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Originally published In Press as doi:10.1074/jbc.M205211200 on June 24, 2002

J. Biol. Chem., Vol. 277, Issue 38, 35642-35649, September 20, 2002
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Upstream of Growth and Differentiation Factor 1 (uog1), a Mammalian Homolog of the Yeast Longevity Assurance Gene 1 (LAG1), Regulates N-Stearoyl-sphinganine (C18-(Dihydro)ceramide) Synthesis in a Fumonisin B1-independent Manner in Mammalian Cells*

Krishnan VenkataramanDagger , Christian RiebelingDagger §, Jacques BodennecDagger , Howard Riezman||, Jeremy C. Allegood**, M. Cameron Sullards**, Alfred H. Merrill Jr.**, and Anthony H. FutermanDagger DaggerDagger

From the Dagger  Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, || Biozentrum, University of Basel, CH-4056 Basel, Switzerland, and ** School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230

Received for publication, May 28, 2002, and in revised form, June 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B1, an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1, upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-3H]sphinganine or L-3-[3H]serine demonstrated that uog1 conferred fumonisin B1 resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids of uog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB1, and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B1 resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays of uog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that the N-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interest in determining the regulatory mechanisms of ceramide metabolism has been stimulated over the past decade by the realization that ceramides formed by turnover of complex sphingolipids, and by de novo synthesis, influence key aspects of cell growth, regulation, differentiation, and death (1-6). Ceramides are formed de novo by N-acylation of sphinganine to dihydroceramide, which is subsequently desaturated by dihydroceramide desaturase (7-9). The N-acyltransferase(s), which are referred to herein as (dihydro)ceramide synthase(s), acylate various long chain bases, including sphinganine, sphingosine, and 4-hydroxysphinganine, utilize a wide spectrum of fatty acyl-CoAs, and are inhibited by the mycotoxin, fumonisin B1 (FB1)1 (10-12). Kinetic evidence has been obtained for multiple (dihydro)ceramide synthases, but no biochemical or molecular evidence has been obtained to prove their existence. One reason for suggesting that multiple (dihydro)ceramide synthases exist is that FB1 suppresses the synthesis of most, but not all, sphingolipids in vivo, and the residual ceramide formed is preferentially channeled into glycosphingolipids (GSLs) rather than sphingomyelin (SM) (13-15). Furthermore, ceramide synthesis is thought to occur primarily in the endoplasmic reticulum (ER) (16, 17), but recent studies (18, 19) have suggested that ceramide can also be made in a mitochondria-enriched fraction. Until more tools are available to study (dihydro)ceramide synthases, the causes of such differences will remain obscure. This issue is further complicated by the ability to bypass FB1 inhibition by formation of ceramide via the reverse activity of an acyl-CoA-independent ceramidase (20).

Recent studies (21) demonstrated that the yeast genes, LAG1 (22) and LAC1, are required for acyl-CoA-dependent ceramide synthesis using very long chain (C26) fatty acids. Yeast strains lacking Lag1p and Lac1p produce greatly reduced levels of ceramides and lack FB1-sensitive ceramide synthase activity (23). Moreover, LAG1 regulates glycosylphosphatidylinositol-anchored protein transport from the ER to the Golgi apparatus in yeast (24), a step that depends on ceramide synthesis (25, 26), supporting the idea that the yeast genes either encode for a catalytic subunit of ceramide synthase or are obligatory activators of ceramide synthase (21, 23). Finally, a tomato gene homolog of LAG1, the Alternaria stem canker locus-1 (ASC1), mediates FB1 resistance in tomato (27), implying that ASC1 might encode a FB1-insensitive (dihydro)ceramide synthase in plants (28).

In the current study, we examine the effect of overexpression of a mouse homolog of LAG1, upstream of growth and differentiation factor-1 (uog1) (29, 30), in mammalian cells. uog1 was originally discovered while screening for transforming growth factor-beta family members, and was found to be expressed in various tissues as part of a bicistronic RNA together with growth/differentiation factor-1 (gdf1) (31). Moreover, it functionally complemented the lethality of a LAG1 and LAC1 double deletion in yeast (29). We now show that transfection with uog1 increased, in a FB1-insensitive manner, (dihydro)ceramide synthesis, which was subsequently preferentially channeled into neutral but not acidic GSLs. Unexpectedly, the ceramide and neutral GSLs formed were greatly enriched in C18- (stearoyl) fatty acids. Our data establish that UOG1 is involved in the regulation of N-stearoyl-sphinganine synthesis in mammalian cells, and we demonstrate for the first time that cells can synthesize ceramides with a high degree of fatty acid selectivity as a result of the increased expression of a single gene product.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- L-3-[3H]Serine (specific activity of 26 or 31 Ci/mmol) and [14C]methylcholine (specific activity of 56 mCi/mmol) were from Amersham Biosciences. Ceramide, FB1, glucosylceramide (GlcCer), galactosylceramide, SM, palmitic acid, stearic acid, palmitoyl-CoA, Red Taq polymerase, defatted bovine serum albumin, phenylmethylsulfonyl fluoride, leupeptin, antipain, and aprotinin were from Sigma. Stearoyl-CoA was from Laroden Lipid Biochemicals (Malmö, Sweden). D-erythro-Sphinganine, D-erythro-sphingosine, D-erythro-sphinganine 1-phosphate, D-erythro-sphingosine 1-phosphate, D-erythro-stearoyl-sphingosine, a neutral GSL standard mixture, a mono-sialo and di-sialo GSL standard mixture, and (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol were from Matreya (Pleasant Gap, PA). Reverse phase RP-18 columns were from Supelco (Bellefonte, PA). pcDNA 3.0 was from Invitrogen. A pGEMT cloning kit was from Promega Corp. (Madison, WI). Vent polymerase was from New England Biolabs (Beverly, MA). LipofectAMINE was from Invitrogen. Restriction enzymes and DNA-modifying enzymes were either from MBI Fermentas (Vilnius, Lithuania) or from New England Biolabs. A one-step reverse transcriptase-PCR kit was from Qiagen GmbH (Hilden, Germany). A rat anti-HA monoclonal antibody (clone 3F10) was from Roche Molecular Biochemicals; a rabbit anti-protein-disulfide isomerase (PDI) antibody was from Stressgen (Victoria, Canada), and MitoTracker® Deep Red was from Molecular Probes (Eugene, OR). Silica Gel 60 thin layer chromatography (TLC) plates were from Merck. All solvents were of analytical grade and were purchased from Biolab (Jerusalem, Israel).

Cloning of uog1-- The coding sequences of the human and mouse gdf1 genes (GenBankTM accession numbers M62302 and M62301) were subject to a Blast search against human and mouse expression sequence tag data bases. Two cDNA clones (GenBankTM accession numbers AU080088 (MNCb-5211) and AU080131 (MNCb-5265)) from mouse brain showed the highest sequence identity with the cDNA sequence of uog1 and were obtained from the National Institute of Infectious Diseases, Japanese Collection of Research Bioresources Gene Bank, Japan.

Restriction digestion analysis of both MNCb-5211 and MNCb-5265 demonstrated that the constructs had two distinct open reading frames coding for uog1 and gdf1. The uog1 fragment (1.3 kilobases) was released by restriction digestion using EcoRI and AvrII sites, sub-cloned into a pcDNA 3.0 vector at EcoRI and XbaI sites, and subjected to nucleotide sequencing. Both MNCb-5211 and MNCb-5265 had identical nucleotide sequences, and the derived amino acid sequence from pcDNA-UOG1(5211) and pcDNA-UOG1(5265) demonstrated that there was one amino acid change, from serine to arginine, at position 334 compared with the derived amino acid sequence from mouse UOG1 (31); because the amino acid sequence was not conserved at this position between human and mouse UOG1, the coding sequence of MNCb-derived clones was not modified. Subsequently, the pcDNA-UOG1(5211) clone derived from MNCb-5211 was used for overexpression studies.

Sequence-specific primers with unique restriction sites were designed for the 5' and 3' ends of uog1 cDNA, and the uog1 cDNA was amplified by PCR to obtain the 1.08-kb PCR product. The PCR product was sub-cloned into a pGEMT vector, and the positive clones were identified, according to instructions provided in the technical manual for the pGEMT easy vector system. The cDNA insert was also sub-cloned into tagged vectors to create vectors carrying hemagglutinin epitope (HA) tags at the N and C termini of UOG1. The coding sequences were confirmed by nucleotide sequencing.

Cell Culture and Transfection-- COS-7 cells, grown on glass coverslips, and human embryonic kidney 293T cells (HEK-293T), grown in Nunc tissue culture dishes, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. COS-7 cells were transfected using LipofectAMINE according to the manufacturer's instructions, and HEK-293T cells were transfected by the calcium phosphate method (32).

Immunolocalization-- Eighteen hours after transfection, COS-7 cells were incubated with MitoTracker® Deep Red (25 nM) for 15 min and chased for 3 min in fresh medium. The cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline (20 min, 37 °C), and subsequently permeabilized with 0.1% (v/v) Triton X-100 in phosphate-buffered saline, 1% bovine serum albumin for 30 min. Primary antibodies (rabbit anti-PDI, dilution of 1:200; rat anti-HA, 1:500) were applied for 2 h at room temperature. A rhodamine-labeled goat anti-mouse secondary antibody (which cross-reacts with the rat anti-HA primary antibody) was diluted 1:200, and a fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody was diluted 1:500. After a 1-h incubation, slides were mounted in Fluoromount G and viewed by confocal laser scanning microscopy using an Olympus Fluoview FV500 imaging system. Fluorescein isothiocyanate, rhodamine fluorescence, and MitoTracker® Deep Red were viewed using an argon and two helium-neon laser with excitation wavelengths of 488, 543, and 633 nm, respectively. Images were acquired in sequential mode and analyzed using Fluoview 3 imaging software.

Metabolic Labeling-- 24 h after transfection, HEK-293T cells were incubated with or without 20 µM FB1. Subsequently, cells were metabolically labeled either with [4,5-3H]sphinganine or with L-3-[3H]serine and [14C]methylcholine.

Labeling with [4,5-3H]Sphinganine-- 9 µCi of [4,5-3H]sphinganine, synthesized by reduction of D-erythro-sphingosine with NaB[3]H4 (11 Ci/mmol) (16), was added to the culture medium 45 h after transfection. Three hours later, cells were washed with phosphate-buffered saline and removed from the dishes by scraping with a rubber policeman. After homogenization by sonication, protein concentration was determined (33), and lipids were extracted (34) and dried under N2. Lipids were subjected to alkaline hydrolysis in chloroform, 0.6 N NaOH in methanol (1:1) (37 °C, 2 h) to degrade glycerolipids (35). Samples were subsequently washed three times with water to remove the NaOH, and the [4/5-3H]sphingolipids2 obtained from the chloroform phase were separated by TLC using chloroform, methanol, 9.8 mM CaCl2 (60:35:8, v/v) as the developing solvent.

Labeling with L-3-[3H]Serine and [14C]Methylcholine-- Cells were incubated with L-3-[3H]serine (30 µCi per dish) and [14C]methylcholine (3 µCi per dish) for 24 h. Lipids were then extracted (36) and dissolved in benzene/methanol (1:1, v/v). 3H- and 14C-labeled phospholipids were resolved by two-dimensional TLC using tetrahydrofurane/acetone/methanol/water (50:20:40:6, v/v) as the first developing solvent, and chloroform/acetone/methanol/acetic acid/water (50:20:10:15:5, v/v) as the second developing solvent (35). Phospholipids were visualized using iodine and recovered from TLC plates by scraping the silica directly into scintillation vials. One ml of methanol and 5 ml of Optima Gold scintillation fluid (Packard Instrument Co.) were added to each vial for scintillation counting, and radioactivity was determined in a Packard 2100 beta radiospectrometer equipped with the Transformed Spectral Index of the External Standard/Automatic Efficiency Control (tSIE/AEC) program for quench correction and counting of double radiolabeled samples.

Another aliquot of the lipid extract was used for analysis of [3H]sphingolipids. After alkaline hydrolysis (see above), the lipids were obtained from the chloroform phase, and after drying were reconstituted in 1:1 benzene/methanol (1:1, v/v). [3H]Sphingolipids were separated by TLC using the following developing solvents. (i) [3H](Dihydro)ceramide was separated using chloroform:methanol (50:3.5, v/v). (ii) [3H]GlcCer and [3H]galactosylceramide were separated on a sodium borate-coated TLC plate using chloroform/methanol/water (24:7:1, v/v). (iii) Neutral [3H]GSLs were separated on two-dimensional TLC using tetrahydrofurane/acetone/methanol/water (50:20:40:6, v/v) and chloroform/acetone/methanol/acetic acid/water (50:20:10:15:5, v/v). For analysis of acidic [3H]GSLs, the aqueous phase of the lipid extract (36) was loaded onto Supelco RP-18 disposable cartridges (37) and washed with water. Lipids were eluted using chloroform/methanol (1:1, v/v), dried under N2, mixed with authentic ganglioside standards, and separated by TLC using chloroform/methanol/9.8 mM CaCl2 (60:35:8, v/v).

Ceramide Synthase Assay-- HEK-293T cells were homogenized in 20 mM Hepes, pH 7.4, 25 mM KCl, 250 mM sucrose and 2 mM MgCl2, containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml antipain, 1 µg/ml leupeptin, and 100 kIU/ml aprotinin). Homogenates or microsomal fractions (16, 38) (both 200 µg of protein) were incubated with 1.48 µCi of [4,5-3H]sphinganine, 15 µM sphinganine, 20 µM defatted bovine serum albumin (16, 38). The reaction was initiated by addition of either 10 µM free fatty acid (palmitate or stearate) or 50 µM palmitoyl-CoA or stearoyl-CoA. Lipids were extracted (34) and [4/5-3H](dihydro)ceramide synthesis was analyzed by TLC using chloroform, methanol, 2 M ammonium chloride (40:10:1, v/v).

Electrospray Tandem Mass Spectrometry-- HEK-293T cells were grown in 60-mm plastic culture dishes and transfected by the calcium phosphate method. After 24 h, cells were incubated with or without 20 µM FB1 for a further 24 h. Cells were harvested by trypsinization and collected by centrifugation. The cell pellet was washed once with ice-cold phosphate-buffered saline and lyophilized. The cell pellets were extracted and analyzed for sphingolipids using essentially the conditions described (39, 40), which utilize normal phase high pressure liquid chromatography to separate the sphingolipid classes followed by electrospray tandem mass spectrometry (ESI-MS/MS) on a PE-Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ionspray source. Dry N2 was used as the nebulizing gas at a flow rate of 6 liters/min. The ionspray needle was held at 5500 V, and the orifice and ring voltages were kept low (40 and 220 V, respectively) to prevent collisional decomposition of molecular ions prior to entry into the first quadrupole, and the orifice temperature was set to 500 °C. N2 was used to collisionally induce dissociations in Q2, which was offset from Q1 by 40-50 V. Q3 was then set to pass molecularly distinctive product ions (N ions) of m/z 264.2. Multiple reaction monitoring scans were acquired by setting Q1 and Q3 to pass the precursor and product ions of the most abundant sphingolipid molecular species. For example, for the ceramides, these transitions occur at m/z 538.7/264.4, 566.5/264.4, 622.7/264.4, 648.7/264.4, 650.7/264.4, which corresponds to ceramides with a d18:1 sphingoid base (sphingosine) and C16:0, C18:0, C22:0, C24:1, and C24:0 fatty acids, respectively. The dwell time was 25 ms for each transition. Quantitation was achieved by spiking the samples prior to extraction with the C12 fatty acid homologs of ceramide and SM along with the C8 fatty acid homolog of GlcCer.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Subcellular Localization of uog1-- uog1 was sub-cloned into a pcDNA vector or into a pcDNA-HA vector to give constructs expressing UOG1 with an HA tag at the N (HA-UOG1) or C terminus (UOG1-HA). When the HA tag was at the N terminus of UOG-1, UOG1 was localized in reticular-like structures that were confirmed to correspond to the ER by almost complete co-localization with the ER marker, PDI, in both HEK-293T cells (not shown) and in COS cells (Fig. 1); due to their size and morphology, much better resolution of intracellular organelles was obtained with COS cells. In contrast, when the tag was at the C terminus, the Golgi apparatus was strongly labeled and there was less co-localization with PDI. Because UOG1 has a C-terminal K(X)KXX motif (41), this suggests that the presence of a tag at the C terminus blocks the recycling and retrieval of UOG1 from the Golgi apparatus to the ER. Neither HA-UOG1 nor UOG1-HA showed any co-localization with the mitochondria-specific dye MitoTracker® (Fig. 1) or with an anti-cytochrome c oxidase IV antibody (not shown). Similar results were obtained with green fluorescent protein-tagged UOG1 constructs.


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  		Fig. 1.   Intracellular localization of UOG1. The localization of HA-tagged UOG1 constructs in COS-7 cells was compared with the ER marker, PDI, and to the mitochondria-specific dye MitoTracker®. The merged view is shown in pseudocolor for the sake of clarity with anti- HA in red, anti-PDI in blue, and MitoTracker® in green. Areas of co-localization of anti-HA with anti-PDI are in pink, anti-HA with MitoTracker in yellow, and anti-PDI with MitoTracker® in white. Bar = 10 µm.

Metabolic Labeling of uog1-transfected HEK-293T Cells-- To determine whether UOG1 is involved in regulating (dihydro)ceramide synthesis in mammalian cells, uog1-transfected HEK-293T cells were metabolically labeled with [4,5-3H]sphinganine (15, 42) or with L-3-[3H]serine, a substrate for the first enzyme in the pathway of sphingolipid biosynthesis, serine palmitoyltransferase (7). There was a 2.8-fold increase in [4/5-3H](dihydro)ceramide synthesis from [4,5-3H]sphinganine (Fig. 2A) and a small increase in L-3-[3H]serine incorporation into [3H]ceramide (Fig. 2B) in uog1-transfected cells compared with mock-transfected cells. As expected, FB1 inhibited (dihydro)ceramide synthesis in mock-transfected cells using both labeling protocols. However, FB1 only inhibited [4/5-3H](dihydro)ceramide synthesis from [4,5-3H]sphinganine by ~35% in uog1-transfected cells (Fig. 2A) and, surprisingly, increased levels of [3H]ceramide synthesis from [3H]serine (Fig. 2B). Thus, by using both [4,5-3H]sphinganine and L-3-[3H]serine, UOG1 overexpression elevates levels of (dihydro)ceramide synthesis and confers FB1 resistance.3


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  		Fig. 2.   UOG1 elevates ceramide synthesis and confers FB1 resistance in HEK-293T cells. A, HEK-293T cells were transfected with pcDNA-UOG1 for 24 h and incubated with 20 µM FB1 for a further 24 h and with [4,5-3H]sphinganine (9 µCi) for the last 3 h of the incubation with FB1. Results are means ± S.D. for three independent experiments. The inset shows an example of [4/5-3H](dihydro)ceramide synthesis from a typical TLC plate. To confirm that these bands are indeed dihydroceramide/ceramide, and not another lipid with a similar Rf, alkaline hydrolysis was performed on the band (16), resulting in production of [4/5-3H]sphingosine and [4,5-3H]sphinganine (16). B, HEK-293T cells were transfected with pcDNA-UOG1 for 24 h and then incubated for 24 h with 30 µCi of L-3-[3H]serine in the presence or absence 20 µM FB1. Results are means ± S.D. of six experiments.

The fate of the (dihydro)ceramide synthesized in uog1-transfected HEK-293T cells was determined by metabolic labeling with L-3-[3H]serine (Table I). In uog1-transfected cells a significant increase in the synthesis of the neutral GSLs, GlcCer, ceramide dihexosides, ceramide trihexosides, and globosides was observed compared with mock-transfected cells (Table I), but there was no elevation of ganglioside synthesis. In FB1-treated uog1-transfected cells, neutral GSL synthesis was elevated to an even greater extent, with ceramide trihexoside and globoside synthesis elevated 10-11-fold; similar to uog1-transfected cells that were not treated with FB1, no increase in ganglioside synthesis was observed. The ability of FB1 to augment the increase in neutral GSL synthesis in uog1-transfected cells is consistent with the changes in neutral GSL synthesis observed in Swiss 3T3 cells incubated with FB1 (15), in which the coordinate up-regulation of the glycosyltransferases in the pathway of ceramide trihexoside synthesis results in increased ceramide trihexoside synthesis (15). Similarly, ceramide trihexoside and globoside synthesis was elevated in FB1-treated mock-transfected HEK-293T cells (Table I). Together these data suggest that the (dihydro)ceramide synthesized by UOG1-overexpressing cells is preferentially incorporated into neutral rather than acidic GSLs.

                              
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Table I
GSL synthesis in uog1-transfected cells
HEK-293T cells were transfected with pcDNA (mock) or with pcDNA-UOG1 and incubated 24 h later with 30 µCi of L-3-[3H]serine in the presence or absence of 20 µM FB1 for a further 24 h. Results are means ± S.D. for three independent experiments. Numbers in parentheses show the percent change compared with untreated mock-transfected cells.

In contrast, SM synthesis was not elevated in uog1-transfected cells, and FB1 inhibited SM synthesis, although to a slightly lower extent than in mock-transfected cells (Table II). There were no changes in levels of glycerolipid synthesis, analyzed by metabolic labeling with [14C]methylcholine and L-3-[3H]serine, with the exception of phosphatidylethanolamine, whose levels increased after FB1 treatment in both mock- and uog1-transfected cells (Table II). This is presumably due to the metabolism of sphinganine, which accumulates upon FB1 treatment (12, 43) by sphinganine kinase and sphinganine phosphate lyase, yielding ethanolamine phosphate, which is incorporated into phosphatidylethanolamine (13, 44, 45).

                              
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Table II
Phospholipid synthesis in uog1-transfected cells
HEK-293T cells were transfected with pcDNA (mock) or with pcDNA-UOG1 and incubated 24 h later with 30 µCi of L-3-[3H]serine and 3 µCi of [14C]methylcholine in the presence or absence of 20 µM FB1 for a further 24 h. Results are means ± S.D. for three independent experiments. Numbers in parentheses show the percent change compared with untreated mock-transfected cells. PS, phosphatidylserine, PE, phosphatidylethanolamine.

Sphingolipid Amounts and Fatty Acid Composition in uog1-transfected Cells-- We next examined amounts of sphingolipids in uog1-transfected cells by ESI-MS/MS. An ~2-fold increase in ceramide and GlcCer mass was observed in uog1-transfected cells, with a smaller fold increase (1.4-fold) in SM (Table III). Upon incubation of mock-transfected cells with FB1, sphinganine, but not sphingosine levels increased by ~6-fold, and levels of ceramide, GlcCer, and SM decreased (Table III), similar to that reported in many other cells (12, 46) and consistent with the metabolic labeling data (see above). In FB1-treated uog1-transfected cells, sphinganine levels were also elevated, presumably due to inhibition of the endogenous FB1-sensitive ceramide synthase (10).

                              
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Table III
Amounts of long chain bases and sphingolipids in uog1-transfected cells
HEK-293T cells were transfected with pcDNA (mock) or with pcDNA-UOG1 and after 24 h incubated with or without 20 µM FB1 for a further 24 h. Data are means of two independent analysis with <10% variability between the individual analyses.

Remarkably, there were significant changes in the fatty acid composition of sphingolipids in uog1-transfected cells. Levels of C18 fatty acids (stearate) were increased in ceramide, GlcCer (monohexosyl ceramides), and SM, but there were essentially no changes in C16 (palmitate) or any of the other major fatty acids (Fig. 3). Thus, stearic acid was the predominant fatty acid in ceramide and GlcCer in uog1-transfected cells, whereas it was present at similar levels to palmitic acid in mock-transfected cells (Fig. 3). Incubation with FB1 preferentially reduced levels of palmitate in ceramide and GlcCer in both mock- and uog1-transfected cells but further enhanced stearate levels in uog1-transfected cells. Similar changes in C16 and C18 fatty acids in lactosylceramide were also obtained (not shown), but palmitate remained the predominant fatty acid in SM under all conditions, although there were increases in the amounts of C18-SM (Fig. 3). These data suggest that UOG1 preferentially uses stearic acid, in contrast to the endogenous FB1-sensitive (dihydro)ceramide synthase(s) that preferentially incorporate other fatty acids (C16, C22:0, C24:0, C24:1); moreover, the stearoyl-containing sphingolipids produced by UOG1 are more enriched in neutral ceramides and monohexosyl ceramides than in SM.


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  		Fig. 3.   Fatty acid composition of sphingolipids in uog1-transfected cells. Fatty acid composition was determined by ESI-MS/MS for ceramide (A), monohexosylceramides (B), and SM (C) in mock- or uog1-transfected cells treated with or without FB1 as in Table III. Results are means ± S.D. of two independent analysis in which the variability was <10%.

Analysis of (Dihydro)ceramide Synthase in Vitro-- The same selectivity toward fatty acids was obtained upon analysis of (dihydro)ceramide synthase activity in vitro as was observed in the sphingolipids of cells after uog1 transfection (16, 38). Thus, (dihydro)ceramide synthase activity was increased 2.5-fold in microsomes from uog1-transfected cells using stearoyl-CoA as substrate, but there was no increase using palmitoyl-CoA (Fig. 4). This strongly suggests that UOG1 is a (dihydro)ceramide synthase that preferentially uses stearoyl-CoA as the fatty acyl donor. To exclude the possibility that UOG1 might form (dihydro)ceramide by the reverse activity of a ceramidase (20), microsomes were incubated with N-stearoyl-[4,5-3H]sphinganine (C18-[4,5-3H]dihydroceramide) (38); no difference in the rate of hydrolysis was detected (mock-transfected cells hydrolyzed 232 fmol of N-stearoyl-[4,5-3H]sphinganine/min/mg and uog1-transfected cells 241 fmol/min/mg). Together with the similar effect of the ceramidase inhibitor, (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol on (dihydro)ceramide levels in mock- and uog1-transfected cells (not shown), the lack of sequence homology to any known ceramidase, and the increase in ceramide amount (see above), these data demonstrate that UOG1 is either a bona fide (dihydro)ceramide synthase or that it regulates the activity of an endogenous (dihydro)ceramide synthase that preferentially uses stearoyl-CoA as fatty acyl donor.


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  		Fig. 4.   In vitro activity of (dihydro)ceramide synthase. Microsomes (200 µg of protein) were prepared from mock- or uog1-transfected cells and incubated with or without FB1 (20 µM). (Dihydro)ceramide synthase was assayed using either 50 µM palmitoyl- or stearoyl-CoA. Results are means ± S.D. of three experiments.

In contrast to the FB1 resistance observed in vivo in uog1-transfected cells, FB1 inhibited (dihydro)ceramide synthase activity in homogenates (not shown) and in microsomes (Fig. 4) using both palmitoyl-CoA and stearoyl-CoA (IC50 of inhibition for FB1 was 0.3-0.5 µM using 50 µM stearoyl-CoA/15 µM sphinganine in both mock- and uog1-transfected cells), although the extent of inhibition was always somewhat higher toward stearoyl-CoA. A number of possibilities could explain the differences between the FB1 resistance observed in vivo and the FB1 sensitivity observed in vitro. First, we examined whether FB1 might induce a change in the intracellular location of UOG1 thus rendering it inaccessible to FB1 in vivo; however, no changes were detected in the localization of HA constructs of UOG1 (not shown). Second, HEK-293T cells were incubated with long chain sphingoid bases whose levels change upon FB1 incubation (40). In mock-transfected cells, the long chain bases had no effect on [3H]ceramide synthesis (Table IV), but sphingosine 1-phosphate consistently increased [3H]ceramide synthesis in uog1-transfected cells, although with some variability and to a smaller extent than FB1. However, sphingosine 1-phosphate, when added directly to the (dihydro)ceramide synthase in vitro assay, had no effect on the Vmax or Km value of UOG1 toward sphinganine (not shown). The inability of long chain bases and of FB1 to up-regulate the UOG1-dependent (dihydro)ceramide synthase in vitro suggests that additional metabolites may be involved in modulating UOG1 activity in vivo by FB1. That this mechanism involves a post-translational mechanism of regulation is supported by the observation that levels of uog1 RNA are not transcriptionally up-regulated upon FB1 treatment in two cell types that express endogenous uog1, HEK-293T cells and cultured hippocampal neurons (47) (not shown).4

                              
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Table IV
Effect of exogenously added long chain bases on ceramide synthesis in HEK-293T cells
HEK-293T cells were transfected with pcDNA (mock) or with pcDNA-UOG1 and incubated 24 h later with 5 µM of the long chain bases (53), or with 20 µM FB1, together with 30 µCi of L-3-[3H]serine. Results are means ± S.D. for six independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The major finding of the current study is that overexpression of UOG1 in mammalian cells preferentially stimulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis, which is subsequently selectively channeled into the pathway of neutral GSL but not ganglioside synthesis. This is the first report of (dihydro)ceramide formation in mammalian cells with such a high degree of fatty acid selectivity. In addition, the increase in N-stearoyl-sphinganine synthesis, and in ceramide and neutral GSL amounts, is insensitive to FB1 in intact cells, suggesting that uog1 may encode a (dihydro)ceramide synthase that is distinct from the FB1-sensitive (dihydro)ceramide synthase, or, alternatively, may modify, in vivo, the activity of the FB1-sensitive (dihydro)ceramide synthase rendering it FB1-resistant.

Sphingolipids contain four kinds of fatty acids, namely saturated long chain fatty acids, very long chain fatty acids, monoenoic fatty acids, and alpha -hydroxy very long chain fatty acids (reviewed in Refs. 48 and 49). This fatty acid profile presumably reflects the fatty acid specificity of (dihydro)ceramide synthase(s), although fatty acid re-modeling could also account for some of the variability. Because(dihydro)ceramide synthase has not been purified to homogeneity, determination of its fatty acid specificity is based on analysis of partially purified enzyme or on its activity in microsomal fractions, where it is enriched (16, 17). In the case of a partially purified bovine liver mitochondria-enriched (dihydro)ceramide synthase, the fatty acid specificity is stearoyl-CoA (C18:0) > palmitoyl-CoA (C16:0) = oleoyl-CoA (C18:1) > behenoyl-CoA (C22:0), in a ratio of 4:3:3:1 (18), whereas in microsomal fractions, the specificity is stearoyl-CoA > lignoceroyl-CoA (C24:0) > palmitoyl-CoA > oleoyl-CoA, in a ratio of 60:12:3:1 (50), or stearoyl-CoA = lignoceroyl-CoA > palmitoyl-CoA, in a ratio of 7:7:1 (13) (reviewed in Ref. 11). This in itself does not prove that more than one distinct (dihydro)ceramide synthase exists, but kinetic evidence would imply this to be so, because the properties of the activities that utilize stearoyl-CoA and lignoceroyl-CoA appear to differ considerably from one another (51).

Our current data support but do not definitively prove the existence of more than one distinct (dihydro)ceramide synthase, based on the shift in fatty acid incorporation into (dihydro)ceramide upon uog1 overexpression and based on the differences in FB1 sensitivity (see below). Interestingly, uog1 is expressed at high levels in adult human brain, skeletal muscle, and testis as well as in fetal human brain (29). Although no data are available in human brain, rat brain contains a bimodal distribution of fatty acids in ceramide, with high levels of stearic and behenic acid (52), which approximately reflects the relative distribution of these fatty acids in brain GSLs. Thus, UOG1 may be involved in the tissue (brain)-specific synthesis of N-stearoyl-containing (dihydro)ceramides. In contrast, LAG1 and LAC1 utilize very long chain fatty acids (21, 23), which may reflect or be responsible for the unique fatty acid composition of yeast sphingolipids. The molecular basis for this difference is not known and awaits characterization of the reaction mechanism of mammalian and yeast (dihydro)ceramide synthases.

The reason for the difference in the FB1 sensitivity of LAG1 compared with uog1 and ASC1 is also not known. However, the ability of ASC1 to confer FB1 resistance in plants (27, 28) is entirely consistent with the FB1 resistance that we detect in uog1-overexpressing mammalian cells. The expression of endogenous uog1 in brain may also provide an explanation for the incomplete inhibition of (dihydro)ceramide synthesis observed in cultured cerebellar granule cells (13) and in cultured hippocampal neurons (14) upon FB1 treatment, in which residual levels of sphingolipid synthesis were consistently detected.

In summary, we have demonstrated that UOG1 regulates C18-(dihydro)ceramide synthesis in mammalian cells (Fig. 5). In cells that do not express UOG1, ceramide is synthesized via an FB1-sensitive (dihydro)ceramide synthase(s) that apparently uses various fatty acyl donors, whereas cells that express UOG1 selectively synthesize (dihydro)ceramides containing stearate. The C18-(dihydro)ceramide that is formed has a major impact on the amounts of neutral GSLs that are formed (Fig. 5) but less of an impact on SM and no effect on gangliosides. This differential effect is augmented upon FB1 treatment, presumably due to the coordinate up-regulation of the glycosyltransferases involved in neutral GSL synthesis, as observed previously (15) upon FB1 treatment of fibroblasts. Future studies will aim to clarify the mechanism by which the fatty acid selectivity of UOG1 is regulated and the mechanism of up-regulation of C18-(dihydro)ceramide synthesis upon FB1 incubation of uog1-overexpressing cells.


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  		Fig. 5.   Hypothetical scheme to explain the effect of uog1 overexpression on sphingolipid metabolism. UOG1 preferentially uses C18 fatty acids rather than C16 fatty acids, and the C18-(dihydro)ceramide thus formed is preferentially channeled (blue) into the pathway of neutral GSL synthesis, to some extent into SM, but not into gangliosides. Upon incubation of mock- and uog1-transfected cells with FB1 (red), sphinganine accumulates due to inhibition of the FB1-dependent (dihydro)ceramide synthase, which is metabolized to sphinganine 1-phosphate and then to ethanolamine phosphate and hexadecanal. One of these, or another unrelated metabolite, may be involved in the activation in vivo of UOG1 upon FB1 treatment.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the Japanese Collection of Research Bioresources Gene Bank, Japan, for the cDNA clones, the Forchheimer Plasmid Bank at the Weizmann Institute of Science for some of the vectors, Dr. Walt Shaw, Avanti Polar Lipids (Alabaster, AL) for some of the internal standards for the ESI-MS/MS analyses, Elaine Wang (Georgia Institute of Technology) for help with the ESI-MS/MS analyses, and members of our laboratories for many helpful suggestions.

    FOOTNOTES

* This work was supported in part by the Minerva Foundation, Munich, Germany (to A. H. F.) and by National Institutes of Health Grant GM46368 (to A. H. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Research Training Network Fellowship HPRN-CT-2000-00077 from the European Union.

Supported by a Koschland Scholar award.

Dagger Dagger To whom correspondence should be addressed: Dept. of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-9342704; Fax: 972-8-9344112; E-mail: tony.futerman@weizmann.ac.il.

Published, JBC Papers in Press, June 24, 2002, DOI 10.1074/jbc.M205211200

2 [4,5-3H]Sphinganine is metabolized to [4,5-3H]dihydroceramide and subsequently to [3H]ceramide, which cannot be differentiated by the TLC solvent used in this experiment. [3H]Ceramide consists of a mixture of [4-3H]ceramide and [5-3H]ceramide (16, 42). The resulting lipid is therefore referred to as [4/5-3H](dihydro)ceramide.

3 The reason for the difference in the extent of FB1 resistance or of FB1-induced elevation of (dihydro)ceramide synthesis using the two different metabolic labeling protocols (i.e. [4,5-3H]sphinganine and L-3-[3H]serine) is not known but may be due to the different times of labeling (3 versus 24 h), the different amounts of label added, or the different sites of incorporation of L-3-[3H]serine and [4,5-3H]sphinganine into the sphingolipid metabolic pathway.

4 Reverse transcriptase-PCR was performed using sequence-specific primers (5'-TCAACTGGTTCCCGCTCAAG and 5'-CTCAGTGGCTTCTCGGCTTT) from the highly conserved region between the mouse uog1 and human uog1 coding sequences.

    ABBREVIATIONS

The abbreviations used are: FB1, fumonisin B1; ER, endoplasmic reticulum; ESI-MS/MS, electrospray tandem mass spectrometry; GlcCer, glucosylceramide; GSL, glycosphingolipid; HA, hemagglutinin; PDI, protein-disulfide isomerase; SM, sphingomyelin; HEK, human embryonic kidney.

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RESULTS
DISCUSSION
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