Kentaro Hanada

Molecular machinery for non-vesicular trafficking of ceramide

Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.

CERT mediates intermembrane transfer of various molecular species of ceramides.

Ceramide produced at the endoplasmic reticulum is transported to the Golgi apparatus for conversion to sphingomyelin. The main pathway of endoplasmic reticulum-to-Golgi transport of ceramide is mediated by CERT, a cytosolic 68-kDa protein, in a nonvesicular manner. CERT contains a domain that catalyzes the intermembrane transfer of natural C16-ceramide. In this study, we examined the ligand specificity of CERT in detail by using a cell-free assay system for intermembrane transfer of lipids. CERT did not mediate the transfer of sphingosine or sphingomyelin at all. The activity of CERT to transfer saturated and unsaturated diacylglycerols, which structurally resemble ceramide, was 5–10% of the activity toward C16-ceramide. Among four stereoisomers of C16-ceramide, CERT specifically recognized the natural d-erythro isomer. CERT efficiently transferred ceramides having C14, C16, C18, and C20 chains, but not longer acyl chains, and also mediated efficient transfer of C16-dihydroceramide and C16-phyto-ceramide. Binding assays showed that CERT also recognizes short chain fluorescent analogs of ceramide with a stoichiometry of 1:1. Moreover, (1R,3R)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecamide, which inhibited the CERT-dependent pathway of ceramide trafficking in intact cells, was found to be an antagonist of the CERT protein. These results indicate that CERT can mediate transfer of various types of ceramides that naturally exist and their close relatives.

Localization, Topology, and Function of the LCB1 Subunit of Serine Palmitoyltransferase in Mammalian Cells

Serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the biosynthesis of sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1 has a single highly hydrophobic domain near the N terminus. Chinese hamster ovary cell mutant LY-B cells are defective in SPT activity because of the lack of expression of an endogenous LCB1 subunit. Stable expression of LCB1 having an epitope tag at either the N or C terminus restored SPT activity of LY-B cells, suggesting that the epitope tag did not affect the localization or topology of LCB1. Indirect immunostaining showed that the N- and C-terminal epitopes are oriented toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in wild-type cells, and the amount of LCB2 in LY-B cells was restored to the wild-type level by transfection with LCB1 cDNA. In addition, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit. These results indicated that the LCB1 subunit is most likely an integral protein having a single transmembrane domain with a lumenal orientation of its N terminus in the endoplasmic reticulum and that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells.

Hereditary sensory neuropathy type 1 mutations confer dominant negative effects on serine palmitoyltransferase, critical for sphingolipid synthesis.

Hereditary sensory neuropathy type 1 (HSN1) is a dominantly inherited degenerative disorder of the peripheral nerves. HSN1 is clinically and genetically heterogeneous. One form arises from mutations in the gene SPTLC1 encoding long-chain base 1 (LCB1), one of two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step of sphingolipid synthesis. We have examined the effects of the mutations C133Y and C133W, which we have identified in two HSN1 families, on the function of SPT. Although in HSN1 lymphoblasts, the C133Y and C133W mutations do not alter the steady-state levels of LCB1 and LCB2 subunits, they result in reduced SPT activity and sphingolipid synthesis. Moreover, in a mutant Chinese hamster ovary (CHO) cell strain with defective SPT activity due to a lack of the LCB1 subunit, these mutations impair the ability of the LCB1 subunit to complement the SPT deficiency. Furthermore, the overproduction of either the LCB1C133Y or LCB1C133W subunit inhibits SPT activity in CHO cells despite the presence of wild-type LCB1. In addition, we demonstrate that in CHO cells the mutant LCB1 proteins, similar to the normal LCB1, can interact with the wild-type LCB2 subunit. These results indicate that the HSN1-associated mutations in LCB1 confer dominant negative effects on the SPT enzyme.

A novel inhibitor of ceramide trafficking from endoplasmic reticulum to the site of sphingomyelin synthesis

Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1R,3S)-HPA-12, [corrected] which resembles natural ceramide stereochemically, was found to be the most active, although (1R,3S)-HPA-12 [corrected] did not affect ER-to-Golgi trafficking of protein. Interestingly, (1R,3S)-HPA-12 [corrected] inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1R,3S)-HPA-12 [corrected] inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway.

Reconstitution of ATP- and cytosol-dependent transport of de novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells.

Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment, where sphingomyelin (SM) synthase exists, was reconstituted within semi-intact Chinese hamster ovary cells. When [3H]ceramide that had been produced from [3H]sphingosine at 15 °C in perforated cells was chased at 37 °C, [3H]ceramide-to-[3H]SM conversion occurred in a cytosol-dependent manner. In various aspects (i.e. kinetics, ATP dependence, and temperature dependence), [3H]ceramide-to-[3H]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a Chinese hamster ovary cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [3H]ceramide-to-[3H]SM conversion in perforated wild-type cells, whereas the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgi apparatus were merged, no longer required cytosol for conversion of [3H]ceramide to [3H]SM. These results indicated that the assay of [3H]ceramide-to-[3H]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. In addition, conversion of [3H]ceramide to [3H]glucosylceramide in semi-intact cells was little dependent on cytosol, suggesting that ceramide reached the site of glucosylceramide synthesis by a cytosol-independent (or less dependent) pathway.

Ceramide sensitizes astrocytes to oxidative stress: protective role of cannabinoids

Cannabinoids induce apoptosis on glioma cells via stimulation of ceramide synthesis de novo, whereas they do not affect viability of primary astrocytes. In the present study, we show that incubation with Delta9-tetrahydrocannabinol did not induce accumulation of ceramide on astrocytes, although incubation of these cells in a serum-free medium (with or without cannabinoids) led to stimulation of ceramide synthesis de novo and sensitization to oxidative stress. Thus treatment with H2O2 induced apoptosis of 5-day-serum-deprived astrocytes and this effect was abrogated by pharmacological blockade of ceramide synthesis de novo. The sensitizing effect of ceramide accumulation may depend on p38 mitogen-activated protein kinase activation rather than on other ceramide targets. Finally, a protective role of cannabinoids on astrocytes is shown as a long-term incubation with cannabinoids prevented H2O2-induced loss of viability in a CB1 receptor-dependent manner. In summary, our results show that whereas challenge of glioma cells with cannabinoids induces accumulation of de novo -synthesized ceramide and apoptosis, long-term treatment of astrocytes with these compounds does not stimulate this pathway and also abrogates the sensitizing effects of ceramide accumulation.

Reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells

Our recent studies have shown that the de novo sphingolipids play a role in apoptosis of photosensitized cells. To elucidate the involvement of the de novo sphingolipids in reactive oxygen species (ROS) production and mitochondrial depolarization during apoptosis, the stress inducer photodynamic therapy (PDT) with the photosensitizer Pc 4 was used. In Jurkat cells PDT-triggered ROS production or mitochondrial membrane potential (deltapsi(m)) loss was not prevented by the de novo sphingolipid synthesis inhibitor ISP-1. However, PDT + C16-ceramide led to enhanced mitochondrial depolarization and DEVDase activation. The superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected Jurkat cells from ROS generation and apoptosis, but not from deltapsi(m) reduction. Sphinganine or C16-ceramide counteracted MnTBAP-induced protection from apoptosis in Jurkat, as well as CHO cells. In LY-B cells, CHO-derived mutants deficient in serine palmitoyltransferase (SPT) activity and the de novo sphingolipid synthesis, mitochondrial depolarization, but not ROS generation, was suppressed post-PDT. In LY-B cells transfected with the SPT component LCB1, deltapsi(m) collapse post-PDT was restored. The data support the following hypotheses: MnTBAP protects against apoptosis via steps downstream of deltapsi(m) loss; de novo sphingolipids are not required for ROS generation, but can play a role in deltapsi(m) dissipation in photosensitized apoptotic cells.

D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis

Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L‐serine to produce 3‐ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [3H]KDS from L‐[3H]serine was performed using purified SPT. D‐Serine inhibited [3H]KDS production as effectively as non‐radioactive L‐serine, whereas neither D‐alanine nor D‐threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1‐14C]palmitoyl coenzyme A and D‐serine did not produce [14C]KDS, while the control incubation with L‐serine did. These results suggest that D‐serine competes with L‐serine for the amino acid recognition site of SPT, but that D‐serine is not utilized by this enzyme to produce KDS.

Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases

Sphingosylphosphocholine (SPC), the N‐deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC‐phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 and nSMase2, human neutral sphingomyelinases (SMases), are capable of hydrolyzing SPC efficiently under detergent‐free conditions. Bacterial and plasmodial neutral SMases also showed SPC‐PLC activity. The substrate specificity of neutral SMases that hydrolyze SM, SPC, and monoradyl glycerophosphocholine, but not diradyl glycerophosphocholine, suggested that a hydrogen‐bond donor at the C‐2 or sn‐2 position in the substrate is required for recognition by the enzymes.