Akiko Tsuchida

SIMPLE SYNTHESIS OF SIALYLLACTOSE-CARRYING POLYSTYRENE AND ITS BINDING WITH INFLUENZA VIRUS

Glycoconjugate polystyrenes bearing sialyllactose moieties were prepared via a simple method from a mixture of α2-6 and α2-3 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.

Identification and expression of a sialyltransferase responsible for the synthesis of disialylgalactosylgloboside in normal and malignant kidney cells: downregulation of ST6GalNAc VI in renal cancers

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.

pp-GalNAc-T13 induces high metastatic potential of murine Lewis lung cancer by generating trimeric Tn antigen

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.

Glycoside Hydrolase Family 89 α-N-acetylglucosaminidase from Clostridium perfringens Specifically Acts on GlcNAcα1,4Galβ1R at the Non-reducing Terminus of O-Glycans in Gastric Mucin

In mammals, α-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. α-N-Acetylglucosaminidases (αGNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal αGNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of αGNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human αGNase, by chemically synthesizing a series of disaccharide substrates containing α-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAcα1,4Galβ1pMP and GlcNAcα1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAcα1,2Galβ1pMP, GlcNAcα1,3Galβ1pMP, GlcNAcα1,6Galβ1pMP, and GlcNAcα1,4GlcAβ1pMP substrates, this enzyme may represent a specific glycosidase required for degrading α-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAcα1,4Galβ1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered α-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.

Trimeric Tn antigen on Syndecan-1 produced by ppGalNAc-T13 enhances cancer metastasis via a complex formation with integrin α5β1 and matrix metalloproteinase 9

Background: ppGalNAc-T13 is up-regulated in high metastatic murine lung cancer cell lines. Results: Trimeric Tn on Syndecan 1 forms a complex with integrin α5β1 and MMP-9, leading to cell adhesion and cancer metastasis. Conclusion: Reduction of GM1 induces ppGalNAc-T13 expression, and its product trimeric Tn enhances metastasis via integrins. Significance: A novel key molecule in cancer metastasis is identified. We demonstrated previously that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in a DNA microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)3 on Syndecan 1 in highly metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer metastasis. Here, we analyzed the 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 matrix metalloproteinase 9 and that these molecules shifted to a glycolipid-enriched microdomain/rafts along with increased metastatic potential in T13-transfectant cells. We also identified a 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 suggest that high expression of the ppGalNAc-T13 gene generates tTn antigen on Syndecan 1 under reduced expression of GM1, leading to enhanced invasion and metastasis via the formation of a molecular complex consisting of integrin α5β1, Syndecan 1, and MMP-9 in the glycolipid-enriched microdomain/rafts.

A Quartz-crystal microbalance study of adsorption behaviors of artificial glycoconjugate polymers with different saccharide chain lengths and with different backbone structures

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 (K a = 10 4 ∼ 10 6 M -1 in molarity of the monomeric unit) and maximum adsorption amounts (Δm max ) on 1-octadecanethiol SAMs and polystyrene cast films were estimated from the Langmuir adsorption isotherms. These values depended on the side-chain oligosaceharide 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 amont 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.

Roles of GalNAc-disialyl Lactotetraosyl Antigens in Renal Cancer Cells

GalNAc-disialyl Lc4 (GalNAc-DSLc4) was reported as a novel antigen that associated with malignant features of renal cell cancers (RCCs). To clarify roles of GalNAc-DSLc4 in malignant properties of RCCs, we identified B4GalNAc-T2 as a responsible gene for the synthesis of GalNAc-DSLc4, and prepared stable transfectants of GalNAc-T2 cDNA using VMRC-RCW cells, resulting in the establishment of high expressants of GalNAc-DSLc4. They showed increased proliferation and invasion, and specific adhesion to laminin. In the transfectants, PI3K/Akt signals were highly activated by serum stimulation or adhesion to laminin. GalNAc-DSLc4 was co-localized in lipid rafts with integrin β1 and caveolin-1 in both immunoblotting of fractionated detergent extracts and immunocytostaining, particularly when stimulated with serum. Masking of GalNAc-DSLc4 with antibodies as well as PI3K inhibitor suppressed malignant properties of the transfectants. These results suggested that GalNAc-DSLc4 is involved in malignant properties of RCCs by forming a molecular complex with integrins in lipid rafts.

Synthesis of 1,5-Anhydro-d-fructose derivatives and evaluation of their inflammasome inhibitors

Synthesis of several 1,5-Anhydro-d-fructose (1,5-AF) derivatives to evaluate inhibitory activities of the inflammasome was carried out. Recently, 1,5-AF reported to suppress the inflammasome, although with only low activity. We focused on the hydration of 2-keto form of 1,5-AF and speculated that this hydration was the cause of low activity. Therefore, we synthesized some 1,5-AF derivatives that would not be able to form the dimer conformation and can be expected to have high activity against inflammasome, and then evaluated their inhibitory activities with respect to the NLRP3 inflammasome by using mouse bone marrow-derived macrophages and human THP-1 cells. As a result, some synthesized 2-keto form compounds had much higher inhibitory activities with respect to the NLRP3 inflammasome than did 1,5-AF.

Glycosyltransferases Involved in the Biosynthesis of Glycolipids

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.