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Yasunori Kozutsumi

Publications and source records attributed to Yasunori Kozutsumi.

At least 19 recordsLinked to original sources
PubMed2006

Dihydroceramide:sphinganine C-4-hydroxylation requires Des2 hydroxylase and the membrane form of cytochrome b5.

Des2 (degenerative spermatocyte 2) is a bifunctional enzyme that produces phytoceramide and ceramide from dihydroceramide. The molecular mechanism involved in C-4-hydroxylation has not been studied in detail. In the present paper, we report that C-4-hydroxylation requires an electron-transfer system that includes cytochrome b5 and that the hydroxylase activity is reconstituted in an in vitro assay with purified recombinant Des2. FLAG-tagged mouse Des2 was expressed in insect Sf9 cells and was purified by solubilization with digitonin and anti-FLAG antibody affinity column chromatography. The activity of dihydroceramide:sphinganine C-4-hydroxylase was reconstituted with the purified FLAG-Des2, mb5 (the membrane form of cytochrome b5) and bovine erythrocyte membrane. The apparent K(m) and V(max) of Des2 for the substrate N-octanoylsphinganine were 35 microM and 40 nmol x h(-1) x mg of protein(-1) respectively. The K(m) of the hydroxylase for mb5 was 0.8 microM. Interestingly, mb5 was not replaced with the soluble form of cytochrome b5, which lacks the C-terminal membrane-spanning domain. The erythrocyte membrane was separated into Triton X-100-soluble and -insoluble fractions, and the detergent-soluble fraction was replaced by the soluble or membrane form of b5R (NADH-cytochrome b5 reductase). The Triton-X-100-insoluble fraction contained trypsin-resistant factors. The Des2 protein is found in the endoplasmic reticulum and is assumed to have three membrane-spanning domains. The findings of the present study indicate that the hydroxylation requires complex formation between Des2 and mb5 via their membrane-spanning domains and electron transfer from NADH to the substrate via the reduction of mb5 by b5R.

Animals↗
PubMed2006

Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis.

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.

Animals↗
PubMed2006

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells.

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Caco-2 Cells↗
PubMed2006

Aberrant expression of N-acetylglucosaminyltransferase-IVa and IVb (GnT-IVa and b) in pancreatic cancer.

The goal of this study was to identify glycosyltransferases that are specifically expressed in pancreatic cancer. To investigate the gene expression of glycosyltransferases between pancreatic cancer and normal pancreatic tissues, we performed DNA-microarray (involving about 1000 oligosaccharide-related genes) using RNA mixtures of pancreatic cancer cells and normal pancreatic tissues. Eighty-six genes were up-regulated and thirty-two were down-regulated in pancreatic cancer, compared to normal pancreatic tissue. Among these changes, it is noteworthy that the expression of GnT-IVa was decreased and the expression of GnT-IVb was increased in pancreatic cancer, compared to normal pancreatic tissues. Although GnT-IVa and -IVb are involved in the same reaction as a glycosyltransferase, their chromosomal localization is different. When 5 cases of pancreatic cancer tissues were examined using the real-time RT-PCR method, the expression of GnT-IVb was dominant in tumor tissues and the expression of GnT-IVa was dominant in the surrounding normal tissues. The expression of GnT-IVa was increased in all 3 cell lines that had been treated with 5-aza-C and butyrate. These results suggest that the down-regulation of GnT-IVa in pancreatic cancer cells is due to an epigenetic abnormality in the gene.

Antimetabolites, Antineoplastic↗
PubMed2006

Down-regulation of caveolin-1 in mouse Lewis lung cancer P29 is a causal factor for the malignant properties in a high-metastatic subline.

In order to analyze molecular mechanisms for cancer metastasis, we established a high-metastatic subline H7-Lu from a subline H7 of mouse Lewis lung cancer (P29) by repeated injection into tail veins. H7-Lu exhibited increased proliferation and invasion activity. Analysis of gene expression profiles between the parent H7 and H7-Lu revealed that several genes were down-regulated in H7-Lu. One of them, caveolin-1, was a component of lipid/rafts. After confirming the down-regulation of caveolin-1 mRNA by real-time RT-PCR and reduction of the protein by immunoblotting, respectively, H7 was transfected with siRNA for caveolin-1 to examine the role of caveolin-1 in H7-Lu. mRNA of the caveolin-1 gene was suppressed to approximately one third of the original level in H7 cells transfected with siRNA. The transfectant cells showed significantly increased cell proliferation and motility when analyzed by MTT assay and scratching wound healing assay, respectively. In the siRNA-transfectant cells, both ERK1/2 and Akt showed stronger phosphorylation than the mock-transfectant cells indicating that both of these signaling pathways were activated in caveolin-1-suppressed cells. These situations seem to reflect some aspects of the cellular changes in the high metastatic subline H7-Lu. Thus, down-regulation of caveolin-1 in a high-metastatic subline of Lewis lung cancer as defined by DNA array is really a causal factor for the increased malignant properties.

Animals↗
PubMed2005

The requirement for the hydrophobic motif phosphorylation of Ypk1 in yeast differs depending on the downstream events, including endocytosis, cell growth, and resistance to a sphingolipid biosynthesis inhibitor, ISP-1.

ISP-1 inhibits de novo sphingolipid biosynthesis and induces growth defects in both mammals and yeast (Saccharomyces cerevisiae). In our previous study, YPK1/SLI2 was identified as one of multicopy suppressor genes for ISP-1 in yeast. Ypk1 is proposed to be a downstream serine/threonine kinase of the sphingolipid signaling pathway in yeast. Other than resistance against ISP-1, Ypk1 is involved in at least two downstream events, namely cell growth and endocytosis. In this study, the effect of mutants of Ypk1 on these three downstream events was investigated. Among Ypk1 mutants, no 'kinase-dead' mutants complemented the defects in any of these three downstream events in the ypk1 null strain. One of the hydrophobic motif phosphorylation-deficient mutants of Ypk1, Ypk1(T662A) had the moderate kinase activity compared with the wild-type Ypk1. Ypk1(T662A) and the wild-type Ypk1 completely restored the slow-growth phenotype and fluid-phase endocytosis defect of the ypk1 null strain. However, unlike the wild-type Ypk1, Ypk1(T662A) lost the ability for the recovery of the ISP-1 resistance in the ypk1 null strain. Furthermore, the expression of Ypk1(T662A) in the wild-type strain showed a dominant-negative effect on the ISP-1-resistance activity. On the other hand, the cell growth revertant of the ypk1 null strain still showed the hypersensitive phenotype to ISP-1. These data suggest that the ISP-1-resistance pathway is under the regulation of the hydrophobic motif phosphorylation and is separated from the other pathways downstream of Ypk1.

Amino Acid Motifs↗
PubMed2005

GM1 ganglioside-mediated accumulation of amyloid beta-protein on cell membranes.

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta is the key step in the development of Alzheimer's disease. Liposomal studies proposed that Abeta specifically recognizes a cholesterol-dependent cluster of monosialoganglioside GM1 and a conformationally altered form of Abeta promotes the aggregation of the protein. In this study, the accumulation of Abeta on living cells was investigated for the first time. The interaction of fluorescein-labeled Abeta (FL-Abeta) with rat pheochromocytoma PC12 cells was visualized using confocal laser microscopy. FL-Abeta was found to colocalize with GM1-rich domains on cell membranes and to accumulate in a concentration- and time-dependent manner, leading to cytotoxicity. Cholesterol depletion significantly reduced Abeta accumulation. These observations corroborate the GM1-mediated Abeta accumulation model.

Amyloid beta-Peptides↗
PubMed2005

Disturbance of sphingolipid biosynthesis abrogates the signaling of Mss4, phosphatidylinositol-4-phosphate 5-kinase, in yeast.

The functional relationships between phosphoinositides and sphingolipids have not been well characterized to date. ISP-1/myriocin is a potent inhibitor of sphingolipid biosynthesis and induces severe growth defects in eukaryotic cells because of the sphingolipid deprivation. We characterized a novel multicopy suppressor gene of ISP-1-mediated cell death in yeast, MSS4. MSS4 encodes a phosphatidylinositol-4-phosphate 5-kinase that synthesizes phosphatidylinositol (4,5)-bisphosphate (PI4,5P(2)). We demonstrate here that ISP-1 treatment of yeast causes defects both in the activity and subcellular localization of Mss4. The effect of the Mss4 defect on the downstream signaling was examined, because interaction between the Mss4 product, PI4,5P(2), and the pleckstrin-homology domain of Rom2 mediates recruitment of Rom2 to the membrane, which is the crucial step for subsequent Rho1/2 activation. Indeed, failure of Rom2 recruitment was observed in ISP-1-treated cells as well as in csg2-deleted cells, which have reduced mannosylated inositolphosphorylceramide. These data suggested that proper sphingolipids are required for the signaling pathway involving Mss4.

Fatty Acids, Monounsaturated↗
PubMed2004

Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

Antineoplastic Agents↗
PubMed2004

SLI1 (YGR212W) is a major gene conferring resistance to the sphingolipid biosynthesis inhibitor ISP-1, and encodes an ISP-1 N-acetyltransferase in yeast.

ISP-1 (myriocin) is a potent inhibitor of serine palmitoyltransferase, the primary enzyme of sphingolipid biosynthesis, and is a useful tool for studying the biological functions of sphingolipids in both mammals and yeast (Saccharomyces cerevisiae). In a previous study, we cloned yeast multicopy suppressor genes for ISP-1, and one of these, YPK1/SLI2, was shown to encode a serine/threonine kinase which is a yeast homologue of mammalian SGK1 (serum/glucocorticoid-regulated kinase 1). In the present study, another gene, termed SLI1 (YGR212W; GenBank accession number CAA97239.1), was characterized. Sli1p has weak similarity to Atf1p and Atf2p, which are alcohol acetyltransferases. Although a sli1-null strain grew normally, the IC50 of ISP-1 for the growth of this strain was markedly decreased compared with that for the parental strain, indicating that Sli1p is a major contributor to ISP-1 resistance in yeast. On a sli1-null background, the increase in resistance to ISP-1 induced by YPK1 gene transfection was almost abolished. These data indicate that Sli1p co-operates with Ypk1p in mediating resistance to ISP-1 in yeast. Sli1p was found to convert ISP-1 into N-acetyl-ISP-1 in vitro. Furthermore, N-acetyl-ISP-1 did not share the ability of ISP-1 to inhibit the growth of yeast cells, and the serine palmitoyltransferase inhibitory activity of N-acetyl-ISP-1 was much lower than that of ISP-1. These data suggest that Sli1p inactivates ISP-1 due to its N-acetyltransferase activity towards ISP-1.

Acetylation↗
PubMed2004

Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates.

Cancer cells undergo distinct metabolic changes to cope with their hypoxic environment. These changes are achieved at least partly by the action of transcriptional factors called hypoxia-inducible factors (HIFs). We investigated gene expression in cultured human colon cancer cells induced by hypoxic conditions with special reference to cell-adhesion molecules and carbohydrate determinants having cell-adhesive activity by using DNA-microarray and RT-PCR techniques. Hypoxic culture of colon cancer cells induced a marked increase in expression of selectin ligands, the sialyl Lewis x and sialyl Lewis a determinants at the cell surface, which led to a definite increase in cancer cell adhesion to endothelial E-selectin. The transcription of genes for fucosyltransferase VII (FUT7), sialyltransferase ST3Gal-I (ST3O), and UDP-galactose transporter-1 (UGT1), which are all known to be involved in the synthesis of the carbohydrate ligands for E-selectin, was significantly induced in cancer cells by hypoxic culture. In addition, a remarkable induction was detected in the genes for syndecan-4 (SDC4) and alpha5-integrin (ITGA5), the cell-adhesion molecules involved in the enhanced adhesion of cancer cells to fibronectin. The transcriptional induction by hypoxia was reproduced in the luciferase-reporter assays for these genes, which were significantly suppressed by the co-transfection of a dominant-negative form of HIF. These results indicate that the metabolic shifts of cancer cells partly mediated by HIFs significantly enhance their adhesion to vascular endothelial cells, through both selectin- and integrin-mediated pathways, and suggest that this enhancement further facilitates hematogenous metastasis of cancers and tumor angiogenesis.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2004

Interaction between amyloid beta-protein aggregates and membranes.

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. Therefore, extensive studies have been carried out on the mechanisms involved in Abeta aggregation and the characterization of Abeta aggregates formed in aqueous solutions mimicking biological fluids. On the other hand, several investigators pointed out that membranes play an important role in Abeta aggregation. However, it remains unclear whether Abeta aggregates formed in solution and membranes are identical and whether the former can bind to membranes. In this study, using a dye-labeled Abeta-(1-40) as well as native Abeta-(1-40), the properties of Abeta aggregates formed in buffer and raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin were compared. Fourier transform infrared spectroscopic measurements suggested that Abeta aggregates formed in buffer and in membranes have different beta-sheet structures. Fluorescence experiments revealed that Abeta aggregated in buffer did not show any affinity for membranes.

Alzheimer Disease↗
PubMed2003

Formation of a membrane-active form of amyloid beta-protein in raft-like model membranes.

The conversion of soluble, nontoxic amyloid beta-protein (A beta) to aggregated, toxic A beta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. We have proposed that the aggregation proceeds in the lipid raft containing a ganglioside cluster, the formation of which is facilitated by cholesterol and for which A beta shows a specific affinity. In this study, using fluorescence resonance energy transfer, we found that after A beta binds to raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin (1/1/1), the protein can translocate to the phosphatidylcholine membranes to which soluble A beta does not bind. Furthermore, self-quenching experiments using fluorescein-labeled A beta revealed that the translocation process competes with the oligomerization of the protein in the raft-like membranes. These results suggest that the lipid raft containing a ganglioside cluster serves as a conformational catalyst or a chaperon generating a membrane-active form of A beta with seeding ability.

Alzheimer Disease↗
PubMed2002

Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid.

GM1 ganglioside-bound amyloid beta-protein (GM1-Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) plaques, has been suggested to accelerate amyloid fibril formation by acting as a seed. We have previously found using dye-labeled Abeta that Abeta recognizes a GM1 cluster, the formation of which is facilitated by cholesterol [Kakio, A., Nishimoto, S., Yanagisawa, K., Kozutsumi, Y., and Matsuzaki, K. (2001) J. Biol. Chem. 276, 24985-24990]. In this study, we investigated the ganglioside species-specificity in its potency to induce a conformational change of Abeta, by which ganglioside-bound Abeta acts as a seed for Abeta fibrillogenesis, using a major ganglioside occurring in brains (GM1, GD1a, GD1b, and GT1b) in raft-like membranes composed of cholesterol and sphingomyelin. Abeta recognized ganglioside clusters, the density of which increased with the number of sialic acid residues. Interestingly, however, mixing of gangliosides inhibited cluster formation. In contrast, the affinities of the protein for the clusters were similar irrespective of lipid composition and of the order of 10(6) M(-)(1) at 37 degrees C. Abeta underwent a conformational transition from an alpha-helix-rich structure to a beta-sheet-rich structure with the increase in protein density on the membrane. Ganglioside-bound Abeta proteins exhibited seeding abilities for amyloid formation. GM1-Abeta exhibited the strongest seeding potential, especially under beta-sheet-forming conditions. This study suggested that lipid composition including gangliosides and cholesterol strictly controls amyloid formation.

Alzheimer Disease↗
PubMed2002

Sphingolipids involved in the induction of multinuclear cell formation.

In this review, we focus on sphingolipids as potential regulators of the induction of multinuclear cell formation through the inhibition of cytokinesis. A sphingolipid, psychosine (Psy) (galactosylsphingosine), was demonstrated to be a trigger lipid for the inhibition of cytokinesis and the induction of multinuclear giant cells associated with a sphingolipid metabolic disease, globoid cell leukodystrophy (GLD). Indeed, Psy is known to accumulate in the patients' brains. Interestingly, inhibition of sphingolipid biosynthesis also induced multinuclear cells. When cells were treated with a new immunosuppressant, ISP-1/myriocin, which inhibits serine palmitoyltransferase, the first step enzyme of sphingolipid biosynthesis, the cells underwent multinucleation and apoptosis. At present, a definitive model of the function of sphingolipids as to the induction of multinuclear cell formation is not available due to the rudimentary information but possible mechanisms are discussed.

Animals↗