Base Exchange Enzyme Research Articles

BST1 regulates nicotinamide riboside metabolism via its glycohydrolase and base-exchange activities

Nicotinamide riboside (NR) is one of the orally bioavailable NAD+ precursors and has been demonstrated to exhibit beneficial effects against aging and aging-associated diseases. However, the metabolic pathway of NR in vivo is not yet fully understood. Here, we demonstrate that orally administered NR increases NAD+ level via two different pathways. In the early phase, NR was directly absorbed and contributed to NAD+ generation through the NR salvage pathway, while in the late phase, NR was hydrolyzed to nicotinamide (NAM) by bone marrow stromal cell antigen 1 (BST1), and was further metabolized by the gut microbiota to nicotinic acid, contributing to generate NAD+ through the Preiss–Handler pathway. Furthermore, we report BST1 has a base-exchange activity against both NR and nicotinic acid riboside (NAR) to generate NAR and NR, respectively, connecting amidated and deamidated pathways. Thus, we conclude that BST1 plays a dual role as glycohydrolase and base-exchange enzyme during oral NR supplementation.

Presence of Phosphatidylserine Synthesizing Enzymes in Triton Insoluble Floating Fractions from Cerebrocortical Plasma Membranes

Mammals synthesize phosphatidylserine (PS), a binding PKC molecule, by exchanging the nitrogen base of phosphatidylethanolamine or phosphatidylcholine with free serine. Serine base exchange enzyme (SBEE) was found in Triton insoluble floating fractions (TIFFs) from rat cerebellum which contained PKC. Consequently, SBEE might modulate PS levels in the PKC binding area (Buratta et al., J Neurochem 103:942-951, 2007). In the present study, we determined whether SBEE and PKC were localised in rat cerebral cortex TIFFs (cx-TIFFs) and in rat cerebrocortical plasma membrane-TIFFs (PM-TIFFs) which are more directly involved in signal transduction than intracellular membranes. Cx-and PM-TIFFs expressed SBEE activity and contained PKC. SBEE used ethanolamine as free exchanging base which may modulate PS level in the PKC binding area, transforming PS into PE and vice versa. The slight decrease in [(14)C]serine incorporation in the presence of choline indicated the existence of a SBEE isoform which may play a peculiar role in this brain area.

Purification and characterization of human phosphatidylserine synthases 1 and 2

PS (phosphatidylserine) in mammalian cells is synthesized by two distinct base-exchange enzymes, PSS1 (PS synthase 1) and PSS2, which are responsible for the conversion of PC (phosphatidylcholine) and PE (phosphatidylethanolamine) respectively into PS in intact cells. The PS synthesis in cultured mammalian cells is inhibited by exogenous PS, and this feedback control occurs through inhibition of PSSs by PS. In the present study, we purified epitope-tagged forms of human PSS1 and PSS2. The purified PSS2 was shown to catalyse the conversion of PE, but not PC, into PS, this being consistent with the substrate specificity observed in intact cells. On the other hand, the purified PSS1 was shown to catalyse the conversion of both PC and PE into PS, although PSS1 in intact cells had been shown not to contribute to the conversion of PE into PS to a significant extent. Furthermore, we found that the purified PSS2, but not the purified PSS1, was inhibited on the addition of PS to the enzyme assay mixture, raising the possibility that there was some difference between the mechanisms of the inhibitory actions of PS towards PSS1 and PSS2.

Thematic Review Series: Glycerolipids. Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are two aminophospholipids whose metabolism is interrelated. Both phospholipids are components of mammalian cell membranes and play important roles in biological processes such as apoptosis and cell signaling. PS is synthesized in mammalian cells by base-exchange reactions in which polar head groups of preexisting phospholipids are replaced by serine. PS synthase activity resides primarily on mitochondria-associated membranes and is encoded by two distinct genes. Studies in mice in which each gene has been individually disrupted are beginning to elucidate the importance of these two synthases for biological functions in intact animals. PE is made in mammalian cells by two completely independent major pathways. In one pathway, PS is converted into PE by the mitochondrial enzyme PS decarboxylase. In addition, PE is made via the CDP-ethanolamine pathway, in which the final reaction occurs on the endoplasmic reticulum and nuclear envelope. The relative importance of these two pathways of PE synthesis has been investigated in knockout mice. Elimination of either pathway is embryonically lethal, despite the normal activity of the other pathway. PE can also be generated from a base-exchange reaction and by the acylation of lyso-PE. Cellular levels of PS and PE are tightly regulated by the implementation of multiple compensatory mechanisms.

Defining the Importance of Phosphatidylserine Synthase-1 (PSS1): Unexpected Viability of PSS1-Deficient Mice

Phosphatidylserine (PS) is a quantitatively minor, but physiologically important, phospholipid in mammalian cells. PS is synthesized by two distinct base-exchange enzymes, PS synthase-1 (PSS1) and PS synthase-2 (PSS2), that are encoded by different genes. PSS1 exchanges serine for choline of phosphatidylcholine, whereas PSS2 exchanges ethanolamine of phosphatidylethanolamine for serine. We previously generated mice lacking PSS2 (Bergo, M. O., Gavino, B. J., Steenbergen, R., Sturbois, B., Parlow, A. F., Sanan, D. A., Skarnes, W. C., Vance, J. E., and Young, S. G. (2002) J. Biol. Chem. 277, 47701-47708) and found that PSS2 is not required for mouse viability. We have now generated PSS1-deficient mice. In light of the markedly impaired survival of Chinese hamster ovary cells lacking PSS1 we were surprised that PSS1-deficient mice were viable, fertile, and had a normal life span. Total serine-exchange activity (contributed by PSS1 and PSS2) in tissues of Pss1(-/-) mice was reduced by up to 85%, but except in liver, the PS content was unaltered. Despite the presumed importance of PS in the nervous system, the rate of axonal extension of PSS1-deficient neurons was normal. Intercrosses of Pss1(-/-) mice and Pss2(-/-) mice yielded mice with three disrupted Pss alleles but no double knockout mice. In Pss1(-/-)/Pss2(-/-) and Pss1(-/-)/Pss2(-/-) mice, serine-exchange activity was reduced by 65-91%, and the tissue content of PS and phosphatidylethanolamine was also decreased. We conclude that (i) elimination of either PSS1 or PSS2, but not both, is compatible with mouse viability, (ii) mice can tolerate as little as 10% of normal total serine-exchange activity, and (iii) mice survive with significantly reduced PS and phosphatidylethanolamine content.

Synthesis of phosphatidylserine by base exchange in Triton‐insoluble floating fractions from rat cerebellum

Phosphatidylserine (PS), which is synthesized in mammalian tissues by the exchange between free serine and the nitrogen bases present in membrane glycerophospholipids, is strictly required for protein kinase C (PKC) activity. PKC, as other molecules involved in signal transduction, is present in lipid rafts, considered as a platform for molecular signaling. Membrane microdomains enriched in components of rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C and their low density in sucrose density gradient. This study demonstrates the existence of serine base exchange enzyme (SBEE) in Triton-insoluble floating fractions containing associated PKC. Using two fractions of detergent-resistant membranes from rat cerebellum, we observed a correlation between the level of SBEE activity and that of membrane-associated PKC. This suggests that SBEE, synthesizing PS in the binding area for PKC, participates to signal transduction. The capability of SBEE to utilize not only serine but also ethanolamine, as free exchanging base, suggests a mechanism for modulating in loco PS concentration.

Phospholipid biosynthesis in mammalian cells.

Identification of the genes and gene products involved in the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine has lagged behind that in many other fields because of difficulties encountered in purifying the respective proteins. Nevertheless, most of these genes have now been identified. In this review article, we have highlighted important new findings on the individual enzymes and the corresponding genes of phosphatidylcholine synthesis via its two major biosynthetic pathways: the CDP-choline pathway and the methylation pathway. We also review recent studies on phosphatidylethanolamine biosynthesis by two pathways: the CDP-ethanolamine pathway, which is active in the endoplasmic reticulum, and the phosphatidylserine decarboxylase pathway, which operates in mitochondria. Finally, the two base-exchange enzymes, phosphatidylserine synthase-1 and phosphatidylserine synthase-2, that synthesize phosphatidylserine in mammalian cells are also discussed.

Cerebrocortical plasma membrane serine base exchange enzyme activity in hypoxia

Phosphatidylserine (PtdSer) is involved in signal transduction and in apoptosis. Peculiarities of its metabolism (1) are relevant for this study. N2 treatment stimulates the incorporation of [14C]serine (Ser) into PtdSer of cerebrocortical slices and in cerebrocortical plasma membranes (PM) serine base exchange enzyme (SBEE) activity is lowered by G protein activator. PtdSer radioactivity of PM from slices incubated with [14C]Ser was greater in hypoxia than in controls. SBEE activity of PM from slices, incubated in a similar manner and assayed at pH 8.2 and pH 7.4 based on a preliminary study, was lower in hypoxia. The hypoxia induced incorporation of [14C]Ser into slice PtdSer was due to stimulation of the SBEE isoform that exchanges Ser and ethanolamine and that in brain membrane is activated by fatty acids. We suggest the presence of different SBEE isoforms in PM differently influenced by hypoxia.

Metabolism and functions of phosphatidylserine in mammalian brain.

Phosphatidylserine (PtdSer) is involved in cell signaling and apoptosis. The mechanisms regulating its synthesis and degradation are still not defined. Thus, its role in these processes cannot be clearly established at molecular level. In higher eukaryotes, PtdSer is synthesized from phosphatidylethanolamine or phosphatidylcholine through the exchange of the nitrogen base with free serine. PtdSer concentration in the nervous tissue membranes varies with age, brain areas, cells, and subcellular components. At least two serine base exchange enzymes isoforms are present in brain, and their biochemical properties and regulation are still largely unknown because their activities vary with cell type and/or subcellular fraction, developmental stage, and differentiation. These peculiarities may explain the apparent contrasting reports. PtdSer cellular levels also depend on its decarboxylation to phosphatidylethanolamine and conversion to lysoPtdSer by phospholipases. Several aspects of brain PtdSer metabolism and functions seem related to the high polyunsaturated fatty acids content, particularly docosahexaenoic acid (DHA).

Serine base-exchange in rat liver nuclei

It has been shown that the incorporation of [ 14C]serine into phosphatidylserine (PS) in isolated rat liver nuclei is intrinsic to this organelle as attested by marker enzyme activity. Serine incorporation into PS was the highest in nuclei depleted of the outer membrane of the nuclear envelope (nucleoplasts) and negligible in the outer membrane. Trypsin treatment of nucleoplasts caused a strong inactivation of PS synthesis and only a moderate one of the NAD pyrophosphorylase activity, the marker enzyme of the inner nuclear membrane. We suggest that the serine base-exchange enzyme is located in the inner membrane of the nuclear envelope and accessible from the periplasmic surface of this membrane.

Alternative pathways for phospholipid synthesis in different brain areas during aging.

Morphological and biochemical changes take place in the membrane of aged brain. In particular, studies on aged rats report alterations in brain phospholipid synthesis and in phospholipid-specific fatty acid composition. However, no significant changes in main phospholipid class content have been reported in aged brain, possibly owing to alterations in the alternative pathways for phospholipid synthesis during aging. Therefore, the present study was designed to determine the effect of aging on the enzyme activities responsible for phospholipid synthesis by alternative pathways. Indifferent brain areas of adult (3.5-month-old) and aged (28.5-month-old) rats we examined: 1) the activity of base exchange enzymes, which is a calcium-dependent, energy-independent and calcium stimulated enzymatic pathway; 2) phosphatidylethanolamine (PE) synthesis by phosphatidylserine decarboxylase activity (PSD); 3) phosphatidylcholine (PC) synthesis by transfer of methyl groups to endogenous PE by phosphatidylethanolamine N-methyltransferase activity (PEMT); 4) the synthesis of phosphatidylglycerol (PG) through phospholipase D (PLD) activity. Because the dependence on and the stimulation by calcium of base-exchange reactions is a well known mechanism and alterations in calcium levels in rat brain have been reported, we decided to investigate PS synthesis in the presence of endogenous and exogenous calcium (2.5mM). PS synthesis increased in cerebral cortex (CC) and cerebellum (CRBL) of aged rats with respect to adult rats in basal conditions (without the addition of exogenous calcium), but more significant changes were observed in serine base exchange activity during aging when exogenous calcium was added. PEMT activity in aged CC increased by 100%, the principal modification being observed in the first methylated product of the sequential reaction. Furthermore, the transphosphatidyl reaction was higher in aged brain as indicated by the increased PG synthesis. Our findings allow us to conclude that age affects some alternative pathways for phospholipid synthesis in the central nervous system, and indicate the presence of a compensatory mechanism to provide a pool of phospholipid classes for the maintenance of cellular membrane lipid composition during aging.

Regulation of phosphatidylserine exposure at the cell surface by the serine–base exchange enzyme system during CD95-induced apoptosis

Early in the apoptotic process, CD95 induces a translocation of phosphatidylserine (PtdSer) from the inner to the outer leaflet of the cellular plasma membrane. In mammalian cells, PtdSer is only synthesized through a calcium-dependent exchange of the polar head group of pre-existing phospholipids, either phosphatidylcholine or phosphatidylethanolamine, by a serine. Using a pharmacological approach, we examined the influence of PtdSer synthesis on CD95-induced PtdSer exposure at the surface of Jurkat cells. We found that CD3/TCR triggering or thapsigargin treatment of Jurkat cells was accompanied both by a decreased PtdSer synthesis and by a strong reduction of CD95-induced PtdSer at the cell surface, as monitored by fluorescence-activated cell sorting (FACS) analysis of annexin V–fluorescein isothiocyanate (FITC)-labeled cells. PtdSer synthesis through the serine–base exchange enzyme system thus appeared as one of the mechanisms implicated in the recently discovered CD3/TCR-induced down-regulation of CD95-induced apoptosis. Conversely, increasing the activity of the serine–base exchange enzyme system with different drugs, either the K + channel blocker quinine, the cationic amphiphil stearylamine, or three different calmodulin antagonists, chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), resulted in an increased appearance of PtdSer at the surface of CD95-treated cells. Both PtdSer synthesis and CD95-induced annexin V–FITC reactivity were abrogated in ATP-depleted cells. Also, modifying the membrane potential with valinomycin (hyperpolarization) or either gramicidin or KCl (depolarization) demonstrated a strong relationship between PtdSer synthesis and annexin V–FITC reactivity in CD95-treated cells. Together, our results indicate that CD95-induced exposure of PtdSer at the cell surface could be regulated by the activity of the serine–base exchange enzyme system.

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AlF4- and different types of heparin.

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca2+ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60 degrees C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45 degrees C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.

Dexamethasone increases the incorporation of [3H]serine into phosphatidylserine and the activity of serine base exchange enzyme in mouse thymocytes: a possible relation between serine base exchange enzyme and apoptosis.

The exposure of phosphatidylserine toward the external surface of the membrane is a well-established event of programmed cell death. The possibility that an apoptotic stimulus influences the metabolism of this phospholipid could be relevant not only in relation to the previously mentioned event but also in relation to the capability of membrane phosphatidylserine to influence PKC activity. The present investigation demonstrates that treatment of mouse thymocytes with the apoptotic stimulus dexamethasone, enhances the incorporation of [3H]serine into phosphatidylserine. Cell treatment with dexamethasone also enhanced the activity of serine base exchange enzyme, assayed in thymocyte lysate. Both the effects were observed at periods of treatment preceding DNA fragmentation. The addition of unlabelled ethanolamine, together with [3H]serine to the medium containing dexamethasone-treated thymocytes lowered the radioactivity into phosphatidylserine. Serine base exchange enzyme activity was influenced by the procedure used to prepare thymocyte lysate and was lowered by the addition of fluoroaluminate, that is widely used as a G-protein activator. The increase of serine base exchange enzyme activity induced by dexamethasone treatment was observed independently by the procedure used to prepare cell lysate and by the presence or absence of fluoroaluminate.

Differential incorporation of precursor moieties into cerebral cortex and cerebellum glycerophospholipids during aging.

The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [3H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [3H]choline was inhibited. However, the incorporation of [14C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([3H]palmitic) and polyunsaturated ([3H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [3H]palmitic, [3H]oleic and [3H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 month old) and adult (3.5 month old) rats. [3H]oleic acid incorporation into neutral lipids (NL) and [3H]arachidonic acid incorporation into PC, PE and phosphatidylinositol (PI) were increased in aged rats with respect to adult rats. Present results show the ability and avidity of aged brain tissue in vitro to incorporate unsaturated fatty acids when they are supplied exogenously. They also suggest a different handling of choline and serine by base exchange enzyme activities to synthesize PC and PS during aging.

Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2

Phosphatidylserine (PtdSer) is synthesized in mammalian cells by two base-exchange enzymes: PtdSer synthase (PSS)-1 primarily uses phosphatidylcholine as a substrate for exchange with serine, whereas PSS2 uses phosphatidylethanolamine (PtdEtn). We previously expressed murine PSS1 in McArdle hepatoma cells. The activity of PSS1 in vitro and the synthesis of PtdSer and PtdSer-derived PtdEtn were increased, whereas PtdEtn synthesis from the CDP-ethanolamine pathway was inhibited [Stone, Cui and Vance (1998) J. Biol. Chem. 273, 7293-7302]. We have now cloned and stably expressed a murine PSS2 cDNA in McArdle cells and M.9.1.1 cells [which are ethanolamine-requiring mutant Chinese hamster ovary (CHO) cells defective in PSS1]. Expression of the PSS2 in M.9.1.1 cells reversed the ethanolamine auxotrophy. However, the PtdEtn content was not normalized unless the culture medium was supplemented with ethanolamine. In both M.9.1.1 and hepatoma cells transfected with PSS2 cDNA the rate of synthesis of PtdSer and PtdSer-derived PtdEtn did not exceed that in parental CHO cells or control McArdle cells respectively, in contrast to cells expressing similar levels of murine PSS1. These observations suggest that PtdSer synthesis via murine PSS2, but not PSS1, is regulated by end-product inhibition. Moreover, expression of murine PSS2 in McArdle cells did not inhibit PtdEtn synthesis via the CDP-ethanolamine pathway, whereas expression of similar levels of PSS1 activity inhibited this pathway by approx. 50%. We conclude that murine PSS1 and PSS2, which are apparently derived from different genes, independently modulate phospholipid metabolism. In addition, mRNAs encoding the two synthases are differentially expressed in several murine tissues, supporting the idea that PSS1 and PSS2 might perform unique functions.

Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2

Phosphatidylserine (PtdSer) is synthesized in mammalian cells by two base-exchange enzymes: PtdSer synthase (PSS)-1 primarily uses phosphatidylcholine as a substrate for exchange with serine, whereas PSS2 uses phosphatidylethanolamine (PtdEtn). We previously expressed murine PSS1 in McArdle hepatoma cells. The activity of PSS1 in vitro and the synthesis of PtdSer and PtdSer-derived PtdEtn were increased, whereas PtdEtn synthesis from the CDP-ethanolamine pathway was inhibited [Stone, Cui and Vance (1998) J. Biol. Chem. 273, 7293-7302]. We have now cloned and stably expressed a murine PSS2 cDNA in McArdle cells and M.9.1.1 cells [which are ethanolamine-requiring mutant Chinese hamster ovary (CHO) cells defective in PSS1]. Expression of the PSS2 in M.9.1.1 cells reversed the ethanolamine auxotrophy. However, the PtdEtn content was not normalized unless the culture medium was supplemented with ethanolamine. In both M.9.1.1 and hepatoma cells transfected with PSS2 cDNA the rate of synthesis of PtdSer and PtdSer-derived PtdEtn did not exceed that in parental CHO cells or control McArdle cells respectively, in contrast to cells expressing similar levels of murine PSS1. These observations suggest that PtdSer synthesis via murine PSS2, but not PSS1, is regulated by end-product inhibition. Moreover, expression of murine PSS2 in McArdle cells did not inhibit PtdEtn synthesis via the CDP-ethanolamine pathway, whereas expression of similar levels of PSS1 activity inhibited this pathway by approx. 50%. We conclude that murine PSS1 and PSS2, which are apparently derived from different genes, independently modulate phospholipid metabolism. In addition, mRNAs encoding the two synthases are differentially expressed in several murine tissues, supporting the idea that PSS1 and PSS2 might perform unique functions.

Positive feedback between ethanolamine-specific phospholipid base exchange and cytochrome P450 activities in rat liver microsomes. The effect of clofibric acid

The results of the present investigation relate the effects of the nutritional state and administration of clofibric acid (CLA), a hypolipidaemic drug and peroxisomal proliferator, on phosphatidylethanolamine (PE) synthesis in rat liver and fatty acid metabolism. Fasting and CLA treatment of animals causes an increase in the amount of PE in endoplasmic reticulum (ER) membranes and mitochondria, as well as in the PE/phosphatidylcholine (PC) ratio. Moreover, the activity of the ethanolamine-specific phospholipid base exchange (PLBE) enzyme in liver ER membranes of fasted animals was enhanced by 75% in comparison to that of animals fed ad libitum. The effect of CLA treatment was additive to that of starvation; PE synthesis tested in vitro via the Ca 2+-sensitive PLBE reaction increased 3-fold in comparison to rats fed ad libitum. This is confirmed by an increased V max for the reaction, but the affinity of the enzyme for ethanolamine was not significantly changed. These effects were accompanied by an enhanced expression of cytochrome P450 CYP4A1 isoform and elevated activity of the enzyme upon CLA administration. The stimulatory effect of CLA administration on the efficiency of the ethanolamine-specific PLBE reaction can be explained by elimination of lauric acid, a known inhibitor of de novo PE synthesis, during the course of ω-hydroxylation catalysed by CYP4A1, and by increased expression of the PLBE enzyme. The products of ω-hydroxylation of lauric acid, which are then converted by dehydrogenase to 1,12-dodecanedioic acid, did not significantly affect the in vitro synthesis of PE.

Occurrence of an Unusual Phospholipid, Phosphatidyl-l-threonine, in Cultured Hippocampal Neurons: EXOGENOUS l-SERINE IS REQUIRED FOR THE SYNTHESIS OF NEURONAL PHOSPHATIDYL-l-SERINE AND SPHINGOLIPIDS

We have recently reported that L-serine released from astroglial cells supports the survival and neuritogenesis of hippocampal neurons under a serum- and glia-free culture condition (Mitoma, J., Furuya, S., and Hirabayashi, Y. (1998) Neurosci. Res. 30, 195-199). In this study, we show that exogenous L-serine is required for the synthesis of phosphatidyl-L-serine (PS) and sphingolipids in hippocampal neurons. When hippocampal neurons were maintained under an astroglial cell-free condition, the levels of sphingolipids and phosphatidyl-L-serine in the neurons were greatly reduced in the absence of external L-serine or glycine. Instead, a novel phospholipid appeared just ahead of PS on TLC. This novel lipid was determined to be phosphatidyl-L-threonine by TLC blotting/negative secondary ion mass spectrometry and amino acid analysis. Biochemical studies on rat brain microsomes have indicated that phosphatidyl-L-threonine is synthesized by the base exchange enzyme that is involved in PS synthesis with much lower affinity, that is, approximately (1)/(150) of L-serine. Addition of L-serine or glycine to the culture medium restored the synthesis of PS and sphingolipids in the neurons. These observations show that hippocampal neurons require exogenous L-serine for the synthesis of PS and sphingolipids in the absence of astroglial cells and suggested that astroglial cells contribute to neuronal lipid synthesis through the supply of L-serine.

Serine Base Exchange Enzyme Activity Is Modulated by Sphingosine and Other Amphiphilic Compounds: Possible Role of Positive Charge in Increasing the Synthesis of Phosphatidylserine

It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) have a stimulatory, and cholesterol 3-sulfate (an amphiphilic anion), an inhibitory, effect on [14C]serine incorporation into phosphatidylserine in glioma C6 and rat liver microsomes. In glioma intact cells sphingosine stimulates phosphatidylserine synthesis in a process independent of protein kinase C, but suppressed by thapsigargin. We suggest that the stimulation of the enzyme occurs by the interaction of amphiphilic cations with the membrane cosubstrate phospholipids, leading to a charge redistribution on their phosphate groups, and hence facilitating the enzyme action. A new hypothesis concerning the mechanism of the serine base exchange reaction is discussed.