論文 - 林 康広
-
Murakami C., Hoshino F., Sakai H., Hayashi Y., Yamashita A., Sakane F.
Journal of Biological Chemistry 295 ( 10 ) 2932 - 2947 2020年3月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro. Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.
-
Hayashi Y., Nemoto-Sasaki Y., Matsumoto N., Hama K., Tanikawa T., Oka S., Saeki T., Kumasaka T., Koizumi T., Arai S., Wada I., Yokoyama K., Sugiura T., Yamashita A.
Journal of Biological Chemistry 293 ( 45 ) 17505 - 17522 2018年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1–GCS complex, resulting in a significant reduction in SM synthesis in vivo. In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo. These results suggest that formation of the SMS1–GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1–GCS complex was confirmed by CRISPR/Cas9 –mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.
-
Yamashita A., Hayashi Y., Matsumoto N., Nemoto-Sasaki Y., Koizumi T., Inagaki Y., Oka S., Tanikawa T., Sugiura T.
Biology 6 ( 2 ) 23 2017年6月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Biology
The coenzyme A (CoA)-independent transacylation system catalyzes fatty acid transfer from phospholipids to lysophospholipids in the absence of cofactors such as CoA. It prefers to use C20 and C22 polyunsaturated fatty acids such as arachidonic acid, which are esterified in the glycerophospholipid at the sn-2 position. This system can also acylate alkyl ether-linked lysophospholipids, is involved in the enrichment of arachidonic acid in alkyl ether-linked glycerophospholipids, and is critical for the metabolism of eicosanoids and platelet-activating factor. Despite their importance, the enzymes responsible for these reactions have yet to be identified. In this review, we describe the features of the Ca2+-independent, membrane-bound CoA-independent transacylation system and its selectivity for arachidonic acid. We also speculate on the involvement of phospholipase A2 in the CoA-independent transacylation reaction.
-
Hayashi Y., Nemoto-Sasaki Y., Matsumoto N., Tanikawa T., Oka S., Tanaka Y., Arai S., Wada I., Sugiura T., Yamashita A.
Journal of Biological Chemistry 292 ( 3 ) 1122 - 1141 2017年1月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
Sphingomyelin synthase (SMS) is the key enzyme for crosstalk between bioactive sphingolipids and glycerolipids. In mammals, SMS consists of two isoforms: SMS1 is localized in the Golgi apparatus, whereas SMS2 is localized in both the Golgi and plasma membranes. SMS2 seems to exert cellular functions through protein-protein interactions; however, the existence and functions of quaternary structures of SMS1 and SMS2 remain unclear. Here we demonstrate that both SMS1 and SMS2 form homodimers. The SMSs have six membrane-spanning domains, and the Nand Ctermini of both proteins face the cytosolic side of the Golgi apparatus. Chemical cross-linking and bimolecular fluorescence complementation revealed that the N-and/or C-terminal tails of the SMSs were in close proximity to those of the other SMS in the homodimer. Homodimer formation was significantly decreased by C-terminal truncations, SMS1-αC22 and SMS2-αC30, indicating that the C-terminal tails of the SMSs are primarily responsible for homodimer formation. Moreover, immunoprecipitation using deletion mutants revealed that the C-terminal tail of SMS2 mainly interacted with the C-terminal tail of its homodimer partner, whereas the C-terminal tail of SMS1 mainly interacted with a site other than the C-terminal tail of its homodimer partner. Interestingly, homodimer formation occurred in the endoplasmic reticulum (ER) membrane before trafficking to the Golgi apparatus. Reduced homodimerization caused by C-terminal truncations of SMSs significantly reduced ER-to-Golgi transport. Our findings suggest that the C-terminal tails of SMSs are involved in homodimer formation, which is required for efficient transport from the ER.
-
Klotho-Related Protein KLrP: Structure and Functions 査読あり
Hayashi Y., Ito M.
Vitamins and Hormones 101 1 - 16 2016年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Vitamins and Hormones
Klotho (KL) family proteins share one or two glycoside hydrolase (GH) motifs homologous to GH family 1. However, the biological significance of GH motifs in KL family proteins remains elusive. We describe here that KL-related protein (KLrP), which is composed of a single GH motif, is a cytosolic β-glucocerebrosidase (GCase, EC 3.2.1.145). We detected a neutral conduritol B epoxide (CBE)-insensitive glucosylceramide (GlcCer)-degrading activity in the cytosol fractions of human fibroblasts, rat brains, and zebrafish embryos. KL family proteins emerged as a potent candidate for the neutral GCase using a bioinformatics approach. Recombinant human KLrP, but not α-KL, β-KL, or KLPH, exhibited GCase activity with a neutral pH optimum in the presence of CBE. We solved the crystal structures of KLrP and a KLrP mutant (E165Q) in complex with glucose, which indicate that KLrP forms a (β/α)8TIM barrel structure with the double-displacement mechanism of the retaining β-glycosidase. Furthermore, knockdown of endogenous KLrP in CHOP cells using small interfering RNA (siRNA) decreased the CBE-insensitive neutral GCase activity and increased the cellular levels of GlcCer, which suggests that KLrP is involved in a novel GlcCer catabolism pathway. A KLrP D106N mutant was discovered in patients with severe Gaucher disease; however, this mutation did not affect the GCase activity of KLrP. © 2016 Elsevier Inc.
-
Das D., Maeda K., Hayashi Y., Gavande N., Desai D.V., Chang S.B., Ghosh A.K., Mitsuya H.
Antimicrobial Agents and Chemotherapy 59 ( 4 ) 1895 - 1904 2015年4月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Antimicrobial Agents and Chemotherapy
The cellular entry of HIV-1 into CD4<sup>+</sup> T cells requires ordered interactions of HIV-1 envelope glycoprotein with C-X-C chemokine receptor type 4 (CXCR4) receptors. However, such interactions, which should be critical for rational structure-based discovery of new CXCR4 inhibitors, remain poorly understood. Here we first determined the effects of amino acid substitutions in CXCR4 on HIV-1<inf>NL4-3</inf> glycoprotein-elicited fusion events using site-directed mutagenesis-based fusion assays and identified 11 potentially key amino acid substitutions, including D97A and E288A, which caused >30% reductions in fusion. We subsequently carried out a computational search of a screening library containing∼604,000 compounds, in order to identify potential CXCR4 inhibitors. The computational search used the shape of IT1t, a known CXCR4 inhibitor, as a reference and employed various algorithms, including shape similarity, isomer generation, and docking against a CXCR4 crystal structure. Sixteen small molecules were identified for biological assays based on their high shape similarity to IT1t, and their putative binding modes formed hydrogen bond interactions with the amino acids identified above. Three compounds with piperidinylethanamine cores showed activity and were resynthesized. One molecule, designated CX6, was shown to significantly inhibit fusion elicited by X4 HIV-1<inf>NL4-3</inf> glycoprotein (50% inhibitory concentration [IC<inf>50</inf>], 1.9 μM), to inhibit Ca2<sup>+</sup> flux elicited by stromal cell-derived factor 1α (SDF-1α) (IC<inf>50</inf>, 92 nM), and to exert anti-HIV-1 activity (IC<inf>50</inf>, 1.5 μM). Structural modeling demonstrated that CX6 bound to CXCR4 through hydrogen bond interactions with Asp97 and Glu288. Our study suggests that targeting CXCR4 residues important for fusion elicited by HIV-1 envelope glycoprotein should be a useful and feasible approach to identifying novel CXCR4 inhibitors, and it provides important insights into the mechanism by which small-molecule CXCR4 inhibitors exert their anti-HIV-1 activities.
DOI： 10.1128/AAC.04654-14
-
Das D., Maeda K., Hayashi Y., Gavande N., Desai D.V., Chang S.B., Ghosh A.K., Mitsuya H.
Antimicrobial Agents and Chemotherapy 59 ( 4 ) 1895 - 1904 2015年4月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Antimicrobial Agents and Chemotherapy
The cellular entry of HIV-1 into CD4<sup>+</sup> T cells requires ordered interactions of HIV-1 envelope glycoprotein with C-X-C chemokine receptor type 4 (CXCR4) receptors. However, such interactions, which should be critical for rational structure-based discovery of new CXCR4 inhibitors, remain poorly understood. Here we first determined the effects of amino acid substitutions in CXCR4 on HIV-1<inf>NL4-3</inf> glycoprotein-elicited fusion events using site-directed mutagenesis-based fusion assays and identified 11 potentially key amino acid substitutions, including D97A and E288A, which caused >30% reductions in fusion. We subsequently carried out a computational search of a screening library containing∼604,000 compounds, in order to identify potential CXCR4 inhibitors. The computational search used the shape of IT1t, a known CXCR4 inhibitor, as a reference and employed various algorithms, including shape similarity, isomer generation, and docking against a CXCR4 crystal structure. Sixteen small molecules were identified for biological assays based on their high shape similarity to IT1t, and their putative binding modes formed hydrogen bond interactions with the amino acids identified above. Three compounds with piperidinylethanamine cores showed activity and were resynthesized. One molecule, designated CX6, was shown to significantly inhibit fusion elicited by X4 HIV-1<inf>NL4-3</inf> glycoprotein (50% inhibitory concentration [IC<inf>50</inf>], 1.9 μM), to inhibit Ca2<sup>+</sup> flux elicited by stromal cell-derived factor 1α (SDF-1α) (IC<inf>50</inf>, 92 nM), and to exert anti-HIV-1 activity (IC<inf>50</inf>, 1.5 μM). Structural modeling demonstrated that CX6 bound to CXCR4 through hydrogen bond interactions with Asp97 and Glu288. Our study suggests that targeting CXCR4 residues important for fusion elicited by HIV-1 envelope glycoprotein should be a useful and feasible approach to identifying novel CXCR4 inhibitors, and it provides important insights into the mechanism by which small-molecule CXCR4 inhibitors exert their anti-HIV-1 activities.
DOI： 10.1128/AAC.04654-14
-
Glycerophosphate/Acylglycerophosphate acyltransferases 査読あり
Yamashita A., Hayashi Y., Matsumoto N., Nemoto-Sasaki Y., Oka S., Tanikawa T., Sugiura T.
Biology 3 ( 4 ) 801 - 830 2014年11月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Biology
Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) and acyl-CoA: 1-acyl-glycerol-3-phosphate acyltransferase (AGPAT) are involved in the de novo synthesis of triacylglycerol (TAG) and glycerophospholipids. Many enzymes belonging to the GPAT/AGPAT family have recently been identified and their physiological or pathophysiological roles have been proposed. The roles of GPAT/AGPAT in the synthesis of TAG and obesity-related diseases were revealed through the identification of causative genes of these diseases or analyses of genetically manipulated animals. Recent studies have suggested that some isoforms of GPAT/AGPAT family enzymes are involved in the fatty acid remodeling of phospholipids. The enzymology of GPAT/AGPAT and their physiological/ pathological roles in the metabolism of glycerolipids have been described and discussed in this review.
-
Hayashi Y., Nemoto-Sasaki Y., Tanikawa T., Oka S., Tsuchiya K., Zama K., Mitsutake S., Sugiura T., Yamashita A.
Journal of Biological Chemistry 289 ( 44 ) 30842 - 30856 2014年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼ 5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor-co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.
-
Genome-wide linkage and exome analyses identify variants of HMCN1 for splenic epidermoid cyst 査読あり
Omer W.H., Narita A., Hosomichi K., Mitsunaga S., Hayashi Y., Yamashita A., Krasniqi A., Iwasaki Y., Kimura M., Inoue I.
BMC Medical Genetics 15 ( 1 ) 2014年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：BMC Medical Genetics
Background: Splenic epidermoid cyst is a benign tumor-like lesion affecting the spleen and sometimes occurs in familial form. The causality of such rare diseases remain challenging, however recently, with the emergence of exome re-sequencing, the genetics of many diseases have been unveiled. In the present study, we performed a combinatorial approach of genome-wide parametric linkage and exome analyses for a moderate-sized Japanese family with frequent occurrence of splenic epidermoid cyst to identify the genetic causality of the disease. Methods: Twelve individuals from the family were subject to SNP typing and exome re-sequencing was done for 8 family members and 4 unrelated patients from Kosovo. Linkage was estimated using multi-point parametric linkage analysis assuming a dominant mode of inheritance. All of the candidate variants from exome analysis were confirmed by direct sequencing. Results: The parametric linkage analysis suggested two loci on 1q and 14q with a maximal LOD score of 2.5 . Exome generated variants were prioritized based on; impact on the protein coding sequence, novelty or rareness in public databases, and position within the linkage loci. This approach identified three variants; variants of HMCN1 and CNTN2 on 1q and a variant of DDHD1 on 14q. The variant of HMCN1 (p.R5205H) showed the best co-segregation in the family after validation with Sanger sequencing. Additionally, rare missense variants (p.A4704V, p.T5004I, and p.H5244Q) were detected in three unrelated Kosovo patients. The identified variants of HMCN1 are on conserved domains, particularly the two variants on calcium-binding epidermal growth factor domain. Conclusions: The present study, by combining linkage and exome analyses, identified HMCN1 as a genetic causality of splenic epidermoid cyst. Understanding the biology of the disease is a key step toward developing innovative approaches of intervention.
-
Yamashita A., Hayashi Y., Nemoto-Sasaki Y., Ito M., Oka S., Tanikawa T., Waku K., Sugiura T.
Progress in Lipid Research 53 ( 1 ) 18 - 81 2014年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Progress in Lipid Research
Over one hundred different phospholipid molecular species are known to be present in mammalian cells and tissues. Fatty acid remodeling systems for phospholipids including acyl-CoA:lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems, are involved in the biosynthesis of these molecular species. Acyl-CoA:lysophospholipid acyltransferase system is involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-dependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. The CoA-dependent transacylation reaction in the rat liver exhibits strict fatty acid specificity, i.e., three types of fatty acids (20:4, 18:2 and 18:0) are transferred. On the other hand, CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids, especially ether-containing lysophospholipids, in the absence of any cofactors. CoA-independent transacylase is assumed to be involved in the accumulation of PUFA in ether-containing phospholipids. These enzymes are involved in not only the remodeling of fatty acids, but also the synthesis and degradation of some bioactive lipids and their precursors. In this review, recent progresses in acyltransferase research including the identification of the enzyme's genes are described.© 2013 Elsevier Ltd. All rights reserved.
-
The actions and metabolism of lysophosphatidylinositol, an endogenous agonist for GPR55 査読あり
Yamashita A., Oka S., Tanikawa T., Hayashi Y., Nemoto-Sasaki Y., Sugiura T.
Prostaglandins and Other Lipid Mediators 107 103 - 116 2013年6月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Prostaglandins and Other Lipid Mediators
Lysophosphatidylinositol (LPI) is a subspecies of lysophospholipid and is assumed to be not only a degradation product of phosphatidylinositol (PI), but also a bioactive lysophospholipid mediator. However, not much attention has been directed toward LPI compared to lysophosphatidic acid (LPA), since the receptor for LPI has not been identified. During screening for an agonist for the orphan G protein coupled receptor GPR55, we identified LPI, 2-arachidonoyl LPI in particular, as an agonist for GPR55. Our efforts to identify an LPI receptor facilitated research on LPI as a lipid messenger. In addition, we also found that DDHD1, previously identified as phosphatidic acid-preferring phospholipase A1, was one of the synthesizing enzymes of 2-arachidonoyl LPI. Here, we summarized the background for discovering the LPI receptor, and the actions/metabolism of LPI. We also referred to the biosynthesis of PI, a 1-stearoyl-2-arachidonoyl species, since the molecule is the precursor of 2-arachidonoyl LPI. Furthermore, we discussed physiological and/or pathophysiological processes involving LPI and GPR55, including the relevance of LPI-GPR55 and cannabinoids, since GPR55 was previously postulated to be another cannabinoid receptor. Although there is no doubt that GPR55 is the LPI receptor, we should re-consider whether or not GPR55 is in fact another cannabinoid receptor. © 2013 Elsevier Inc. All rights reserved.
-
Nomura K.H., Murata D., Hayashi Y., Dejima K., Mizuguchi S., Kage-Nakadai E., Gengyo-Ando K., Mitani S., Hirabayashi Y., Ito M., Nomura K.
Glycobiology 21 ( 6 ) 834 - 848 2011年6月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Glycobiology
Ceramide glucosyltransferase (Ugcg) [uridine diphosphate (UDP)-glucose:N-acylsphingosine d-glucosyltransferase or UDP-glucose ceramide glucosyltransferase (GlcT): EC 2.4.1.80] catalyzes formation of glucosylceramide (GlcCer) from ceramide and UDP-glucose. There is only one Ugcg gene in the mouse genome, which is essential in embryogenesis and brain development. The nematode Caenorhabditis elegans has three Ugcg genes (cgt-1, cgt-2 and cgt-3), and double RNAi of the cgt-1 and cgt-3 genes results in lethality at the L1 larval stage. In this study, we isolated knockout worms for the three genes and characterized the gene functions. Each gene product showed active enzymatic activity when expressed in GM95 cells deficient in glycosphingolipids (GSLs). When each gene function was disrupted, the brood size of the animal markedly decreased, and abnormal oocytes and multinucleated embryos were formed. The CGT-3 protein had the highest Ugcg activity, and knockout of its gene resulted in the severest phenotype. When cgt-3 RNAi was performed on rrf-1 worms lacking somatic RNAi machinery but with intact germline RNAi machinery, a number of abnormal oocytes and multinucleated eggs were observed, although the somatic phenotype, i.e., L1 lethal effects of cgt-1/cgt-3 RNAi, was completely suppressed. Cell surface expression of GSLs and sphingomyelin, which are important components of membrane domains, was affected in the RNAi-treated embryos. In the embryos, an abnormality in cytokinesis was also observed. From these results, we concluded that the Ugcg gene is indispensable in the germline and that an ample supply of GlcCer is needed for oocytes and fertilized eggs to maintain normal membranes and to proceed through the normal cell cycle. © 2011 The Author.
-
Zama K., Hayashi Y., Ito S., Hirabayashi Y., Inoue T., Ohno K., Okino N., Ito M.
Glycobiology 19 ( 7 ) 767 - 775 2009年6月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Glycobiology
We report here a method of simultaneously quantifying glucosylceramide (GlcCer) and galactosylceramide (GalCer) by normal-phase HPLC using O-phtalaldehyde derivatives. Treatment with sphingolipid ceramide N-deacylase which converts the cerebrosides in the sample to their lyso-forms was followed by the quantitative labeling of free NH2 groups of the lyso-cerebrosides with O-phtalaldehyde. Using this method, 14.1 pmol of GlcCer and 10.4 pmol of GalCer, and 108.1 pmol of GlcCer and 191.1 pmol of GalCer were detected in zebrafish embryos and RPMI 1864 cells, respectively, while 22.2 pmol of GlcCer but no GalCer was detected in CHOP cells using cell lysate containing 100 μg of protein. Linearity for the determination of each cerebroside was observed from 50 to 400 μg of protein under the conditions used, which corresponds to approximately 103 to 105 RPMI cells and 5 to 80 zebrafish embryos. The present method clearly revealed that the treatment of RPMI cells with a GlcCer synthase inhibitor P4 resulted in a marked decrease in GlcCer but not GalCer, concomitantly with a significant decrease in the GlcCer synthase activity. On the other hand, GlcCer but not GalCer increased 2-fold when an acid glucocerebrosidase inhibitor CBE was injected into zebrafish embryos. Interestingly, the treatment of CHOP cells with ciclosporin A increased GlcCer possibly due to the inhibition of LacCer synthase. A significant increase in levels of GlcCer in fibroblasts from patients with Gaucher disease was clearly shown by the method. The proposed method is useful for the determination of GlcCer and GalCer levels in various biological samples. © The Author 2009. Published by Oxford University Press. All rights reserved.
-
Hayashi Y., Zama K., Abe E., Okino N., Inoue T., Ohno K., Ito M.
Analytical Biochemistry 383 ( 1 ) 122 - 129 2008年12月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Analytical Biochemistry
The activity of lysosomal acid β-glucocerebrosidase (AGC, EC 3.2.1.45), which hydrolyzes the O-glycosidic linkage between d-glucose and ceramide of glucosylceramide (GlcCer), is a marker for the diagnosis of Gaucher disease because the disease is caused by dysfunction of AGC due to mutations in the gene. The activity of AGC is potently inhibited by conduritol B epoxide (CBE), whereas CBE-insensitive nonlysosomal neutral β-glucocerebrosidase (NGC) activities have been found in various vertebrates, including humans. We report here a new reliable method to determine AGC as well as NGC activities using normal-phase high-performance liquid chromatography (HPLC) and NBD (4-nitrobenzo-2-oxa-1,3-diazole)- or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-labeled GlcCer as a substrate. The reaction products of the enzymes, C6-NBD-ceramide and C12-BODIPY-ceramide, were clearly separated from the corresponding substrates on a normal-phase column within 5 min using a different solvent system. Reaction products could be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol for C6-NBD-ceramide and from 10 fmol to 5 pmol for C12-BODIPY-ceramide. Vmax/Km values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β-d-glucoside (4MU-Glc), which is used prevalently for Gaucher disease diagnosis. As a result, AGC activity was detected quantitatively using fluorescent GlcCer, but not 4MU-Glc, using 5 μl of human serum or 1 × 104 cultured human fibroblasts. The current method clearly showed the decrease of AGC activities in fibroblasts and serum from the patient with Gaucher disease compared with normal individuals, suggesting that the method is applicable for the diagnosis of Gaucher disease. Furthermore, this method was found to be useful for measuring the activities of nonlysosomal NGC of various cells and tissues in the presence of CBE. © 2008 Elsevier Inc. All rights reserved.
-
Crystal structure of the covalent intermediate of human cytosolic β-glucosidase 査読あり
Noguchi J., Hayashi Y., Baba Y., Okino N., Kimura M., Ito M., Kakuta Y.
Biochemical and Biophysical Research Communications 374 ( 3 ) 549 - 552 2008年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Biochemical and Biophysical Research Communications
Human cytosolic β-glucosidase, also known as klotho-related protein (KLrP, GBA3), is an enzyme that hydrolyzes various β-d-glucosides, including glucosylceramide. We recently reported the crystal structure of KLrP in complex with glucose [Y. Hayashi, N. Okino, Y. Kakuta, T. Shikanai, M. Tani, H. Narimatsu, M. Ito, Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase, J. Biol. Chem. 282 (2007) 30889-30900]. Here, we report the crystal structure of a covalent intermediate of the KLrP mutant E165Q, in which glucose was covalently bound to a nucleophile, Glu373. The structure confirms the double displacement mechanism of the retaining β-glucosidase. In addition, the structure suggests that a water molecule could be involved in the stabilization of transition states through a sugar, 2-hydroxyl. © 2008 Elsevier Inc. All rights reserved.
-
Klotho-related protein is a novel cytosolic neutral β- glycosylceramidase 査読あり
Hayashi Y., Okino N., Kakuta Y., Shikanai T., Tani M., Narimatsu H., Ito M.
Journal of Biological Chemistry 282 ( 42 ) 30889 - 30900 2007年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a β-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl- glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k cat/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6 Å resolution revealed that KLrP is a (β/α)8 TIM barrel, in which Glu165 and Glu 373 at the carboxyl termini of β-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
-
Abe E., Hayashi Y., Hama Y., Hayashi M., Inagaki M., Ito M.
Journal of Biochemistry 140 ( 2 ) 247 - 253 2006年8月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biochemistry
Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3- phosphocholine." © 2006 The Japanese Biochemical Society.
DOI： 10.1093/jb/mvj145
-
Hayashi Y., Horibata Y., Sakaguchi K., Okino N., Ito M.
Analytical Biochemistry 345 ( 2 ) 181 - 186 2005年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Analytical Biochemistry
Glucosylceramide synthase (GlcT) and lactosylceramide synthase (GalT) are key enzymes for the synthesis of major glycosphingolipids of vertebrates. In this article, we report a new reliable method to determine GlcT and GalT activities using the fluorescent acceptor substrates C6-4-nitrobenzo-2-oxa-1,3- diazole (NBD)-ceramide and C6-NBD-glucosylceramide, respectively, and a normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-glucosylceramide for GlcT and C6-NBD-lactosylceramide for GalT, could be separated from the corresponding acceptor substrates within 6 min under the conditions used. Reaction products were able to be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol, making it possible to determine both activities using the lysate from 1 × 10 4 cultured CHOP cells (Chinese hamster ovary cells expressing polyoma LT antigen) and one zebrafish embryo. This method was used successfully to evaluate the degree of knockdown of GlcT and GalT during zebrafish embryogenesis after injection of the morpholino-oligo-based antisense into one- to four-cell embryos. These results indicate that the fluorescence-based HPLC method is a highly sensitive, rapid, and reproducible assay for determining GlcT and GalT activities and is useful for evaluating the activities in gene knockdown experiments. © 2005 Elsevier Inc. All rights reserved.