The SPTLC3 Subunit of Serine Palmitoyltransferase Generates Short Chain Sphingoid Bases^*

Cell Lines

HEK293 cells were obtained from American Type Culture Collection and cultured in full medium (Dulbecco's modified Eagle's medium, Sigma) containing 10% fetal bovine serum (Fisher) and penicillin/streptomycin (100 units/ml and 0.1 mg/ml).

HEK293 cells were transfected with Lipofectamine 2000 (Invitrogen) and selected for neomycin resistance (G418, 400 μg/ml) to generate a pool of stably expressing cells. The expression of each of the SPT subunits in the three cell lines was confirmed by Western blots.

Fumonisin B1-dependent Accumulation of Sphingoid Bases

Fumonisin B1 (Sigma) was added to the media of exponentially growing cells at a final concentration of 10 μg/ml. As a negative control, myriocin (10 μg/ml, Sigma) was added together with fumonisin B1. 24 h after fumonisin B1 addition cells were washed twice with phosphate-buffered saline, harvested, and counted (Coulter® Z2, Beckman Coulter). Synthetic C17 sphingosine (Avanti Polar Lipids) was added to each sample as an internal extraction standard.

SPT Activity Assay

Cells were grown in 10-cm dishes to ∼80% confluency. Medium was removed, and the cells were washed 2 times with phosphate-buffered saline and harvested in 1 ml of phosphate-buffered saline by scraping. The suspension was transferred into a 1.5-ml reaction tube. Cells were pelleted by centrifugation (2500 × g, 2 min at 4 °C) and resolved in assay buffer (50 mm Hepes, pH 8.0, 0.5 mm MnCl₂). The protein concentration was adjusted to 2 mg/ml. Protein concentrations of the cell lysates were determined using the Bradford Assay (Bio-Rad). Albumin was used as calibration standard.

The reaction mixture for measuring in vitro SPT activity was composed of 400 μg of total lysate protein, 50 mm Hepes, pH 8.0, 0.5 mm l-serine, 0.05 mm palmitoyl-CoA, 20 μm pyridoxal-5′-phosphate, 0.5 mm MnCl₂, and 0.1 μCi of l-[U-¹⁴C]serine (Amersham Biosciences) in a total volume of 200 μl. The assay was performed at 37 °C for 60 min. For the negative controls SPT activity was specifically blocked by the addition of the SPT inhibitor myriocin (40 μm, Sigma). The reaction was stopped by adding 0.5 ml of methanolic-KOH, CHCl₃ (4:1) to the mixture. Methanolic KOH was prepared by dissolving 0.7 g of KOH pellets in 100 ml of MeOH. Lipids were extracted at 37 °C under steady agitation for 30 min. Subsequently, 500 μl of CHCl₃, 500 μl of alkaline water (100 μl of NH₄ (2 n) in 100 ml of H₂O), and 100 μl of NH₄ (2 n) was added in this order. Phases were separated by centrifugation (13,000 × g, 5 min), and the upper phase was discarded. The lower phase was washed three times with alkaline water. Finally, the lower organic phase was transferred to a scintillation vial, and the CHCl₃ was evaporated under a stream of N₂. After the addition of scintillation mixture, the radioactivity was quantified on a Scintillation Analyzer (Packard Liquid 1900TR).

Quantitative Reverse Transcription-PCR

The mRNA was extracted using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) and transcribed to cDNA using oligo(dT) primers and Superscript III (Invitrogen) according to manufacturer's instructions. Specific primers for the different SPT subunits were designed using the Oligo6.0 software (Molecular Biology Insights, Cascade, CO). Light Cycler PCR was performed using the DNA Master Kit (Roche Diagnostics) according to the manufacturer's instructions. The following primer pairs were used (0.4 μm each): huSPTLC3fw, 5′-TGCAGCCAAGTATGATGAGTCTA-3′; huSPTLC3rv: 5′-GCAGATGCACGATGGAAC-3′; moSPTLC3fw: 5′-GGCTTGCAGGGAAATATG-3′; moSPTLC3rv: 5′-GGATGACTGAAGTGTGGTTA-3′. Amplification was carried out for SPTLC3: 50 cycles, each consisting of 10 s at 95 °C, 10 s at 61 °C, 20 s at 72 °C. The linearity of the assays was determined by serial dilutions of the templates for each primer set separately.

Separation of Plasma Lipoproteins

Plasma was isolated from healthy normolipidemic donors after overnight fasting. 3 ml of plasma was fractionated on a four-step density gradient essentially as described (14). Ultracentrifugation was performed in a Beckman SW-40 swinging bucket rotor for 24 h at 41,000 rpm at 15 °C. Fractions (1 ml) were collected from the top of the centrifuge tube and analyzed for triglyceride, cholesterol, and other lipids.

Lipid Extraction and Hydrolysis

Total lipids were extracted and, before analysis, either base- or acid/base-hydrolyzed (15). Briefly, cells were resuspended in 200 μl of phosphate-buffered saline, and lipids were extracted in 1 ml of extraction buffer (2 volumes of methanol/1 volume of chloroform + 0.15 μl/ml C₁₇-sphingosine (C₁₇-SO; 1 mm in EtOH). 100 μl of NH₄ (2 n) was added, and the lipids were extracted under constant agitation (1 h, 37 °C). Subsequently 0.5 ml of chloroform was added, and samples were centrifuged (12,000 × g, 5 min) to separate the organic from the water phase. The upper (water) phase was discarded, and the lower phase was washed twice with 1 ml of alkaline water (1 ml of NH₄ (2 n) in 100 ml of water) and dried under N₂. For acid hydrolysis, the dried lipids were resuspended in 200 μl of methanolic HCl (1 n HCl, 10 m water in methanol) and kept at 65 °C for 12–15 h. The solution was neutralized by the addition of 40 μl of KOH (5 m) and subsequently subjected to 0.5 ml of extraction buffer (4 volumes of 0.125 m KOH in methanol + 1 volume of chloroform) and mixed. Subsequently, 0.5 ml of chloroform, 0.5 ml of alkaline water, and 100 μl of NH₄ (2 n) were added in this order. Liquid phases were separated by centrifugation (12,000 × g, 5 min). The upper phase was aspirated, and the lower phase was washed twice with alkaline water. Finally, the lipids were dried by evaporation of the chloroform phase under N₂ and subjected to LC-MS analysis. Plasma lipids were analyzed from 100 μl of human EDTA-treated plasma which was treated in the same manner.

LC-MS

Extracted lipids were solubilized in 56.7% methanol, 33.3% ethanol, 10% water and derivatized with ortho-phthaldialdehyde (Sigma) (15). The lipids were separated on a C₁₈ column (Uptispere 120 Å, 5 μm, 125 × 2 mm, Interchim, France) and analyzed by a serial arrangement of a fluorescence detector (HP1046A, Hewlett Packard) followed by a MS detector (LCMS-2010A, Shimadzu). Atmospheric pressure chemical ionization was used for ionization. Non-natural C17 sphingosine (Avanti Polar Lipids) was used as the internal standard.

SPTLC3-expressing Cells Generate C₁₆ Sphingoid Bases

The human SPT subunits SPTLC1, SPTLC2, and SPTLC3 were cloned into a pcDNA3.1 expression vector and expressed in HEK293 cells as reported earlier (12). HEK293 cells were chosen because they express low endogenous levels of SPTLC3 mRNA (see Fig. 4A). All subunits were expressed at comparable levels as demonstrated by Western blot analysis and showed no signs of degradation (12).

To determine whether SPTLC3-expressing cells generated more or other sphingoid bases, we compared the spectrum of de novo-synthesized sphingoid bases between SPTLC3-deficient (HEK^Cnt) and SPTLC3-overexpressing cells (HEK^SPTLC3). This was done by blocking the de novo synthesis pathway at the step of the ceramide synthase with fumonisin B1 (FB1). The inhibition of ceramide synthase leads to a time-dependent accumulation of its substrate sphinganine (SA). Hence, potential side products of the SPT reaction also accumulate under these conditions (7). The accumulated lipids were extracted 24 h after the addition of FB1, derivatized with ortho-phthaldialdehyde, and analyzed on a C18 reverse phase column with a serially arranged fluorescence and MS detector (15).

In the presence of FB1 we observed a significant accumulation of sphinganine (Fig. 1A), whereas no SA accumulation was seen when SPT activity was blocked with myriocin. In parallel, we observed the appearance of a second unknown peak which eluted before SA after 8.2 min. Because of similar retention times, this peak was partly overlaid by the internal standard (C₁₇-SO), which was added for normalization (Fig. 1B). Accumulation of SA but also of this unknown peak was not observed when SPT activity was inhibited with myriocin. This indicates that the unknown metabolite is a product of the SPT reaction. Subsequent MS analysis revealed that the unidentified metabolite had an m/z of 450.3 (as an ortho-phthaldialdehyde derivate). For this mass the single ion chromatogram revealed a single peak with the same retention time as seen in the fluorescence chromatogram (Fig. 1B). This signal was not seen in the presence of myriocin (Fig. 1C). The unknown metabolite (m/z 450.3) showed a mass difference to sphinganine (m/z 478.3) of 28 Da, which equals the mass of a CH₂CH₂ group and suggests that the unknown metabolite is dihydrosphingosine (SA) with a C₁₆, instead of a C₁₈, carbon chain (C₁₆-SA). This metabolite could be formed by the conjugation of l-serine with myristoyl-CoA instead of palmitoyl-CoA.

SPTLC3 Has Higher Activity with C₁₂ and C₁₄ Acyl-CoA

To test this hypothesis we compared the in vitro SPT activity with various acyl-CoA substrates in extracts from control and SPTLC1-, SPTLC2-, and SPTLC3-overexpressing cells. All acyl-CoAs were used at the same concentration (50 μm). In comparison, the SPTLC3-expressing cells showed a significantly higher activity with lauryl-CoA and myristoyl-CoA than did the control or SPTLC1- or SPTLC2-expressing cells (Fig. 2A). This suggests that the SPTLC3-mediated SPT activity is primarily responsible for the generation of sphingoid bases with a C₁₄ or C₁₆ backbone, whereas the SPTLC2 subunit seems to be more specific for longer acyl-CoAs, thereby forming C₁₈- and C₂₀-sphingoid bases.

C₁₆-SA Generation Is Stimulated by Serine

The addition of l-serine to the cell culture medium generally stimulates SPT activity and SA de novo synthesis (7). To determine whether l-serine also stimulated SPTLC3-mediated C₁₆-SA generation, we compared the accumulation of C₁₆-SA in FB1-treated HEK^Cnt and HEK^SPTLC3 cells, which were cultured either in regular medium or in medium that was supplemented with l-serine (10 mm). The normally cultured HEK^SPTLC3 cells showed a clear accumulation of C₁₆-SA in the presence of FB1, whereas HEK^Cnt cells did not show any accumulation under these conditions (Fig. 2B). In the cells which were cultured at elevated serine levels, the accumulated sphingoid bases increased significantly. For HEK^SPTLC3 cells we observed a 3-fold increase in SA accumulation (data not shown) and a 4-fold increase in the accumulation of C₁₆-SA (Fig. 2B). Even in HEK^Cnt cells a small accumulation of C₁₆-SA was detected under these conditions.

Kinetic Analysis of SPTLC3 Activity

To obtain further insight into the enzymatic mechanism, we compared the kinetics for myristoyl-CoA and palmitoyl-CoA in HEK^Cnt and HEK^SPTLC3 cells. For palmitoyl-CoA, the enzyme followed a Michaelis-Menten kinetics up to a concentration of about 0.1 mm (Fig. 3A). The kinetics with palmitoyl-CoA was essentially the same in HEK^Cnt and HEK^SPTLC3 cells. At palmitoyl-CoA concentrations above 0.15 mm, substrate inhibition was observed in accordance to earlier reports (16, 17). Also, the Hanes-Woolf plot (18) showed a linear relationship between [S] and 1/v up to a palmitoyl-CoA concentration of 0.1 mm (Fig. 3A). For myristoyl-CoA we observed an about 5-fold higher activity in HEK^SPTLC3 cells compared with HEK^Cnt cells (Fig. 3B). As for palmitoyl-CoA, we also observed an inhibitory effect of myristoyl-CoA at higher concentrations. For both substrates the optimal activity was in a concentration range of 0.1–0.125 mm. K_m and V_max values (Fig. 3C) were deduced from the Hanes-Woolf plots and confirmed by a hyperbolic regression analysis (19). Both methods gave similar results.

For palmitoyl-CoA the kinetic parameters V_max and K_m were similar in HEK^Cnt and HEK^SPTLC3 cells, whereas for myristoyl-CoA both cell lines showed significant differences. Because of the low activity in HEK^Cnt cells, a precise determination of V_max and K_m for myristoyl-CoA was not possible. Nevertheless, it was obvious that the V_max for myristoyl-CoA is significantly lower in HEK^Cnt compared with HEK^SPTLC3 cells. The K_m values for palmitoyl-CoA and myristoyl-CoA in HEK^SPTLC3 cells were comparable, whereas the maximal velocity for myristoyl-CoA was about 50% lower than for palmitoyl-CoA (Fig. 3C).

SPTLC3 mRNA Levels Correlate with C₁₆-SA Accumulation in FB1-blocked Cells

To further confirm that the generation of C₁₆-SA is linked to the expression of SPTLC3, we analyzed the SPTLC3 mRNA levels in various human and non-human cell lines (Fig. 4A). Some cell lines like HEK293 or the human monocytic cell line THP1 showed very low SPTLC3 mRNA expression, whereas intermediate levels were found in HepG2, COS, and NIH/3T3 cells. The highest SPTLC3 mRNA levels were in the trophoblast lines JAR and JEG-3 as reported earlier (12). The SPTLC3 mRNA levels showed a close correlation with the levels of accumulated C₁₆-SA in these cells (Fig. 3B), providing further evidence that C₁₆-sphingoid bases are primarily generated by SPTLC3.

C₁₆-SA Is Metabolized to C₁₆-SO

The observation that C₁₆-SA accumulates in the presence of the ceramide synthase inhibitor FB1 indicates that C₁₆-SA is also a substrate for ceramide synthase. Consequently, the generated C₁₆-SA is further metabolized to C₁₆-dihydroceramide and to C₁₆-ceramide. The degradation of C₁₆-ceramide by ceramidase would finally lead to the generation of C₁₆-SO.

To test whether the SPTLC3-expressing cells also produce C₁₆-SO, we compared the total sphingoid base content between HEK^Cnt and HEK^SPTLC3 cells. In view of the great variety of fatty acids and possible head groups that can be attached to the sphingoid backbone, a complete analysis of all possible C₁₆-ceramide variants is a demanding task and will be addressed in future studies. To simplify the analysis we, therefore, subjected the extracted lipids to acid and base hydrolysis. Under acidic conditions the amide bond of the conjugated fatty acid is released, whereas the O-linked phospho-ester and carbohydrate moieties of the sphingoid base head group are removed under basic conditions. This procedure allowed the quantification of the total C₁₆- and C₁₈-sphingoid base levels in the cells. The molecular mass of SO and SA differs because of the Δ4 double bond by 2 daltons. As described above we observed a significant FB1-dependent accumulation of C₁₆-SA (m/z 450.3) in HEK^SPTLC3 cells (Fig. 5A). No signal in the single mass chromatogram for C₁₆-SO (m/z 448.3) was seen because the metabolism of the de novo-formed C₁₆-SA was blocked in the presence of FB1. In contrast, analysis of the acid/base-treated extract from HEK^SPTLC3 cells showed a pronounced peak with the m/z ratio of C₁₆-SO (448.3). Interestingly, HEK^Cnt cells also contained small but detectable amounts of C₁₆-SO but did not show the accumulation of C₁₆-SA in the presence of FB1. This could be possibly explained by an uptake of C₁₆-ceramide from the medium as an external source. Mammalian serum, including fetal calf serum, contains considerable amounts of C₁₆-based sphingolipids (see also Fig. 6).

To further demonstrate that the identified metabolite is C₁₆-SO, we compared its retention time with respect to other sphingoid bases with different carbon chains. On a C₁₈ reverse phase column, sphingoid bases show a logarithmic correlation between retention time and their carbon chain length (Fig. 5B). Regression analysis of this function revealed a theoretical retention time for C₁₆-SO of 6.5 min, which is in agreement with the observed retention time (6.45 min) of the identified metabolite (Fig. 3, A and B). Our studies also show that the C16-SO levels of acid/base-treated cell lines closely correlate with C16-SA (Fig. 5C).

Up to 15% of Human Plasma Sphingolipids Are Based on a C₁₆ Backbone

The observation that SPTLC3 is responsible for the generation of C₁₆-sphingoid bases raised the questions of whether these lipids are also present in human plasma and how they are transported in plasma.

We analyzed plasma samples of 20 healthy donors. The plasma was acid/base-treated and analyzed by LC-MS. The analysis showed that the majority of plasma sphingolipids are based on a C₁₈-sphingosine backbone. Nevertheless, an astonishingly high fraction of plasma sphingolipids was found with a C₁₆-SO backbone (Fig. 6A). The proportion of C₁₆-SO in total plasma SO may be as high as 15%. The fraction of plasma sphingolipids which contained an SA backbone was much lower. About 4% of the plasma sphingolipids contained a sphingosine backbone, whereas 15–20% of the total SA contained a C₁₆ backbone.

The analysis of lipoprotein fractions which were isolated by ultracentrifugation demonstrated that both the C₁₆- and the C₁₈-sphingolipids are present in the LDL and HDL fractions but not in the VLDL fraction (Fig. 6B). In comparison to total cholesterol and triglyceride, the sphingolipids showed a shoulder in fractions 9 and 10, indicating that they are also part of a denser HDL subfraction.