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1.  Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons 
Nature Communications  2014;5:5514.
Sphingosine is a major storage compound in Niemann–Pick type C disease (NP–C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP–C patient fibroblasts and NP–C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome–lysosome fusion in NP–C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP–C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP–C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP–C neurons where defective SphK activity is due to impaired VEGF levels.
Sphingosine is abnormally accumulated in Niemann–Pick type C disease (NP–C), but the causes of this accumulation have not been fully characterized. Here the authors show that sphingosine kinase activity is reduced in NP–C patient fibroblasts and NP–C mouse neurons due to defective vascular endothelial growth factor levels, suggesting therapeutic avenues.
PMCID: PMC4263144  PMID: 25417698
2.  Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine 
PLoS ONE  2014;9(8):e102377.
N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38∶6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38∶6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44∶12)] and DHA-DHA-PE [PE(44∶12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.
PMCID: PMC4121067  PMID: 25090090
3.  Pseudomonas-Derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate 
PLoS ONE  2014;9(2):e89402.
Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed “3D keratinocytes”), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.
PMCID: PMC3934885  PMID: 24586752
4.  Inhibition of GM3 Synthase Attenuates Neuropathology of Niemann-Pick Disease Type C by Affecting Sphingolipid Metabolism 
Molecules and Cells  2014;37(2):161-171.
In several lysosomal storage disorders, including Niemann-Pick disease Type C (NP-C), sphingolipids, including glycosphingolipids, particularly gangliosides, are the predominant storage materials in the brain, raising the possibility that accumulation of these lipids may be involved in the NP-C neurodegenerative process. However, correlation of these accumulations and NP-C neuropathology has not been fully characterized. Here we derived NP-C mice with complete and partial deletion of the Siat9 (encoding GM3 synthase) gene in order to investigate the role of ganglioside in NP-C pathogenesis. According to our results, NPC mice with homozygotic deletion of GM3 synthase exhibited an enhanced neuropathological phenotype and died significantly earlier than NP-C mice. Notably, in contrast to complete depletion, NP-C mice with partial deletion of the GM3 synthase gene showed ameliorated NP-C neuropathology, including motor disability, demyelination, and abnormal accumulation of cholesterol and sphingolipids. These findings indicate the crucial role of GM3 synthesis in the NP-C phenotype and progression of CNS pathologic abnormality, suggesting that well-controlled inhibition of GM3 synthesis could be used as a therapeutic strategy.
PMCID: PMC3935629  PMID: 24599001
GM3; neuropathology; Niemann-pick type C disease; sphingolipids
5.  Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids 
A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neor), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neor marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neor mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C20:3n-6) and eicosatetraenoic acid (C20:4n-3), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C20:4n-6) and eicosapentaenoic acid (C20:5n-3), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
PMCID: PMC3346472  PMID: 22344656
6.  Increase of Eicosapentaenoic Acid in Thraustochytrids through Thraustochytrid Ubiquitin Promoter-Driven Expression of a Fatty Acid Δ5 Desaturase Gene▿† 
Applied and Environmental Microbiology  2011;77(11):3870-3876.
Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C16:0), n − 6 docosapentaenoic acid (DPA) (C22:5n − 6), and docosahexaenoic acid (DHA) (C22:6n − 3), with eicosapentaenoic acid (EPA) (C20:5n − 3) and arachidonic acid (AA) (C20:4n − 6) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C20:4n − 3) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C20:3n − 6) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs.
PMCID: PMC3127612  PMID: 21478316
7.  Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage 
Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known.
Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically.
These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin.
PMCID: PMC3089780  PMID: 21489308
8.  Purification, crystallization and preliminary crystallographic characterization of the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 
Crystallization of the α2,6-sialyltransferase from Photobacterium.
Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids. The newly cloned α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 (from the Vibrionaceae family) is composed of two domains: an unknown N-terminal domain and a catalytic C-terminal domain which shares significant homology with the Pasteurella multocida multifunctional sialyltransferase. The putative mature form of JT-ISH-224 α2,6-sialyltransferase was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystal belonged to space group P3121 or P3221, with unit-cell parameters a = b = 90.29, c = 204.33 Å. X-ray diffraction data were collected to 2.5 Å resolution.
PMCID: PMC2335162  PMID: 17671362
α2,6-sialyltransferase; Photobacterium sp. JT-ISH-224
9.  Molecular Cloning and Expression of Mn2+-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4 
Journal of Bacteriology  2002;184(2):540-546.
We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn2+. The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.
PMCID: PMC139580  PMID: 11751833
10.  Ceramidase Activity in Bacterial Skin Flora as a Possible Cause of Ceramide Deficiency in Atopic Dermatitis 
A marked decrease in the content of ceramide has been reported in the horny layer of the epidermis in atopic dermatitis (AD). This decrease impairs the permeability barrier of the epidermis, resulting in the characteristic dry and easily antigen-permeable skin of AD, since ceramide serves as the major water-holding molecule in the extracellular space of the horny layer. On the other hand, the skin of such patients is frequently colonized by bacteria, most typically by Staphylococcus aureus, possessing genes such as those for sphingomyelinase, which are related to sphingolipid metabolism. We therefore tried to identify a possible correlation between the ceramide content and the bacterial flora obtained from the skin of 25 patients with AD versus that of 24 healthy subjects, using a thin-layer chromatographic assay of the sphingomyelin-associated enzyme activities secreted from the bacteria. The findings of the assay demonstrated that ceramidase, which breaks ceramide down into sphingosine and fatty acid, was secreted significantly more from the bacterial flora obtained from both the lesional and the nonlesional skin of patients with AD than from the skin of healthy subjects; sphingomyelinase, which breaks sphingomyelin down into ceramide and phosphorylcholine, was secreted from the bacterial flora obtained from all types of skin at similar levels for the patients with AD and the healthy controls. The finding that the skin of patients with AD is colonized by ceramidase-secreting bacteria thus suggests that microorganisms are related to the deficiency of ceramide in the horny layer of the epidermis, which increases the hypersensitivity of skin in AD patients by impairing the permeability barrier.
PMCID: PMC95668  PMID: 9874672

Results 1-10 (10)