Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism

Biochimica et Biophysica Acta 1632 (2003) 16 – 30 www.bba-direct.com

Review

Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism Kentaro Hanada * Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku, Tokyo 162-8640, Japan Received 7 January 2003; received in revised form 19 March 2003; accepted 1 April 2003

Abstract The first step in the biosynthesis of sphingolipids is the condensation of serine and palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT) to produce 3-ketodihydrosphingosine (KDS). This review focuses on recent advances in the biochemistry and molecular biology of SPT. SPT belongs to a family of pyridoxal 5V-phosphate (PLP)-dependent a-oxoamine synthases (POAS). Mammalian SPT is a heterodimer of 53-kDa LCB1 and 63-kDa LCB2 subunits, both of which are bound to the endoplasmic reticulum (ER) most likely with the type I topology, whereas other members of the POAS family are soluble homodimer enzymes. LCB2 appears to be unstable unless it is associated with LCB1. Potent inhibitors of SPT structurally resemble an intermediate in a probable multistep reaction mechanism for SPT. Although SPT is a housekeeping enzyme, its activity is regulated transcriptionally and post-transcriptionally, and its up-regulation is suggested to play a role in apoptosis induced by certain types of stress. Specific missense mutations in the human LCB1 gene cause hereditary sensory neuropathy type I, an autosomal dominantly inherited disease, and these mutations confer dominant-negative effects on SPT activity. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Serine palmitoyltransferase; Sphingolipid; Sphingosine; Ceramide; Pyridoxal phosphate; Hereditary sensory neuropathy

1. Introduction Sphingolipids are defined as lipids containing sphingoid bases (1,3-dihydroxy-2-amino-alkane and its derivatives) as a structural backbone. Sphingolipids are ubiquitous constituents of membrane lipids in eukaryotes, and are also distributed to some prokaryotes. Since Johann L.W. Thudichum first described the existence of biological compounds containing a previously unrecognized aliphatic alkaloid called ‘‘sphingosine’’ in the brain more than 100 years ago, studies on the structure, distribution, and metabolism of sphingolipids have advanced greatly [1,2]. In the past decade, sphingolipid metabolites have been revealed to modulate various cellular events including proliferation, differentiation, and apoptosis [3 – 5], and sphingolipids, along with cholesterol, have been shown to be required for the formation of detergent-resistant membrane microdomains [6 –8], which are implicated in signal transduction and membrane trafficking [9]. In addition, crucial roles for cutaneous ceramides in the skin barrier function have been recognized [10]. Accordingly, the molecular mechanisms

* Tel.: +81-3-5285-1111x2126; fax: +81-3-5285-1157. E-mail address: [email protected] (K. Hanada).

underlying sphingolipid metabolism have attracted much attention. The first step involved in sphingolipid biosynthesis is the condensation of serine and palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT) [EC 2.3.1.50] to produce 3-ketodihydrosphingosine (KDS). SPT is suggested to be a key enzyme for the regulation of sphingolipid levels in cells because regulation of sphingolipid synthesis at the SPT step prevents a harmful accumulation of metabolic sphingolipid-intermediates including sphingoid bases and ceramide, while repression of other anabolic steps in the sphingolipid synthetic pathway may cause the intermediates to accumulate. This review summarizes recent advances in the biochemistry and molecular biology of SPT, mainly focusing on the mammalian SPT enzyme.

2. Structure and biosynthesis of sphingolipids There are three major types of sphingoid bases (Fig. 1). Sphingosine is the principal sphingoid base of sphingolipids in mammalian cells, and dihydrosphingosine is the second most abundant type. Phytosphingosine (4-hydroxydihydrosphingosine) is the principal sphingoid base in plants and

1388-1981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1388-1981(03)00059-3

K. Hanada / Biochimica et Biophysica Acta 1632 (2003) 16–30

Fig. 1. Structure of sphingoid bases. Systematic names of the major natural sphingoid bases are also indicated in parentheses.

fungi, although some tissues including the kidney and stomach in mammals also have considerable amounts of phytosphingosine-containing sphingolipids. For natural sphingoid bases, the alkyl chain length is predominantly 18, and the configuration of the chirality at the carbon atom (C)-2 and C-3 positions is D-erythro (2S, 3R) (Fig. 1) [11]. Polar head groups of sphingolipids show marked structural diversity among phylogenetically separate or-

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ganisms, whereas polar heads of glycerophospholipids are structurally conserved from fungi to human. In mammalian cells, choline phosphoceramide (sphingomyelin) accounts for 5– 10% of membrane phospholipids, while various lower animal cells have ethanolamine phosphoceramide and its N-mono or dimethyl derivatives in place of sphingomyelin [12], and fungal and plant cells produce phosphoinositol-containing sphingolipids [13]. For glycosphingolipids, numerous variety in the glycosyl head structure exists even in the same organisms. Moreover, the Gram-negative bacterium Sphingomonas is known to have sphingoid base-containing components in the envelope [14]. Despite such diversity, SPT is commonly responsible for the initial step of synthesis in all organisms producing sphingolipids. As shown in Fig. 2, KDS generated by the SPT reaction is reduced to form dihydrosphingosine. In mammalian cells, dihydrosphingosine is N-acylated to form dihydroceramide, which is then desaturated to form ceramide [15]. Ceramide is converted to sphingomyelin or various glycosphingolipids. In plant and fungal cells, dihydrosphingosine is hydroxylated to generate phytosphingosine, which is Nacylated to produce hydroxyceramide (phytoceramide) [13]. The prevalent acyl chain of fungal phytoceramide is C26 hydroxy fatty acid, whereas typical acyl chains of mammalian ceramide are saturated C16 and C24 fatty acids. In fungal cells, phytoceramide is converted to inositol phosphophytoceramide, which is absent in mammalian cells [13].

Fig. 2. Biosynthetic pathway of sphingolipids in mammalian and fungal cells.

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3. Structure and function of SPT 3.1. Early history of SPT In the early 1950s, chemical analysis of metabolically labeled sphingolipids suggested that C-3 to C-18 of sphingosine were derived from palmitic acid [16], but that C-1 and C-2 were from serine [17]. Based on these results, Sprinson and Coulon [17] hypothesized that the condensation of acyl CoA and serine produced a 3-keto derivative, and Weiss [18] later proposed a Schiff’s base-dependent mechanism for the formation of a 3-keto derivative from serine and palmitoyl CoA. At that time, the first product in the sphingolipid synthesis pathway was believed to be sphingosine. But it was found that dihydrosphingosine formation precedes sphingosine formation in intact cells, and that palmitoyl CoA serves as an efficient precursor for dihydrosphingosine in vitro in the presence of serine and NADPH [19]. In 1968, Braun and Snell [20] and Stoffel et al. [21] demonstrated that incubation of the particulate fraction of the yeast Hansenula ciferri with serine and palmitoyl CoA (in the absence of NADPH) produces KDS, and that KDS is rapidly reduced to dihydrosphingosine in the presence of NADPH. Based on these and other studies [2], the initial step in sphingolipid biosynthesis was eventually revealed to be the formation of KDS, which is catalyzed by the SPT enzyme. Conclusive evidence that SPT activity detected in cell-free systems is actually responsible for the main pathway of sphingolipid synthesis in intact cells was obtained by genetic studies as described below. 3.2. SPT is responsible for de novo synthesis of sphingolipids Wells and Lester [22] and Pinto et al. [23] isolated mutant strains of the yeast Saccharomyces cerevisiae that require an external supply of phytosphingosine for growth and for synthesis of complex sphingolipids, and showed that the mutant strains are defective in SPT activity [24]. For mammalian cultured cells, we isolated a temperature-sensitive Chinese hamster ovary (CHO) cell mutant (strain SPB1) with a thermolabile SPT after selection by in situ colony assay [25]. SPB-1 cells are incapable of synthesizing any sphingolipids de novo at 40 jC and require external sphingolipids for cell growth at a nonpermissive temperature [26]. After selecting variants resistant to a sphingomyelin-binding toxin, we isolated another type of SPTdefective CHO cell mutant (strain LY-B) that lacks the activity regardless of temperature, and demonstrated that LY-B cells require an external supply of sphingolipids for growth at any physiological temperature [27]. As for invertebrates, it has been shown that a complete loss of SPT activity in fruit fly results in embryonic lethality [28]. When the SPT deficiency is partial, mutant flies grow into adults with abnormalities in various external organs, but such

abnormalities are rescued by feeding with sphingosine [28]. These genetic findings represent conclusive evidence that SPT is responsible for the initial step of de novo sphingolipid synthesis in eukaryote cells, and that sphingolipids are essential for the growth of cells and presumably also for the development of animals. 3.3. Genes and molecular structure of SPT Mutations causing a complete loss of SPT activity in yeast cells have been shown to fall into two genetic complementation groups, Lcb1 and Lcb2, and their wildtype alleles (LCB1 and LCB2) were isolated by functional rescue experiments [23,29,30]. Independently, the SCS1 gene, which is identical to LCB2, was isolated as an allele whose mutations suppressed the hypersensitivity of a yeast mutant to high concentrations of calcium [31]. Then, mammalian cDNA homologs of yeast LCB1 and LCB2 were isolated from human, mouse, and CHO cells [32 –34]. For simplicity, LCB1 and LCB2 are used to refer also to mammalian cDNA homologs (and their genomic genes, if necessary), and their products are referred to as LCB1 and LCB2, respectively, although five letters (i.e., SPTLC1 and SPTLC2 in place of LCB1 and LCB2) should be used for mammalian genomic genes. Each type of LCB protein shows f40% identity between yeast and mammals, and f95% identity among mammals at the amino acid level (Fig. 3) [33,34]. In the human genome, the LCB1 (SPTLC1) gene comprises 15 exons spanning f85 kbp in the chromosome 9q21– q22 region, and the LCB2 (SPTLC2) gene comprises 12 exons spanning f110 kbp in the chromosome 14q24.3 – q31 region. Mammalian LCB1 and LCB2 encode 53- and 63-kDa proteins, respectively [33,34]. These subunits have mutual similarity (f20% identity) (Fig. 3) [33,34], and this similarity is probably relevant to the formation of a heterodimer by the two subunits. Both LCB1 and LCB2 have a single highly hydrophobic domain, which represents a transmembrane domain (TMD), in their amino-terminal region without cleavable signal sequences (Fig. 3) [33,34]. Neither LCB1 nor LCB2 appears to be glycosylated [33]. Indirect immunocytochemical analysis with epitope-tagged LCB1 indicated that the N and C termini of LCB1 are oriented to the lumen and cytosol, respectively, in the endoplasmic reticulum (ER) [35]. Subcellular fractionation analysis has also shown enrichment of SPT activity in the ER [36,37]. In addition, protease-sensitivity of SPT in sealed ‘‘right-side out’’ ER membranes has suggested a cytosolic orientation of the catalytic site [37]. Although some programs for the prediction of TMDs assign not only the highly hydrophobic region but also several moderately hydrophobic regions of the LCB proteins as TMDs, the latter regions most likely serve as internal domains of globular parts of the proteins rather than as TMDs, because the moderately hydrophobic regions of the LCB proteins have sequence similarities to corresponding regions of soluble members of an SPT-related

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Fig. 3. Deduced sequences of SPT subunits from various species. Deduced amino acid sequences of LCB1 and LCB2 proteins are compared using an alignment algorithm [123]. Dark shaded boxes indicate identical amino acids. Putative transmembrane domains are indicated by underlines, although the domain in yLCB1 may not be hydrophobic enough to transverse membranes. The N and C termini of LCB proteins are most likely oriented to the lumenal and cytosol side, respectively, of the ER (see text). An asterisk marks the putative PLP-binding lysine residues in the LCB2 protein sequences. The cystein and valine residues that correspond to the LCB1 mutation sites in human hereditary sensory neuropathy type I are marked by open circles. Prefixes represent the origin: h, human; c, CHO cells; f, fruit fly (Drosophila melanogaster); y, yeast (S. cerevisiae). SpSPT1, S. paucimobilis SPT. The fLCB1 sequence was selected from D. melanogaster genome sequences, based on overall sequence homology to mammalian LCB1 sequences. The fLCB1 sequence determined in silico was submitted to the GenBank Third Party Annotation data bank (accession number, TPA:BK000017). The GenBank accession numbers for the other sequences are shown in Fig. 4B.

enzyme family (for this family, see below) as discussed in Refs. [35,38]. Collectively, these results strongly suggest that both LCB1 and LCB2 are type I integral membrane proteins of the ER.

SPT was purified as an active form from CHO cells by affinity-peptide chromatography, and this purified enzyme was shown to consist of LCB1 and LCB2 at a 1:1 ratio [39]. In addition, it was shown that two

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LCB1 molecules are not integrated into one LCB1/ LCB2 complex [35,40]. Gable et al. have shown that, in the yeast S. cerevisiae the 10-kDa product of the Tsc3 gene is associated with the yeast LCB1/LCB2 complex [40]. Tsc3p is not essential for SPT activity, but is required for the optimum activity of SPT in yeast cells [40,41]. No mammalian homolog of Tsc3 has been found so far even by computer search of sequence databases. Thus, mammalian SPT is likely a heterodimer of LCB1/LCB2, although another factor(s) might be associated with the mammalian LCB1/LCB2 complex in intact cells.

3.4. SPT is a member of a subfamily of pyridoxal 5Vphosphate (PLP)-dependent enzymes SPT requires PLP as a co-factor [42,43]. LCB1 and LCB2 have significant similarities to members of a subfamily of PLP-dependent enzymes that includes 5-aminolevulinic acid synthase, 2-amino-3-ketobutyrate ligase (KBL), and 8-amino-7-oxononanoate synthase (AONS) (Fig. 4A and C). These enzymes catalyze condensations of amino acids and carboxylic acid CoA thioesters to produce a-oxoamines (Fig. 4A). This subfamily is hereafter referred to as the PLP-dependent a-oxoamine synthase

Fig. 4. POAS family. (A) Reactions catalyzed by POAS family members. (B) Phylogenetic relationship among members of the POAS family determined by multiple alignment using the Clustal W algorithm [124]. The result is shown as a dendrogram. The GenBank accession number for each member is indicated in parentheses. hALS, human 5-aminolevulinic acid synthase; hKBL, human 2-amino-3-ketobutyrate ligase; EcAONS, Escherichia coli 8-amino-7-oxononanoate synthase. The prefix m and At represent mouse and Arabidopsis thaliana, respectively. For the other proteins, see the legend to Fig. 3. (C) PLP-binding motif in the POAS family. An asterisk marks the PLP-binding lysine residue that was identified in bacterial AONS [44]. Corresponding regions in other members of the POAS family are compared. Dark shaded boxes indicate identical amino acids.

K. Hanada / Biochimica et Biophysica Acta 1632 (2003) 16–30

(POAS) family. Among the members of this family identified to date, only eukaryotic (but not prokaryotic) SPT is a membrane-bound heterodimer enzyme, while all other members are soluble homodimer enzymes. The lysine residue that forms a Schiff’s base with PLP has been identified in bacterial KLB and AONS [44,45]. Alignment of POAS members shows a conserved motif (T[FL][GTS]K[SAG][FLV]G) around the PLP-binding lysine, and this motif is present in LCB2, but not in LCB1 (Fig. 4C), strongly suggesting that PLP binds only to LCB2 [29,30,34]. In contrast to eukaryote cells, the bacteria Sphingomonas paucimobilis produces soluble SPT as a homodimer of a 45-kDa protein [46]. The bacterial SPT,

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which is f30% identical to both mammalian LCB1 and LCB2 at the amino acid level (Fig. 3), has the conserved lysine residue in the PLP-binding motif [46] (Fig. 4B). It is unclear why eukaryotic SPT consists of two different subunits unlike other POAS members, although it has been shown that LCB1 is indispensable for the maintenance of LCB2 as described below. 3.5. Mechanism of SPT reaction The carboxyl group of the substrate serine is removed during the condensation of serine and palmitoyl CoA by SPT [47]. Two different basic mechanisms can be postu-

Fig. 5. A model of the SPT reaction mechanism. Pathway B is suggested to be the predominant pathway of SPT reaction, although pathway A might also operate. PLP binds to the LCB2 subunit in the SPT enzyme complex. B: base. For an explanation of each step in pathway B, see text.

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lated for the condensation reaction: (A) formation of PLPstabilized carbanion by decarboxylation of the substrate Lserine, followed by acylation; (B) formation of the carbanion by removing the a-hydrogen atom of L-serine, followed by acylation and decarboxylation. An early study suggested that the a-hydrogen atom of serine was retained during the condensation, supporting the former mechanism (pathway A in Fig. 5) for SPT [18]. However, the latter mechanism (pathway B in Fig. 5) was supported by a later study showing that the a-hydrogen atom of serine is replaced by a proton from H2O during the KDS formation [48]. The removal of the a-hydrogen atom of the amino acid substrates has also been demonstrated in the condensation reactions by 5-aminolevulinic acid synthase [49] and AONS [50]. Strong sequence similarities among the POAS family suggest that the mechanism of action of SPT is analogous to the well-characterized action mechanism of AONS [45,50 – 52]. In addition, the structure of several potent SPT inhibitors (i.e., sphingofungins and myriocin) resembles the structure of a transient intermediate retaining the carboxyl group of Lserine (this intermediate forms in pathway B but not in pathway A in Fig. 5) as discussed below. Collectively, these results imply that the SPT reaction proceeds predominantly by a multistep mechanism depicted as pathway B in Fig. 5. This pathway composes (1) the formation of a Schiff’s base between the substrate Lserine and PLP in the enzyme; (2) the removal of the ahydrogen atom of serine; (3) the nucleophilic attack of palmitoyl CoA by the a-carbanion to form a transitional acylated intermediate; (4) decarboxylation; (5) reprotonation of the a-carbanion; and (6) the release of KSD from the enzyme. Analysis with purified enzyme suggests that

one molecule of mammalian SPT is capable of catalyzing maximally f80 cycles of these steps per minute [39]. 3.6. Substrate specificity of SPT Among various acyl CoAs, palmitoyl CoA is the best substrate of mammalian SPT in vitro [36,39,43,53]. Pentadecanoyl- and heptadecanoyl-CoAs are also effective, whereas myristoyl- and stearoyl-, palmitoleoyl- and arachidoyl-CoA are far less effective. In mammalian cells, palmitoyl CoA is one of the most abundant acyl-CoA types, while pentadecanoyl- and heptadecanoyl-CoAs are scarce [54 – 57]. Therefore, palmitoyl CoA is the predominant acylCoA substrate of SPT in vivo, and for this reason the chain length of the sphingoid bases from mammalian cells is mainly 18 [39]. SPT strictly utilizes L-serine as its amino acid substrate [39]. Neither L-alanine, L-serinamide, D,L-serinol, nor Lserine methylester serves as a competitor in the formation of [3H]KDS from L-[3H]serine, indicating that all of the hydroxyl, amino, and carboxyl groups of L-serine are responsible for the recognition of the amino acid substrate by the SPT enzyme [39]. Nevertheless, the formation of [3H]KDS from L-[3H]serine is competitively inhibited by D-serine with an IC50 of f0.3 mM, which is a value similar to the Km of L-serine for the SPT reaction, although D-serine is not utilized by SPT to produce KDS [58]. Presumably, the SPT enzyme is able to form Schiff’s base complexes with both enantiomers of serine, and the postulated next step (deprotonation of the serine adduct; see step 2 in Fig. 5) proceeds in the complex with L-serine, but not with D-serine [58]. Since substantial quantities of D-serine are present in discrete areas of the brain [59,60],

Fig. 6. Potent inhibitors of SPT. (A) Structure of potent SPT inhibitors. The absolute structures of sphingofungin B, myriocin (ISP-1/thermozymocidin), lipoxamycin (neoenactin M1), and viridiofungin A are based on Refs. [64,125 – 127], respectively. (B) A model of the formation of intermediate-like adducts of specific SPT inhibitors with PLP in the enzyme. The adducts of sphingofungin B (a), myriocin (b), and lipoxamycin (c) with PLP are expected to mimic a postulated intermediate (d) of the SPT reaction. The lipoxamycin adduct (c) may mimic the product of step 5, rather than step 3, in Fig. 5.

K. Hanada / Biochimica et Biophysica Acta 1632 (2003) 16–30 D-serine might affect de novo sphingolipid synthesis in certain cell types enriched by it.

3.7. Inhibitors of SPT Natural inhibitors of SPT have been discovered (Fig. 6A). Sphingofungins, lipoxamycin (neoenactin M1), and myriocin (ISP-1/thermozymocidin) are potent and highly selective inhibitors of SPT, inhibiting fungal and mammalian SPT in cell-free preparations with IC50 values in the nanomolar range [61 – 63]. These potent inhibitors structurally resemble the postulated transient intermediate (the product of step 3 in pathway B in Fig. 5) that forms in the condensation of L-serine and palmitoyl CoA, suggesting that the formation of intermediate-like adducts of these drugs with PLP in the SPT enzyme underlies the strong inhibitory activity (Fig. 6B). Consistent with this suggestion, the inhibitory activity of sphingofungin B is highly dependent on its stereochemistry [64], and myriocin-linked resins bind the LCB1/LCB2 complex tightly [65]. Viridiofungins are potent inhibitors of mammalian SPT [66], but they also inhibit squalene synthase with IC50 values in the micromolar range [67]. L-Cycloserine and h-chloro-Lalanine, which have sometimes been used to inhibit SPT in intact cells, are wide-range inhibitors of PLP-dependent enzymes [68,69], and caution is needed in the use of these drugs for the specific inhibition of sphingolipid synthesis. Indeed, metabolic bypassing of the SPT function with external sphingosine rescues the growth inhibition of CHO cells by sphingofungin B and myriocin (ISP1/thermozymocidin), but not by L-cycloserine and h-chloro-L-alanine [70]. Interestingly, ISP-1 was initially discovered as a potent immunosuppressant [71]. Very low concentrations (f50 nM) of ISP1 are enough to induce apoptosis of mouse CTLL-2 cells, but not most other cell types [63,72]. The viability of limited types of lymphoid cells might be strongly affected by the perturbation of intracellular sphingolipid levels.

4. Regulation of SPT activity Consistent with the ubiquitous expression of sphingolipids in mammalian cells, SPT activity is detected in many types of tissue and cell preparations as summarized in a previous review [73]. In addition, mRNA for SPT subunits is ubiquitously expressed in various tissues, although the mRNA levels vary depending on tissue type [33,34]. The level of SPT activity also varies among different types of tissues and cells. SPT activity levels are significantly higher in rat lung and kidney microsomes than in heart and testis microsomes [74], and the mRNA levels of SPT subunits are higher in mouse lung and kidney than in heart and testis [33,34]. The levels of SPT activity also depend on the developmental stage of tissues. For example, SPT activity increases progressively from the fetal to neonatal period, and reaches a plateau at the adult stage in rat lung [75].

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Furthermore, the levels of SPT activity in several tissues of animals are affected by diet [76,77]. Although the mechanism underlying the regulation of SPT activity is largely unknown, recent studies have begun to provide some insight into the transcriptional and post-transcriptional regulation of SPT activity. 4.1. Transcriptional and post-transcriptional regulation in response to extracellular stimuli LCB mRNA and SPT activity levels increase in response to several types of inflammatory and stress stimuli (Table 1). Intraperitoneal administration of endotoxin to Syrian hamsters stimulates SPT activity two- to threefold in the liver, spleen, and kidney with a concomitant increase in the level of LCB2 mRNA [78,79]. Similar changes are observed upon administration of interleukin 1h (IL-1), an inflammatory cytokine [78]. Irradiation of epidermal cells with ultraviolet light B (UVB) also affects LCB mRNA levels [80]. SPT activity in epidermal cells appears to be up-regulated in response to barrier requirements of skin [81]. These results, along with the observation that inflammation and UVB stimuli also enhance the synthesis of other lipid types including fatty acids and cholesterol [82,83], raise the possibility that a lipogenic signaling pathway(s) is involved in transcriptional control of LCB genes. Consistent with this possibility, both LCB1 and LCB2 mRNA levels are increased when cultured human keratinocytes are treated with nicotineamide, which also enhances fatty acid and cholesterol synthesis [84]. The expression of LCB1 mRNA is also upregulated in islets of leptin-receptor-deficient obese fa/fa rats, compared to the levels in control islets, and this upregulation is suggested to be a response to an increase in intracellular fatty acid [85]. Cytotoxic accumulation of palmitate induces apoptosis accompanied by an elevation of the intracellular ceramide level in various cell types including the hematopoietic LyD9 cell line [86], fa/fa pancreas islets [85], astrocytes [87], and CHO cells [88]. For most cell types, enhanced de novo synthesis of ceramide via SPT is required for palmitateinduced apoptosis [85,86,88,89], whereas palmitate-induced apoptosis of CHO cells is suggested to occur through a ceramide-independent, but reactive oxygen species-dependent, pathway [88]. Palmitate-induced enhancement of SPT activity in primary astrocytes is prevented by exposure of cells to an activator of AMP-activated protein kinase (AMPK) [89]. The AMPK cascade acts as a metabolic sensor that monitors cellular AMP and ATP levels, and once activated, the cascade down-regulates various ATPconsuming anabolic pathways including fatty acid and cholesterol synthesis [90,91]. The AMPK cascade might also participate in the regulation of sphingolipid synthesis. Certain types of apoptic stimuli appear to activate SPT post-transcriptionally (Table 1). Upon treatment with retinoic acid, a fraction of mouse teratocarcinoma PCC7-Mz1 cells undergoes apoptosis, but the remaining cells begin to

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Table 1 Biological and chemical stimuli that up-regulate SPT activity in mammalian cells Stimulus (tissue or cell types)

SPT

References

Activity mRNA

Protein

LCB1 LCB2 LCB1 LCB2 Fold-increase relative to control UVB (mouse epidermis and cultured human keratinocytes) Endotoxin, IL1 (liver, spleen and kidney of Syrian hamster) Endotoxin, IL1, tumor necrosis factor-a (human HepG cell line) Leptin receptor mutation (rat pancreas islet) Fatty acids (rat pancreas islet) Palmitic acid (rat astrocytes) Nicotineamide (cultured human keratinocytes) Etoposide (human leukemia Molt-4 cell line) Retinoic acid (mouse teratocarcinoma PCC7-Mz1 cell line) D9-Tetrahydrocannabinol (a subline of the rat glioma C6 line) Activation of angiotensin II type 2 receptor (a subline of the rat pheochromocytoma PC12 cell line) N-(4-hydroxyphenyl) retinamide (human neuroblastoma CHLA-90 cell line) Hexachlorobenzene (rat liver) Apolipoprotein E knockout (mouse liver)

1.5

1.5a

2 – 3a nd

2

[80,83]

2–3

nd

2–4

nd

nd

[78,79]

2–3

nd

2–3

nd

nd

[78]

ndb

2–3

nd

nd

nd

[85]

ndb

1.5 – 2 nd

nd

nd

[85]

1.3

nd

nd

nd

1.4

[89]

1.2

1.8

1.8

nd

nd

[84]

2–3

–

–

nd

nd

[93]

3

–

–

nd

nd

[92]

6

1.4

1.1

1.8

–

[95]

2

nd

nd

nd

nd

[128]

2

nd

nd

nd

nd

[129]

1.5 – 2a nd

nd

nd

nd

[130]

2

–

nd

nd

[131]

nd

intracellular ceramide levels without increases in LCB mRNA levels [93] (however, heat shock-induced up-regulation of de novo synthesis of sphingolipids in Molt4 cells has been reported to accompany no increase in SPT activity [94]). During cannabinoid-induced apoptosis, both SPT activity and intracellular ceramide levels increase four- to sixfold without major changes in LCB mRNA or protein levels in a subline of the rat glioma C6 line [95]. Inhibitors of de novo synthesis of sphingolipids repress the enhancement of ceramide levels and apoptosis in response to these stimuli [92,93,95]. Thus, these stimuli are likely to activate SPT post-transcriptionally, thereby enhancing de novo synthesis of ceramide, an elicitor of apoptosis [92,93]. An increase in the sphinganine level in several cell types during photosensitizer-induced apoptosis might also accompany up-regulation of SPT [96]. 4.2. The maintenance of LCB2 is LCB1-dependent The LY-B cell line is a CHO mutant cell line defective in SPT activity, due to the lack of expression of an endogenous LCB1 subunit [27]. Interestingly, the level of the LCB2 subunit is also far lower in LY-B cells than in the wild-type CHO cells (Fig. 7A), although the level of LCB2 mRNA in the mutant cells is quite normal [35]. Stable transfection of LY-B cells with LCB1 cDNA restores the amount of LCB2 subunit to (but never beyond) the wild-type level (Fig. 7A) [35]. Moreover, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit, whereas LCB1 can be overproduced even without overproduction of

– , no increase; nd, not determined. a After stimulation, the levels initially decreased by f50%, but subsequently increased above unstimulated control levels. b De novo sphingolipid synthesis in intact cells was significantly increased.

differentiate in a manner mimicking the early steps of neuronal development. Retinoic acid treatment of PCC7Mz1 stem cells induces an accumulation of ceramide accompanied by an increase in SPT activity without any increases in LCB1 and LCB2 mRNA [92]. Likewise, treatment of Molt-4 human leukemia cells with the chemotherapy agent etoposide elevates both SPT activity and

Fig. 7. LCB1-dependent existence of LCB2. (A) The levels of LCB1 and LCB2 in the membrane fraction (20 Ag protein/well) of various CHO cell lines were examined by Western blotting. CHO-K1, wild-type; LY-B, LCB1-defective mutant; LY-B/cLCB1, stable transformant of LY-B with cLCB1. (B) CHO-K1 cells were transfected with pSV-cLCB1, pSV-cLCB2, and/or the empty vector in the indicated combinations. Lysate (7.5 Ag protein/well) of the transfected cells was subjected to Western blotting. Redrawn from Ref. [35].

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LCB2 (Fig. 7B) [35]. Similar results have also been demonstrated in yeast cells [40]. These results suggest that the LCB2 subunit is unstable unless it is associated with the LCB1 subunit. Based on the LCB1-dependent existence of LCB2, we have proposed the hypothesis that, if a pool of free LCB1 subunit is available, cells can regulate the level of LCB1/ LCB2 complex through transcriptional regulation of the LCB2 gene even without parallel regulation of LCB1 [35]. This scenario might account for the previously reported observation that the change in SPT activity in response to UVB irradiation parallels the change in the LCB2 mRNA level but not the LCB1 mRNA level in cultured human keratinocytes [80]. 4.3. Regulation for homeostasis of sphingolipid levels An external supply of sphingolipids depresses de novo synthesis of sphingolipids in various cell types [26,97 – 104]. This depression is presumably a physiologically important response for homeostasis of intracellular sphingolipid levels. When cultured mouse cerebellar cells are incubated with sphingosine or its analogs, SPT activity in microsomes prepared from the sphingoid base-loaded cells is significantly reduced, while no inhibition of SPT activity is observed when cerebellar cell microsomes are incubated directly with sphingoid bases [98,101]. Puzzlingly, depression of de novo sphingolipid synthesis by exogenous ceramide or its analogs seems not to be accompanied by any inhibition of the activities of anabolic enzymes including SPT [100,102]. This depression might be explained, at least in part, by enhanced degradation of dihydrosphingosine, an early intermediate in the pathway for sphingolipid synthesis [102]. More studies are required to elucidate the mechanism underlying the depression of de novo synthesis by exogenous sphingolipids. 4.4. Is SPT activity regulated by cholesterol? It is not clear whether SPT activity is regulated in response to changes in intracellular cholesterol levels, because depletion or loading of cholesterol affects SPT activity differently depending on cell type and experimental conditions. In fact, a positive correlation [105], no correlation [7,8,106 – 108], and a negative correlation [109,110] have all been reported between the intracellular cholesterol content and SPT activity. Sterol regulatory element-binding proteins (SREBPs) are transcriptional factors that activate genes encoding enzymes for cholesterol and fatty acid synthesis by binding to specific promoter DNA sequences termed sterol regulatory elements [111,112]. Activation of SREBP-2 (a predominant isoform of SREBPs in keratinocytes) does not increase LCB2 mRNA levels in cultured human keratinocytes, while it clearly increases mRNA levels for cholesterol and fatty acid synthesis [105].

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4.5. Other factors that affect the function of SPT in intact cells The rate of sphingolipid synthesis in intact cells is correlated with concentrations of serine and palmitic acid, but not other fatty acid types, in the culture medium [113,114], suggesting that the actual rate of de novo synthesis in intact cells reflects levels of cytosolic serine and palmitoyl CoA. As SPT activity shows a bell-shape dependence on palmitoyl CoA in vitro [39,40,42,43,53], palmitoyl CoA at levels over the optimum concentration may exert negative effects on SPT to prevent sphingolipid overproduction. The inhibitory effect of palmitoyl CoA on SPT activity is remarkable in yeast tsc3 mutant cells, compared to the wild-type control [40], raising the possibility that Tsc3p regulates interaction between palmitoyl CoA and the SPT enzyme, although no animal homolog of the yeast TSC3 has been identified to date. Various cell types in culture undergo a transient but remarkable increase in the de novo synthesis of sphingoid bases upon replacement of the conditioned culture medium with fresh medium [115,116]. Warden et al. [117] have shown that the burst of synthesis of sphingoid bases upon medium change is due to the removal of inhibitory factors accumulated in the conditioned medium, and identified these inhibitory factors as ammonium ion and a novel natural compound named batrachamine. These factors might affect SPT activity directly or indirectly in intact cells.

5. SPT and a genetic disease Abnormalities of the SPT enzyme cause clinical disorders, although a complete lack of SPT activity is predicted to be embryonic lethal. Hereditary sensory neuropathy type I (HSN1) is a dominantly inherited disease involving the progressive degeneration of lower limb sensory and autonomic neurons [118]. HSN1 is a genetically heterogeneous disease, and at least three gene variants are reported. It has recently been revealed that the genetic defect in the HSN1 families linked to the chromosome 9q22 locus is associated with missense mutations in the human LCB1 (SPTLC1) gene, which alter a specific amino acid residue (Cys133 or Val144) in the LCB1 subunit [119,120]. HSN1 mutation was initially suggested to enhance SPT activity because the rate of de novo synthesis of glucosylceramide (but not other sphingolipids) was estimated to be 1.8-fold higher in patient-derived lymphoblasts than in healthy controls in metabolic labeling experiments with radioactive serine. However, more recent investigations demonstrated compelling evidence that HNS1 mutations negatively affect SPT. Bejaoui et al. [121] showed that, in HSN1 lymphoblasts, SPT activity and the rate of de novo synthesis of sphingolipids are substantially reduced, whereas the steady-state levels of LCB1 and LCB2 subunits are normal. The reason for the discrepancy in the results of

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metabolic labeling between the two studies is unknown. The negative effects of HSN1 mutations on SPT activity and sphingolipid synthesis were reproduced in CHO cells [121] and yeast cells [38] as the expression of LCB1 mutants having the HSN1 mutations was shown to inhibit SPT activity and sphingolipid synthesis in these cells. Importantly, the mutated LCB1 proteins remain capable of forming a complex with the LCB2 subunit, and this complex is inactive as SPT [38,121]. It is therefore most likely that HSN1 mutations confer dominant negative effects on SPT, probably due to competition between the mutated and normal LCB1 proteins for interaction with LCB2. The amino acid sequence around Cys133 and Val144 in LCB1 is highly conserved from yeast to mammals (Fig. 3). The tertiary structure of AONS has demonstrated that the catalytic site is formed at the interface of each subunit of this homodimer enzyme [45]. In a tentative model for a tertiary structure of the SPT complex, both Cys133 and Val144 of LCB1 are predicted to be spatially close to the PLP-binding site of LCB2 [38]. Presumably, the amino acid sequence around Cys133 and Val144 in LCB1 is involved in the formation of the catalytic site, and the HSN1 mutant types of LCB1 are unable to contribute to the formation of the active catalytic site. It remains unclear why mutations in a protein widely expressed in all tissues trigger pathology that is highly restricted to specific subsets of cells within a tissue.

6. Future directions Great progress in research on SPT has been made in the past decade as summarized in this review. Nevertheless, more studies are needed to elucidate the molecular mechanisms underlying the function and regulation of SPT. For example, determination of the tertiary structure of this enzyme will be required to elucidate how the two subunits form a complex. Because no report has so far demonstrated direct inhibition of SPT activity by sphingolipid metabolites in vitro, assay conditions that mimic the environment in intact cells might have to be introduced to reconstitute the metabolic regulation of SPT activity. Whether splicing or posttranslational variants of SPT exist also needs to be addressed, since two types of SPT, one which prefers palmitoyl CoA and another which prefers stearoyl CoA, might be expressed in neurons depending on differentiation and aging [122]. Transgenic and mutant animals will also be invaluable tools to investigate roles of SPT in multicellular organisms. The intellectual and material resources that have accumulated over the past decade will facilitate further study of SPT, a key enzyme of sphingolipid metabolism, in the decade to come.

Acknowledgements I thank Dr. Masahiro Nishijima, Dr. Yuzuru Akamatsu, and all other present and past co-workers for invaluable

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