Akiko Tsuchida

Nagoya University, Nagoya-shi, Aichi-ken, Japan

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Publications (15)27.7 Total impact

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    ABSTRACT: We previously demonstrated that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in DNA microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)3 on Syndecan-1 in high metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer metastasis. Here, we analyzed roles of tTn antigen in cancer properties. tTn antigen on Syndecan-1 increased cell adhesion to fibronectin in an integrin-dependent manner. Furthermore, cell adhesion to fibronectin induced phosphorylation of focal adhesion kinase and paxillin in T13-transfectant cells. In the search of Syndecan-1-interacting molecules, it was demonstrated that tTn antigen-carrying Syndecan-1 interacted with integrin α5β1 and MMP-9, and these molecules shifted to glycolipid-enriched microdomain (GEM)/rafts along with increased metastatic potential in T13-transfectant cells. We also identified tTn-substitution site on Syndecan-1, demonstrating that tTn on Syndecan-1 is essential for the interaction with integrin α5β1 as well as for the reaction with mAb MLS128. These data suggested that high expression of ppGalNAc-T13 gene generated tTn antigen on Syndecan-1 under reduced expression of GM1, leading to the enhanced invasion and metastasis via the formation of a molecular complex consisting of integrin α5β1, Syndecan-1 and MMP-9 in GEM/rafts.
    Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
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    ABSTRACT: In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80 kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.
    Biochemical and Biophysical Research Communications 03/2012; 419(1):7-13. · 2.41 Impact Factor
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    ABSTRACT: Assay methods for representative glycosyltransferases were described, covering those involved in the synthesis of glycosphingolipids, N-glycans, O-glycans, and proteoglycan glycosaminoglycans. In addition, intracellular localization of glycosyltransferase was comprehensively summarized. Lastly, complex formation of glycosyltransferase proteins with related molecules including subunits, chaperones, and enzyme regulators, that have been recently reported was also summarized.
    Glycoconjugate Journal 09/2008; 26(8):987-98. · 1.88 Impact Factor
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    ABSTRACT: Molecular mechanisms for both morphogenesis and carcinogenesis have frequently overlapped, and similar signaling pathways are often involved in these processes. Yamamoto et al. identified a novel protein that induces head formation in Xenopus (Yamamoto et al. Cell, 120, 223-225, 2005). This new protein, named Shisa, plays unique roles in head formation by suppressing the maturation processes of receptors for Wnt and FGF at the endoplasmic reticulum. Here, we have identified a human homologue of the shisa gene (hu-shisa-2), and analyzed its expression in various human cancer cell lines by real-time reverse transcription polymerase chain reaction. High levels of mRNA expression were observed in some neuroectoderm-derived human cancer cell lines and small cell lung cancer cell lines. Intracellular localization of hu-Shisa-2 protein was also analyzed, indicating that it is present in the endoplasmic reticulum. Over-expression of hu-Shisa-2 resulted in increased cell growth and invasion, suggesting that hu-Shisa-2 is involved in the evolution and/or progression of human cancers.
    Nagoya journal of medical science 09/2008; 70(3-4):73-82.
  • Koichi Furukawa, Akiko Tsuchida, Keiko Furukawa
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    ABSTRACT: Of the glycosyltransferases involved in the synthesis of glycosphingolipids, those involved in the common steps, those responsible for the synthesis of globo-series glycolipids, and those for the synthesis of lacto/neolacto-series glycolipids were summarized here. The common and fundamental steps consist of reactions catalyzed by Glc-Cer synthase (Ichikawa et al. 1996), lactosylceramide synthase (Nomura et al. 1997), or GalCer synthase (Schulte and Stoffel 1993). Globo-series includes the reactions catalyzed by Gb3/CD77 synthase (Furukawa et al. 2007), Gb4 synthase (Furukawa et al. 2007), Gb5 synthase (Zhou et al. 2000), or sialyl-Gb5 synthase (monosialyl-galactosylgloboside, MSGG) (Saito et al. 2003). Furthermore, DSGG (disialyl-galactosylgloboside) (Furukawa et al. 2007) is synthesized from MSGG by ST6GalNAcVI. Lacto/neolactoseries are generated by various enzymes consisting of amino-CTH synthase (β3GlcNAcTV) (Togayachi et al. 2001), core 1 precursor synthase (β3GalTV) (Isshiki et al. 1999) β4-galactosyltransferase, α2,3/6-sialyltransferase, α1,3/4-fucosyl transferase, ST6GalNAc-VI (α2,6-sialyltransferase) (Furukawa et al. 2007), etc.
    12/2007: pages 53-55;
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    ABSTRACT: Although disialyl glycosphingolipids such as GD3 and GD2 have been considered to be associated with malignant tumours, whether branched-type disialyl glycosphingolipids show such an association is not well understood. We investigated the sialyltransferases responsible for the biosynthesis of DSGG (disialylgalactosylgloboside) from MSGG (monosialylgalactosylgloboside). Among six GalNAc:alpha2,6-sialyltransferases cloned to date, we focused on ST6GalNAc III, V and VI, which utilize sialylglycolipids as substrates. In vitro enzyme analyses revealed that ST6GalNAc III and VI generated DSGG from MSGG with V(max)/K(m) values of 1.91 and 4.16 respectively. Transfection of the cDNA expression vectors for these enzymes resulted in DSGG expression in a renal cancer cell line. Although both ST6GalNAc III and VI genes were expressed in normal kidney cells, the expression profiles of ST6GalNAc VI among 20 renal cancer cell lines correlated clearly with those of DSGG, suggesting that the sialyltransferase involved in the synthesis of DSGG in the kidney is ST6GalNAc-VI. ST6GalNAc-VI and DSGG were found in proximal tubule epithelial cells in normal kidney tissues, while they were downregulated in renal cancer cell lines and cancer tissues. All these findings indicated that DSGG was suppressed during the malignant transformation of the proximal tubules as a maturation arrest of glycosylation.
    Biochemical Journal 04/2007; 402(3):459-70. · 4.65 Impact Factor
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    ABSTRACT: We isolated human ST6GalNAc III cDNA clones. The typical cDNA clones predicted a type II membrane protein of 305 amino acids with a short cytoplasmic transmembrane domain of sixteen amino acids and a catalytic domain of 280 amino acids. A short form clone predicted a protein of 240 amino acids lacking 65 amino acids including the transmembrane portion. The alternative usage of the second exon seemed to generate these two transcripts. Both had two common regions found among sialyltransferases cloned so far, i.e. sialyl motif L and sialyl motif S. Alignments of human, mouse and rat orthologs indicated that high homologies, i.e. 85-95% identity among these species at amino acid levels. We analyzed the expression pattern and substrate specificity of the product, demonstrating a very restricted expression pattern and a high substrate specificity. Northern blotting revealed that hST6GalNAc III is expressed in kidney and brain as a single band at 3.2 kb. In enzyme assay of the long form, the transfer of sialic acid onto alpha2,3-sialylated acceptor substrates, i.e. GM1b and sialyl lactotetraosylceramide, was observed. hST6GalNAc III also showed sialyltransferase activity toward O-glycans (but not N-glycans) in fetuin.
    Journal of Biochemistry 10/2005; 138(3):237-43. · 3.07 Impact Factor
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    ABSTRACT: Asymmetric o- and m-nitrobenzoic acids with a 1,3-benzodioxole skeleton have been synthesized and characterized. The synthesis involved nitration at either the C-5 or C-6 position of 2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylate derived from catechol. These were then resolved with chiral solid-phased HPLC columns into each of enantiomers (>99% ee), which were characterized by circular dichroism. The (S)-enantiomers were also obtained by fractional crystallization of the precursor prior to the nitration. When applied as chiral derivation agents for alcohols and amines, m-nitrobenzoic acid showed notable ability to separate the enantiomers in both HPLC and NMR analyses. On the other hand, 1H NMR and X-ray data of their ester and amide derivatives strongly suggested that the o-nitrobenzoic acid would serve as a fluorescent building block useful for the assembly of β-peptide linkages with a defined conformation.
    Tetrahedron Asymmetry 01/2004; 15(1):159–165. · 2.12 Impact Factor
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    ABSTRACT: Biosynthesis of disialyl Lewis a (Lea) was analyzed using previously cloned ST6GalNAc V and ST6GalNAc VI, which were responsible for the synthesis of alpha-series gangliosides. Among lactotetraosylceramide (Lc4), neolactotetraosylceramide, and their sialyl forms, only sialyl Lc4 was sialylated with ST6GalNAc V and ST6GalNAc VI. The products were confirmed to be disialyl Lea in TLC-immunostaining. Compared with the original substrate GM1b, the synthetic rates of disialyl Lea were 22 and 38% with ST6GalNAc V and ST6GalNAc VI, respectively. Since sialyl Lea could not be converted to disialyl Lea, disialyl Lea was produced only from disialyl Lc4. Therefore, it appears that ST6GalNAc V/VI and fucosyltransferase III (FUT-3) compete for sialyl Lc4, their common substrate. The results of either one transfection or co-transfection of two genes into COS1 cells revealed that both ST6GalNAc VI and FUT-3 contributed in the synthesis of disialyl Lea but partly compete with each other. Many colon cancer cell lines expressed the ST6GalNAc VI gene more or less, and some of them actually expressed disialyl Lea. None of them expressed ST6GalNAc V. These results suggested the novel substrate specificity of ST6GalNAc VI, which is responsible for the synthesis of disialyl Lea but not for alpha-series gangliosides in human colon tissues.
    Journal of Biological Chemistry 07/2003; 278(25):22787-94. · 4.65 Impact Factor
  • Bulletin of The Chemical Society of Japan - BULL CHEM SOC JPN. 01/2002; 75(12):2681-2685.
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    ABSTRACT: Adsorption of glycopolymers is essential for the development of biologically active surfaces employing oligosaccharides as recognition signals. The adsorption behavior has been investigated by quartz-crystal microbalances (QCMs) with use of two different series of glycopolymers: (1) polystyrenes carrying maltooligosaccharides with different saccharide lengths and (2) lactose-carrying polymers with polystyrene and polyphenylacrylamide main chain structures. These glycopolymers were adsorbed on the hydrophobic surfaces of self-assembled monolayers (SAM) of 1-octadecanethiol and cast films of polystyrene from their aqueous solutions, but not on hydrophilic surfaces of SAM of cystamine hydrochloride. The apparent association constants (Ka = 104 ˜ 106 M–1 in molarity of the monomeric unit) and maximum adsorption amounts (Δmmax) on 1-octadecanethiol SAMs and polystyrene cast films were estimated from the Langmuir adsorption isotherms. These values depended on the side-chain oligosaccharide lengths and the main chain chemical structures of these glycopolymers. The adsorption behavior has been discussed in correlation with the desorption of the adsorbed polymer in an aqueous surfactant solution, the partition of the glycopolymer in a water/1-octanol system, and the adsorption amount of bovine serum albumin (BSA) onto the glycopolymer-adsorbed surface. We propose that these polymers are adsorbed in loop-train-tail conformations in which the ratio of the loop-tail portion to the train portion depends on the hydrophilic-hydrophobic property of the polymers.
    Macromolecular Chemistry and Physics 10/2000; 201(17):2245 - 2250. · 2.39 Impact Factor
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    ABSTRACT: Glycoconjugate polystyrenes bearing sialyllactose moieties were prepared via a simple method from a mixture of 2-6 and 2-3 linked sialyllactose isomers of bovine milk origin. The reducing end of sialyllactose was converted to an amino function with ammonium hydrogen carbonate and then coupled with p -vinylbenzoyl chloride. The resulting styrene derivative substituted with sialyllactose via an amide linkage was polymerized with ammonium peroxodisulfate and N,N,N,N -tetramethylethylenediamine in water at 30 C. The interaction of the glycopolymer with influenza A and B viruses was investigated by three different methods. The glycopolymer inhibited the hemagglutination of influenza A virus (PR/8/34) and its activity was 103 times higher than that of the oligosaccharide itself. The cytopathic effect of virus-infected MDCK (Madine-Darby canine kidney) cells was inhibited by the glycopolymer. The homopolymer showed 102 times higher inhibitory activity than naturally-occurring fetuin. It was also found that various viruses could be trapped by the glycopolymer adsorbed on a polystyrene surface. The inhibitory and trapping activities of the glycopolymers were correlated with the sialyl linkage specificities of the virus strains.
    Glycoconjugate Journal 10/1998; 15(11):1047-1054. · 1.88 Impact Factor
  • Chemistry Letters - CHEM LETT. 01/1998;
  • Bulletin of The Chemical Society of Japan - BULL CHEM SOC JPN. 01/1998; 71(12):2973-2977.
  • Kazukiyo Kobayashi, Akiko Tsuchida
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    ABSTRACT: A convenient synthetic route to a new type of artificial glycoconjugate polymer has been designed to develop biomedical materials using oligosaccharide moieties as recognition signals. An amino function was introduced to the reducing end of lactose and N,N‘-diacetylchitobiose with ammonium hydrogen carbonate and then was allowed to react with p-vinylbenzoyl chloride. The N-glycosidation proceeded stereospecifically in one flask to give only the β-glycoside without any protection and deprotection steps. The resulting p-vinylbenzamide glycoside derivatives were homo- and copolymerized with acrylamide using 2,2‘-azobisisobutyronitrile as initiator in dimethyl sulfoxide at 60 °C. The interaction of the glycopolymers with lectins was investigated by means of a two-dimensional immunodiffusion test in agar and inhibition of the hemagglutinating activity. The specificity of lectins with these glycopolymers was similar to that reported for naturally-occurring glycoconjugates. Binding between wheat germ agglutinin lectin (WGA) and poly((p-vinylbenzamido)-β-diacetylchitobiose) was increased by 103 times compared with that of the oligosaccharide itself. The enhancement was attributed to the presence of the hydrophobic phenyl aglycon as well as the high density, multiantennary disaccharide ligands along the polymer chain. The present synthetic method is useful to introduce biologically important, complex oligosaccharides into glycopolymers.
    Macromolecules. 04/1997; 30(7).