Long-chain Base Synthesis Research Articles

Regulation of sphingolipid synthesis by the G1/S transcription factor Swi4

SBF (Swi4/Swi6 Binding Factor) complex is a crucial regulator of G1/S transition in Saccharomyces cerevisiae. Here, we show that SBF complex is required for myriocin resistance, an inhibitor of sphingolipid synthesis. This phenotype was not shared with MBF complex mutants nor with deletion of the Swi4p downstream targets, CLN1/CLN2. Based on data mining results, we selected putative Swi4p targets related to sphingolipid metabolism and studied their gene transcription as well as metabolite levels during progression of the cell cycle. Genes which encode key enzymes for the synthesis of long chain bases (LCBs) and ceramides were periodically transcribed during the mitotic cell cycle, having a peak at G1/S, and required SWI4 for full transcription at this stage. In addition, HPLC-MS/MS data indicated that swi4Δ cells have decreased levels of sphingolipids during progression of the cell cycle, particularly, dihydrosphingosine (DHS), C24-phytoceramides and C24-inositolphosphoryl ceramide (IPC) while it had increased levels of mannosylinositol phosphorylceramide (MIPC). Furthermore, we demonstrated that both inhibition of de novo sphingolipid synthesis by myriocin or SWI4 deletion caused partial arrest at the G2/M phase. Importantly, our lipidomic data demonstrated that the sphingolipid profile of WT cells treated with myriocin resembled that of swi4Δ cells, with lower levels of DHS, IPC and higher levels of MIPC. Taken together, these results show that SBF complex plays an essential role in the regulation of sphingolipid homeostasis, which reflects in the correct progression through the G2/M phase of the cell cycle.

Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1

Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle.

Expression of the ORMDLS, Modulators of Serine Palmitoyltransferase, Is Regulated by Sphingolipids in Mammalian Cells

The relationship between serine palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.

Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.

Synthesis of 4-hydroxysphinganine and characterization of sphinganine hydroxylase activity in corn

Sphinganine and 4-hydroxysphinganine (phytosphingosine) are the predominant free long-chain bases in lipid extracts of plant tissues. While the synthesis of sphinganine in plants has been investigated, the metabolic origin of 4-hydroxysphinganine is not known. Three different approaches utilizing fumonisin B 1, an inhibitor of sphinganine acylation, alone or in combination with β-chloroalanine, an inhibitor of sphinganine synthesis, were used to establish that free 4-hydroxysphinganine is produced in excised corn shoots by the direct hydroxylation of sphinganine and not from the breakdown of complex sphingolipids. Sphinganine hydroxylase activity was characterized in microsomes isolated from corn. The enzyme was found to utilize d- erythro-sphinganine (with half-maximal activity observed at a substrate concentration of approximately 60 μM) and either NADPH ( K m=33 μM) or NADH ( K m=58 μM) as substrates. Ceramide hydroxylation was also demonstrated in corn microsomes, and the lack of competition between ceramide and sphinganine suggests the presence of distinct enzymes responsible for hydroxylating these two substrates. Using marker assays, sphinganine hydroxylase activity was localized to the endoplasmic reticulum. Sphinganine hydroxylase activity in microsomes isolated from corn shoots treated with fumonisin B 1 increased more than 3-fold compared to controls. The results of this study shed light on sphingolipid long-chain base synthesis and modification in plant tissues and suggest a possible contribution of sphinganine hydroxylase in manifesting the effects of fumonisin in plants.

Sphingolipid Biosynthesis de Novo by Rat Hepatocytes in Culture.: CERAMIDE AND SPHINGOMYELIN ARE ASSOCIATED WITH, BUT NOT REQUIRED FOR, VERY LOW DENSITY LIPOPROTEIN SECRETION

Sphingolipids are constituents of liver and lipoproteins, but relatively little is known about their synthesis and secretion. Incubation of rat hepatocytes with [14C]- or [3H]serine labeled the long-chain base backbones of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids were associated with the cells; however, 1-5% (the majority of which was ceramide) was released into the medium as part of very low density lipoproteins (VLDL). Since this is the first report that lipoproteins contain ceramide, lipoproteins were isolated from rat plasma, and the ceramide contents were (per mg of protein): 6.5 nmol for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins (1.018 < d < 1.063), 0.2 nmol for high density lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the albumin fraction; the lipoproteins also contained 0.1-0.4 nmol of free sphingosine/mg of protein. A number of factors affected the secretion of radiolabeled sphingolipids: 1) addition of palmitic acid, but not stearic or oleic acid, enhanced secretion due to an increase in long-chain base synthesis de novo. 2) Choline deficiency caused a 42% reduction in the secretion of radiolabeled sphingomyelin, but this was due to an effect on VLDL secretion rather than a decrease in sphingolipid synthesis. Removal of choline was examined because previous studies (Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004) have shown that choline deficiencies depress phosphatidylcholine synthesis and lipoprotein secretion. 3) Incubation of the cells with fumonisin B1, a mycotoxin inhibitor of sphinganine (sphingosine) N-acyltransferase, reduced overall sphingolipid synthesis and secretion by 90%, but had no effect on the secretion of apoB, phosphatidylcholine, or cholesterol. All together, these findings demonstrate that rat hepatocytes synthesize ceramide and sphingomyelin de novo and incorporate them into both cellular membranes and secreted VLDL, but normal sphingolipid synthesis is not required for lipoprotein secretion.

Cholesterol and sphingomyelin syntheses are regulated independently in cultured human intestinal cells, CaCo-2: role of membrane cholesterol and sphingomyelin content.

There is a presumed association between cellular cholesterol and sphingomyelin metabolism. To study this relationship in the intestine, the activity of the rate controlling enzyme of sphingolipid synthesis, serine palmitoyltransferase (SPT), and the biosynthesis of long-chain bases were characterized in cultured human intestinal cells, CaCo-2. Cells were then incubated with substances known to alter cholesterol biosynthesis, and the effect of these mediators on SPT activity and long-chain base synthesis was determined and compared with their effects on HMG-CoA reductase activity and cholesterol synthesis. The polar sterol, 25-hydroxycholesterol, the squalene epoxide inhibitor, U18666A, and the inhibitor of HMG-CoA reductase, lovastatin, all significantly inhibited the synthesis of cholesterol without altering either SPT activity or long-chain base synthesis. Mevalonate, which increased cholesterol production 3-fold, also had no affect on SPT activity or sphingoid base synthesis. Serine, which significantly increased the synthesis of long-chain bases, did not alter cholesterol biosynthesis. Moreover, the suicide inhibitors of SPT, beta-chloroalanine and cycloserine, did not alter cholesterol synthesis while markedly decreasing long chain base synthesis. Cells were incubated with palmitic, oleic, linoleic, and eicosapentaenoic acids. Only palmitic acid, the preferred substrate for SPT, increased the production of long-chain bases. Both palmitic and oleic acids, however, increased the synthesis of cholesterol. Cells enriched in sphingomyelin had higher rates of synthesis of both cholesterol and long-chain bases compared to their controls. In contrast, cholesterol and long-chain base syntheses were significantly decreased in cells enriched in cholesterol. Control cells incubated with phospholipid liposomes alone had higher rates of synthesis of both lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes).

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.

Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids

The existence of auxotrophic mutants of Saccharomyces cerevisiae having an absolute requirement for the long-chain base (lcb) component of sphingolipids suggests that sphingolipids are crucial for viability and growth. One mutant, termed the lcb1-1 mutant, lacks the activity of serine palmitoyltransferase, the first enzyme in the pathway for long-chain base synthesis. Here, we present evidence that LCB1 has been molecularly cloned. The size of the LCB1 transcript, the direction of transcription, and transcription initiation sites were determined. In addition, the coding region and its 5' and 3' flanking regions were sequenced. Analysis of the DNA sequence revealed a single open reading frame of 1,674 nucleotides, encoding a predicted peptide of 558 amino acids. The hydropathy profile of the predicted peptide suggests a hydrophobic, globular, membrane-associated protein with two potential transmembrane helices. Comparison of the predicted amino acid sequence to known protein sequences revealed homology to 5-aminolevulinic acid synthase and to 2-amino-3-ketobutyrate coenzyme A ligase. These homologies, the similarity of the chemical reactions catalyzed by the three enzymes, and the finding that LCB1 restores serine palmitoyltransferase activity to an lcb1-defective strain indicate that serine palmitoyltransferase or a subunit of the enzyme is the most likely product of LCB1. Homology of the LCB1 predicted protein to the Escherichia coli biotin synthetase was also observed, but the biological significance of this observation is not clear. A role for sphingolipids in sporulation is implicated by our finding that diploids homozygous for lcb1 failed to sporulate.

Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by beta-chloroalanine.

The effects of beta-chloroalanine (beta-Cl-alanine) on serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid (5 mM beta-Cl-alanine caused complete inactivation in 10 min), irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with beta-Cl-L-alanine (not beta-Cl-D-alanine) and was blocked by L-serine. These are characteristics of mechanism-based ("suicide") inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with beta-Cl-alanine (complete inhibition occurred in 15 min with 5 mM), and this treatment inhibited [14C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of beta-Cl-L-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [14C]serine uptake was not blocked, total lipid biosynthesis from [14C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [3H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [3H]leucine, which was only decreased by 14%. Although beta-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that beta-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.

Enzymology of long-chain base synthesis by aorta: induction of serine palmitoyltransferase activity in rabbit aorta during atherogenesis.

Serine palmitoyltransferase [EC 2.3.1.50] initiates the biosynthesis of sphingolipids by catalyzing the condensation of a fatty acyl-CoA with serine to yield the committed intermediate 3-ketosphinganine or one of its homologues. The presence of serine palmitoyltransferase in aorta was established under optimal assay conditions using microsomes from New Zealand White rabbits. Its activity was dependent on microsomal protein, L-serine, pyridoxal 5'-phosphate, and palmitoyl-CoA. Although several different saturated and unsaturated fatty acyl-CoA thioesters were utilized as substrates, maximal activity was with palmitoyl-CoA, suggesting that this enzyme contributes to the predominance of 18-carbon long-chain bases in sphingolipids from aorta. Rabbits, fed a Purina lab chow supplemented with 2% cholesterol, were used to study serine palmitoyltransferase activity in aorta during experimental atherogenesis. An increase in activity from intimal-medial preparations was detectable prior to prominent lipid accumulation or cellular proliferation. Activity continued to elevate over the 12-week duration of feeding concurrent with the increase in serum cholesterol and in proportion to the development of plaques resulting in a 3.7-fold increase in activity (20.7 +/- 2.6 pmol per min per mg microsomal protein +/- SE in the cholesterol-fed group versus 5.6 +/- 1.9 in the pair-fed controls also matched for age and sex; P less than 0.005). Thus, the accumulation of sphingomyelin that occurs in aorta during experimental atherogenesis may be related to increased long-chain base synthesis by serine palmitoyltransferase.

Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway.

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the condensation of L-serine and palmitoyl-CoA to yield 3-ketosphinganine in the first unique reaction of long-chain (sphingoid) base biosynthesis. The kinetic effects of changing the extracellular concentrations of the precursors for this pathway were studied with LM cells by following the incorporation of L-[3-14C]serine into the long-chain base (i.e., sphinganine and sphingenine) backbones of complex sphingolipids. [14C]Serine was taken up by the cells and rapidly reached steady-state concentrations similar to those of the medium. From the cellular [14C]serine concentrations and specific activities, the apparent Vmax [14 pmol min-1 (10(6) cells)-1] and Km (0.23 mM) values for long-chain base synthesis were determined and found to be essentially identical with those for serine palmitoyltransferase assayed in vitro [i.e., 13 pmol min-1 (10(6) cells)-1 and 0.27 mM, respectively]. The other precursor, palmitic acid, was also taken up rapidly and increased long-chain base biosynthesis in a concentration-dependent manner. This effect was limited to palmitic acid and matched the known specificity of serine palmitoyltransferase for saturated fatty acyl-CoA's of 16 +/- 1 carbon atoms. These studies delineate the influence of extracellular precursors on the formation of the sphingolipid backbone and suggest that the kinetic properties of serine palmitoyltransferase govern this behavior of long-chain base synthesis in intact cells.

Enzymology of long-chain base synthesis by liver: Characterization of serine palmitoyltransferase in rat liver microsomes

Serine palmitoyltransferase [palmitoyl-CoA: l-serine C-palmitoyltransferase (decar-boxylating) EC 2.3.1.50]catalyzes the initial and committed step in the biosynthesis of the long-chain bases of sphingolipids. A simple assay, based upon the incorporation of [ 3H]serine into the chloroform-soluble product 3-ketosphinganine, has been developed and demonstrated to be valid for analyzing this enzyme in rat liver microsomes. More than 75% of the serine palmitoyltransferase of rat liver was associated with the microsomal subfraction. The dependencies of activity on the incubation time, pH, temperature, other assay components (e.g., dithiothreitol, EDTA, and pyridoxal 5′-phosphate), and the concentrations of microsomal protein, l-serine, and palmitoyl-CoA were investigated. The requirement of pyridoxal 5′-phosphate for activity was established by formation of the apoenzyme by dialysis against cysteine, and recovery of full activity upon reconstitution with the coenzyme. Activities with fatty acyl-CoA's of varying alkyl chain length were distributed nearly symmetrically around a maximum at 16 carbons (palmitoyl-CoA) for the fully saturated substrates. Less activity was obtained with the CoA thioesters of cis-unsaturated fatty acids, but trans-9-hexadecenoyl-CoA yielded essentially the same activity as palmitoyl-CoA. Hence, this enzyme is capable of initiating the synthesis of the major long-chain bases, as well as compounds that may constitute the unidentified bases reported in analyses of mammalian sphingolipids.

Characterization of serine palmitoyltransferase activity in Chinese hamster ovary cells

Serine palmitoyltansferase (palmitoyl-CoA: l-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50) catalyzes the first unique and regulatory reaction of sphingolipid biosynthesis. Its activity was demonstrated in Chinese hamster ovary cells (CHO-K1) by measuring the incorporation of radiolabel from l-[ 3H] serine into 3-ketosphinganine, which was found to be the predominant chloroform-soluble product under optimal assay conditions. Most of the total activity (14.8 ± 4.2 pmol/min per 10 6 cells, measured with sonicated cells) was recovered in particulate fractions, with the highest percentage (54%) and specific activity (102 pmol/min per mg) in the high-speed (airfuge) pellet. The greatest activity was obtained with palmitoyl-CoA; however, other fatty acyl-CoA thioesters were also utilized. Serine palmitoyltransferase required pyridoxal 5'-phosphate for activity, but was apparently fully saturated with this coenzyme when assayed with sonicated cells. Regardless of whether the CHO cells were grown in culture medium containing whole serum with or without sphinganine addition, lipoprotein-depleted serum, lipid-extracted serum, low-density lipoproteins, or no serum, the activities of this enzyme were identical. This finding was confirmed using human fibroblasts. Hence, these results establish that CHO cells, and probably others, are engaged in long-chain base synthesis de novo and that serine palmitoyltransferase activity is not regulated by the availability of such compounds in the culture media.

Regulation of sphingomyelin long chain base synthesis in human fibroblasts in culture. Role of lipoproteins and the low density lipoprotein receptor.

We have studied regulation of synthesis of long chain bases in human fibroblasts using 3 different radioactive precursors and 2 different hydrolysis and separation procedures. Serum and low density lipoproteins inhibited synthesis. Inhibition of long chain base synthesis by various concentrations of low density lipoproteins paralleled inhibition of cholesterol synthesis. This inhibition was dependent on the low density lipoprotein receptor pathway since fibroblasts from homozygous familial hypercholesterolemic patients did not show the inhibition observed with normal fibroblasts. Incorporation of precursor palmitate into free or total long chain bases was inhibited by low density lipoproteins to the same extent as incorporation into sphingomyelin long chain bases. We thus propose that an enzyme in the pathway leading to sphinganine synthesis, probably palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50, is regulated by low density lipoproteins.