantly stimulated the reverse activity. In contrast, cardiolipin (CL), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine, and phosphatidylinositol inhibited this activity. As shown in Fig. 5B, sphingomyelin (SPM) also strongly inhibited the reverse activity, whereas glucosylceramide and C16-ceramide were less effective inhibitors. Notably, of the lipids tested, several were found to have differential effects on the reverse and hydrolysis reactions. For example, PA stimulated the reverse activity, but was inhibitory toward the hydrolysis reaction. SPM and CL, on the other hand, were very inhibitory toward the reverse reaction but stimulated the hydrolysis reaction.
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Reverse AC Activity in Farber Disease Cells—To further demonstrate that AC carried out the reverse reaction, we next used normal, human lymphoid cells and lymphoid cells from a patient with Farber disease containing mutations in the AC gene. As shown in Fig. 6A, the reverse activity was reduced in lysates prepared from the Farber disease cells to the same extent as ceramide hydrolysis, further demonstrating that both reactions were catalyzed by the same enzyme.
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To investigate whether AC carried out the reverse activity in
situ, as well as in vitro, the lymphoid cell lines were
incubated with NBD-C12-fatty acid and Sph with and without fumonisin B1. The
amount of synthesized NBD-ceramide formed in the cells was then assessed. As
shown in Fig. 6B, in
the absence of fumonisin B1, the FD cells synthesized about 20% less
NBD-ceramide than normal cells. When fumonisin B1 was included to inhibit
ceramide synthesis by acyl CoA-dependent N-acyltransferase (ceramide
synthetase), the reduction in the Farber disease cells as compared with normal
was 30%. Note that there was no difference in the amount of ceramide
synthesized by normal cells with or without fumonisin B1, indicating that
little, if any, NBD-C12-fatty acid received CoA modification and was utilized
by ceramide synthetase.
Localization of AC in Cultured Skin Fibroblasts—We next determined the subcellular location of AC in normal, human skin fibroblasts using anti-human AC antibodies in combination with antibodies specific for several subcellular compartments (Fig. 7). As can be seen, most of the AC staining (red) was punctate and perinuclear, indicating that the majority of the enzyme was present in lysosomes or late endosomes. Co-incubation of the anti-AC antibodies with anti-LAMP-2 antibodies confirmed this observation (see yellow, merged image). Under these normal growth conditions, little or no AC colocalized with early endosomes (EEA1) or the endoplasmic reticulum (BiP).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The genes encoding several ceramidases have been recently cloned from eukaryotic and prokaryotic sources (20, 21, 25–29). Table I compares several of their properties. Among these, all but one (human alkaline ceramidase) can carry out the reverse ceramidase reaction in vitro. The characteristics of these reverse activities have not been fully evaluated, but from the data available some common features are evident: 1) Ceramidase-associated reverse activities proceed in an energy-independent manner and are resistant to fumonisin B1, revealing that the mechanism of the reverse reaction is distinct from that of acyl CoA-dependent N-acyltransferase (ceramide synthetase). 2) The optimum pH of the hydrolysis and reverse reactions are different for each enzyme. In the case of AC, the reverse reaction proceeded at a more alkaline pH than the hydrolysis reaction; for the other ceramidases the reverse reactions preferred a more acidic pH than the hydrolysis reactions. 3) The stereospecificity for the sphingoid base is very strict and the natural, D-erythro form is preferred. 4) In contrast to the sphingoid base, the utilization of fatty acids is quite broad.
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Detailed characterization of the AC-associated reverse reaction revealed
some characteristics that were in common with the AC hydrolysis reaction but
also some that were distinct. For example, the activation effect by NaCl and
the preference for Sph (d18:1), as compared with sphinganine (d18:0), were
shared by the hydrolysis and reverse reactions. In addition, both activities
were inhibited by the addition of ceramide to the reaction mixture. Among the
important differences were the pH optima for the reverse and hydrolysis
reactions (5.5 and 4.5, respectively), suggesting that the two reactions
likely occur in different subcellular compartments. Immunostaining using
anti-AC antibodies and several subcellular markers revealed that under normal
growth conditions most AC was present in lysosomes or late endosomes. In the
future it will be interesting to subject cells to various stress stimuli and
monitor the effects on AC localization, because, as has been shown previously
for the related lipid hydrolase, acid sphingomyelinase, stress stimulation can
lead to dramatic and rapid relocalization of enzymes from lysosomes/late
endosomes to other cellular compartments
(30).
The effect of various lipids on the AC-associated reverse and hydrolysis
reactions also was very revealing. CL, PC, LPC, phosphatidylethanolamine, and
SPM moderately activated the hydrolysis activity but inhibited the reverse
activity at the same concentrations. Indeed, among the various lipids tested,
SPM was the most effective inhibitor of the AC-associated reverse activity. CL
and SPM also inhibited the reverse reaction associated with rat brain
ceramidase (22), and similar
to what we have found with AC, CL activated the hydrolysis reaction of that
enzyme as well. CL is known to be a major lipid in mitochondria, and it has
been suggested that CL might play a role in the regulation of one or more
mitochondria-associated ceramidases. In contrast to CL and SPM, the anionic
glycerophospholipid, PA, stimulated the reverse activity 30%, but
inhibited the hydrolysis reaction. Another anionic glycerophospholipid, PS,
which is known to activate some neutral sphingomyelinases
(31,
32), also activated the
AC-associated reverse reaction to about the same extent as PA.
The differential effects of pH and various lipids on the reverse
versus hydrolysis reactions may explain, in part, how these
activities are regulated in cells. In lysosomes and late endosomes, where the
pH is between 4.0 and 4.5 and a high concentration of SPM likely exists
due to the normal process of membrane turnover, the hydrolysis reaction should
dominate. On the other hand, we found that PA and PS activated the
AC-associated reverse activity. PS is abundant in the inner leaflet of the
plasma membrane, and PA is formed by the action of either phospholipase D or
diacylglycerol kinase during signal transduction. Therefore, it is possible to
speculate that PS- and PA-rich compartments are optimal sites for ceramide
formation via the AC-associated reverse reaction. Although our data show that
under normal growth conditions AC is localized primarily in lysosomes and late
endosomes, where it should function in ceramide breakdown, upon appropriate
stimulation cells might relocate this enzyme to sites where the reverse
reaction is favored. It is also notable that PA, which stimulates the
AC-associated reverse reaction, markedly inhibited the hydrolysis
reaction.
In conclusion, we have shown that, similar to other cloned ceramidases, highly purified, recombinant AC can carry out ceramide synthesis using free fatty acids and sphingosine as substrates. This reaction is distinct from the activity of ceramide synthetase (acyl CoA-dependent N-acyltransferase) based on a number of criteria. We have also demonstrated that the AC-associated reverse reaction can proceed in situ and shown that cells from Farber disease patients are deficient in this activity, as well as ceramide hydrolysis. We also suggest that the differential effects of pH and various lipids on the AC-associated reverse and hydrolysis reactions indicate possible mechanisms by which the two activities might be regulated in cells. We believe that the AC-associated reverse reaction represents a "salvage" pathway for ceramide synthesis that is utilized only following cell stress and/or stimulation of signal transduction pathways requiring ceramide. We further believe that the normal subcellular location of AC in lysosomes/late endosomes might be altered under these conditions, similar to acid sphingomyelinase, moving the enzyme into a compartment that promotes ceramide production versus degradation.
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FOOTNOTES |
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¶ Supported by a postdoctoral fellowship from the Japan Society for the
Promotion of Science.
To whom correspondence should be addressed: Dept. of Human Genetics, Box 1498,
Mount Sinai School of Medicine, 1425 Madison Ave., Rm. 14-20A, New York, NY
10029. Tel.: 212-659-6711; Fax: 212-849-2447; E-mail:
Edward.Schuchman{at}mssm.edu.
1 The abbreviations used are: Sph, sphingosine; NBD,
12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)); AC,
acid ceramidase; CHO, Chinese hamster ovary; CL, cardiolipin; PS,
phosphatidylserine; PA, phosphatidic acid; SPM, sphingomyelin; MES,
2-[N-morpholino]ethanesulfonic acid; PBS, phosphate-buffered saline;
LPC, lysophosphatidylcholine; PC, phosphatidylcholine; BODIPY,
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene.
2 He, X., Okino, N., Dhami, R., Dagan, A., Gatt, S., Schulze, H., Sandhoff,
K., and Schuchman, E. H. (2003) J. Biol. Chem., in press.
3 N. Okino, X. He, S. Gatt, K. Sandhoff, M. Ito, and E. H. Schuchman,
unpublished observation.
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REFERENCES |
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