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J. Biol. Chem., Vol. 277, Issue 29, 25843-25846, July 19, 2002

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MINIREVIEW
De Novo Sphingolipid Biosynthesis: A Necessary, but Dangerous, Pathway^*

Alfred H. Merrill Jr.

From the School of Biology, Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332-0230

	INTRODUCTION

TOP INTRODUCTION Structural Diversity of... De Novo Sphingolipid... Implication of de Novo... Perspectives REFERENCES

Sphingolipids form specialized structures, mediate cell-cell and cell-substratum interactions, modulate the behavior of cellular proteins and receptors, and participate in signal transduction. They are synthesized de novo via a common backbone (sphinganine) that is modified to produce ceramides and more complex phospho- and glycosphingolipids. This minireview summarizes sphingoid base metabolism, function, and perturbation, including the participation of de novo sphingolipid biosynthesis in disease; other minireviews in this series will focus on ceramides (1), sphingosine 1-phosphate (2), complex sphingolipids (3), and sphingolipid trafficking (4).

	Structural Diversity of Sphingoid Bases

TOP INTRODUCTION Structural Diversity of... De Novo Sphingolipid... Implication of de Novo... Perspectives REFERENCES

Sphingoid bases¹ are compounds with structural similarity to sphingosine from the root name ("sphingosin") assigned to this family of alkaloidal lipids by Thudichum (5). They encompass a wide array of 2-amino-1,3-dihydroxyalkanes or -enes with (2S,3R)-erythro stereochemistry, alkyl chain lengths from 14 to 22 carbon atoms, 0 to 2 double bonds, and other modifications, such as hydroxyl group(s) at positions 4 or 6 and branching methyl groups at -l (iso), -2 (anti-iso), or elsewhere (6, 7). Mammals produce mainly the species shown in Fig. 1 plus small amounts of other chain length homologs; yeast have 18- and 20-carbon phytosphingosines and sphinganines (sphingoid bases with double bonds and hydroxyl and/or methyl groups are common in other fungi); and plants have unsaturated bases such as sphing-8-enines, sphing-4,8-dienes, and phytosphing-(8 or 9)-enines.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   De novo biosynthetic pathway for sphingoid bases and complex sphingolipids. The color coding distinguishes the biosynthetic enzymes (with common names in red and green arrows for the reactions catalyzed) and intermediates (in blue) from additional reactions that occur with these intermediates (in black). The dashed line for (N-acyl)phytosphingosine synthesis reflects that in yeast, where this has been best characterized, hydroxylation may occur with both free sphinganine and dihydroceramide.

Some organisms (such as fungi and sponges) produce compounds that are sphingoid base-like, examples of which are shown in Fig. 2. As will be discussed later, at least some of these disrupt sphingolipid metabolism.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Examples of naturally occurring inhibitors for two key enzymes of sphingolipid biosynthesis as well as other sphingoid base-like compounds. More information on these inhibitors is given in the text. Calyxoside (65) and BRS1 (66) were both isolated as bioactive compounds from sponges.

	De Novo Sphingolipid Biosynthesis

TOP INTRODUCTION Structural Diversity of... De Novo Sphingolipid... Implication of de Novo... Perspectives REFERENCES

The capacity for de novo sphingolipid biosynthesis (Fig. 1) is widespread among cell types and tissues. In the absence of an exogenous sphingoid base source, loss of this pathway by mutation of serine palmitoyltransferase (SPT)² (8, 9) or its inhibition by ISP1/myriocin or sphingofungin B (10) affects growth and viability. De novo sphingolipid biosynthesis is probably required for survival in vivo because, although sphingolipids are present in most foods, the sphingoid bases are largely degraded in the mammalian intestine (11).

It is intriguing that this pathway contains so many compounds that affect cell behavior when added exogenously or formed via sphingolipid turnover and that the consequences can be growth arrest and cytotoxicity (ceramide and sphingosine) or growth stimulation or inhibition of apoptosis (sphingosine 1-phosphate) (1, 2). With so many bioactive intermediates, essentially all of the enzymes of sphingolipid metabolism must be efficiently coordinated, with three warranting particular attention: serine palmitoyltransferase, which catalyzes the initial step of the pathway; (dihydro)ceramide synthase, which removes sphingoid bases as well as produces dihydroceramide (or ceramide, if sphingosine is available from sphingolipid turnover or an exogenous source); and dihydroceramide desaturase, which converts relatively inactive dihydroceramides to ceramides.

Serine Palmitoyltransferase-- For mammals and yeast, two gene products (termed SPTLC1 and SPTLC2, or sometimes SPT1 and SPT2) are necessary for this activity (12) and appear to be physically associated (13). A third has been identified in yeast, but there does not appear to be a mammalian homolog (14). The amino acid sequence of SPT2 has homology to other pyridoxal 5'-phosphate-dependent decarboxylases, with Lys³⁷⁷ predicted to be the site of the Schiff base with this cofactor (15). SPT2 may be primarily responsible for catalytic activity because SPT1 lacks this Lys (16); nonetheless, mutations in SPT1 (SPTLC1) cause hereditary sensory neuropathy type I (HSN1), the most common hereditary disorder of peripheral sensory neurons (17, 18). An intrinsic membrane protein, SPT is difficult to study; however, Sphingomonas has a soluble, homodimeric SPT (19).

The regulation of SPT is only beginning to be understood. One of the more straightforward factors that affects SPT activity is the availability of both serine- and palmitoyl-CoA, and because SPT is highly selective for fatty acyl-CoA with 16 ± 1 carbon atoms, other fatty acids can be inhibitory in vivo, possibly by competing for the CoA pool (20). Serine palmitoyltransferase is inhibited by a number of synthetic and naturally occurring agents. As for many pyridoxal 5'-phosphate-dependent enzymes, it undergoes active site-dependent ("suicide") inhibition with -haloalanines and other aldehyde reactive compounds (21, 22). More potent and selective inhibitors have been isolated from microorganisms (sphingofungins, lipoxamycins, and ISP1/myriocin) (Fig. 2) (23, 24). These inhibitors (and particularly ISP1, which is available commercially) have been valuable in identifying the roles of de novo biosynthesis in sphingolipid-mediated cell death (25); however, care must be exerted in using the less specific inhibitors (10). D-Serine inhibits SPT, which may have significance in brain tissue, where this stereoisomer is found (26).

Sphingoid base synthesis can be suppressed by adding lipoproteins or free sphingoid bases to cells in culture (reviewed in Ref. 11) perhaps by down-regulation of SPT by sphingoid base 1-phosphates (27). Regulation at a transcriptional level has been seen with a number of agents, including endotoxin and cytokines (28), UVB irradiation (29), retinoic acid (30), and other agents (31). Induction of both SPT1 and SPT2 occurs in balloon-injured rat carotid artery (32). Activation of SPT occurs post-translationally in response to etoposide (33) and heat shock in yeast (34). The heat shock response in yeast involves mainly eicosasphinganines (i.e. C₂₀ sphingoid bases) (35) and induces changes in amino acid transport (36) and activation of ubiquitin-dependent proteolysis (37).

(Dihydro)ceramide Synthase-- The reduction of 3-ketosphinganine and acylation of sphinganine to dihydroceramide (Fig. 1) both appear rapid in vivo due to lack of accumulation of the intermediates under usual conditions. (Dihydro)ceramide synthase(s) utilize a range of fatty acyl-CoAs (C16:0 to C26:0) and probably represent a family of isozymes. The recent identification of yeast genes essential for acyl-CoA-dependent ceramide synthesis (38) should lead to isolation of the counterpart(s) in other organisms. Ceramides can also be synthesized by the reverse reaction of ceramidase(s) (39, 40), and in yeast this route has allowed cloning of an alkaline ceramidase based on resistance to fumonisin B₁ (40).

(Dihydro)ceramide synthesis is the target of a number of fungal inhibitors (11) such as fumonisin B₁ (FB₁) (Fig. 2). Structure-function investigations suggest that the aminoalkyl backbone competes with the sphingoid base binding site of (dihydro)ceramide synthase, and the anionic tricarballylic side chains interfere with utilization of the co-substrate fatty acyl-CoA; thus, compounds with the aminopentol backbone alone (AP₁ in Fig. 2) are both substrates and inhibitors (41).

Dihydroceramide Desaturase-- The last step of ceramide synthesis is insertion of a 4,5-trans-double bond into dihydroceramide as shown in Fig. 1 (42). This is an important reaction because ceramides (but much less so dihydroceramides) are active in inducing apoptosis (1). This reaction can be reproduced in vitro using either dihydroceramide or dihydrosphingomyelin (42, 43). Sphingolipid desaturases have been cloned from plants (44), leading to the recent identification of the 4-desaturase genes of Homo sapiens, Mus musculus, Drosophila melanogaster, and Candida albicans and a bifunctional 4-desaturase/C-4-hydroxylase from M. musculus (45). The deuterium isotope effect for C-H bond cleavage suggests that the desaturase initially oxidizes C-4 (46), which is consistent with the finding that one of the desaturases catalyzes both ceramide and phytoceramide synthesis (45). A cyclopropene analog of ceramide potently inhibits the desaturase (47). Genes responsible for phytosphingosine synthesis have been identified in yeast (48, 49), plants (50), and M. musculus (45). In vitro assays suggest that hydroxylation can occur with both the free sphingoid base and dihydroceramide, at least in yeast (49) (dashed arrow in Fig. 1).

Other Reactions-- The enzymes that remove ceramide (ceramidases and synthases for complex sphingolipids), the sphingoid base kinases (as well as the phosphatases that reverse this reaction and the lyase that cleaves sphingoid base 1-phosphates to a fatty aldehyde and ethanolamine phosphate) (Fig. 1) will be discussed in the accompanying minireviews (1-4).

	Implication of de Novo Sphingolipid Biosynthesis in Cell Death

TOP INTRODUCTION Structural Diversity of... De Novo Sphingolipid... Implication of de Novo... Perspectives REFERENCES

Alteration of de novo sphingolipid biosynthesis can be toxic, as was first shown for the fumonisins (51). Fumonisins are mycotoxin contaminants of maize that cause a spectrum of disease: cancer (rats and humans), leukoencephalomalacia (equines), pulmonary edema (pigs), liver and kidney toxicity (multiple species), and other disease (52). By inhibiting (dihydro)ceramide synthase, fumonisins cause the accumulation of sphinganine (Fig. 3) in tissues, serum, and urine, which is widely used as a biomarker of fumonisin exposure (51). The accumulation of sphinganine appears to be responsible for most of the deleterious effects of these mycotoxins, although depletion of complex sphingolipids impairs the function of some membrane proteins, such as the folate transporter (53), and may contribute to neural tube disease (67).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   A scheme that depicts the bioactive intermediates of de novo sphingolipid biosynthesis and some factors that influence their amounts and fates. Shown are some of the intermediates from Fig. 1: the incorporation of palmitoyl-CoA (Pal-CoA) into 3-ketosphinganine (KetoSa), which is converted to sphinganine (Sa), dihydro- (DH) ceramide (Cer), or sphinganine 1-phosphate (Sa-1-P). In hepatocytes, ceramide is not only incorporated into more complex sphingolipids but also into nascent very low density lipoproteins (VLDL), which are secreted. The sites of action of commonly used inhibitors (ISP1/myriocin and fumonisin) are also shown. The subcellular locations of these reactions are indicated only for a general context; there are likely to be other sites where some of these reactions occur (for example, ceramide formation is thought to occur in the endoplasmic reticulum (ER), but in unpublished studies we have recently found these activities in mitochondrial-associated membranes (MAM)). GSL, glycosphingolipid; SM, sphingomyelin.

Fumonisins can significantly elevate sphinganine 1-phosphate (54) and production of ethanolamine phosphate (55) (Figs. 1 and 3). Because sphingoid base 1-phosphates are mitogenic and anti-apoptotic (2), this may account for (or at least contribute to) the seemingly paradoxical stimulation of growth by fumonisins in some cells (56) and the oft cited "protection of cells from apoptosis because of de novo synthesized ceramide"³ in some cases where fumonisins are used. This cautionary statement notwithstanding, it is clear that de novo sphingolipid biosynthesis participates in cell death induced by a wide variety of agents. First noted by Kolesnick and collaborators (57) in studies of daunorubicin-induced apoptosis, activation of (dihydro)ceramide synthase may also be involved in some aspects of the toxicities of phorbol esters and radiation (58, 59), angiotensin II and cannabinoids (60, 61), and elevations in palmitoyl-CoA because of excess production or impaired removal by mitochondrial oxidation or other metabolism (62) (Fig. 3). This has implications for a wide range of disorders and has been articulated as one of the mechanisms for "lipotoxic" disease (63).

	Perspectives

TOP INTRODUCTION Structural Diversity of... De Novo Sphingolipid... Implication of de Novo... Perspectives REFERENCES

Why would nature risk producing compounds with such a diverse spectrum of biological effects and toxicities unless these compounds have functions beyond just being pathway intermediates? One suspects that both de novo sphingolipid biosynthesis and turnover are used for cell regulation. Thus, pathologies arise from malfunctions in these pathways, and (as often occurs in nature) organisms exploit them for their own purposes, as in the case with fumonisins, which allow the fungus to kill its host.

Some of the advantages of forming highly bioactive compounds via both complex sphingolipid turnover and de novo biosynthesis are as follows. 1) The amounts of the backbones (sphingoid bases and ceramides) can be raised to higher levels than by sphingolipid turnover alone because palmitoyl-CoA and serine are plentiful; 2) the bioactive sphingolipid backbone(s) can be formed with minimal perturbation of the cellular status/utilization of complex sphingolipids; 3) the bioactive compounds may be targeted more directly to the intracellular membranes where they are needed; 4) the formation and removal of these species could be integrated with other cell states, such as whether or not the mitochondria are active and utilizing palmitoyl-CoA (Fig. 3); and 5) the molecular subspecies (such as the type of ceramide) can be modified to activate/inhibit downstream targets more selectively.

It should be evident that understanding de novo sphingolipid biosynthesis and turnover under normal and abnormal conditions necessitates examination of all of these bioactive species as well as when and where they are made and removed (64). This is literally an "-omic" field ("sphingolipidomics"), as foreshadowed by Thudichum (5) in declaring that lipids are "the center, life, and chemical soul of all bioplasm whatsoever, that of plants as well as animals."

	ACKNOWLEDGEMENTS

I thank colleagues in my laboratory and others who have contributed findings that cannot be cited because of space limitations.

	FOOTNOTES

^* This minireview will be reprinted in the 2002 Minireview Compendium, which will be available in December, 2002. Pertinent findings from this laboratory were supported by National Institutes of Health Grants GM46368 and ES09204. This is the first article of five in the "Sphingolipid Metabolism and Signaling Minireview Series."

To whom correspondence should be addressed. Tel.: 404-385-2842; Fax: 404-385-2917 or 404-894-0519; E-mail: al.merrill@biology.gatech.edu.

Published, JBC Papers in Press, May 13, 2002, DOI 10.1074/jbc.R200009200

¹ Names in use for common sphingoid bases with those recommended by the IUPAC (7) shown in brackets are: sphingosine [(2S,3R,4E)- 2-aminooctadec-4-ene-1,3-diol and (E)-sphing-4-enine]; dihydrosphingosine [(2S,3R)-2-aminooctadecane-1,3-diol and sphinganine]; phytosphingosine and 4-hydroxysphinganine [(2S,3S,4R)-2-aminooctadecane-1,3,4-triol]. When alkyl chain length is not specified, it is assumed to be 18-carbon atoms; other lengths can be designated by a name or number prefix (such as icosasphingosine or C₂₀-sphingosine for a sphingosine with 20 carbon atoms).

³ As an inhibitor of the first step of this pathway, ISP1 (myriocin) can be used to block ceramide synthesis without elevating sphinganine and other intermediates (30).

	ABBREVIATIONS

The abbreviation used is: SPT, serine palmitoyltransferase.

	REFERENCES

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