Masashi Hyuga

Simultaneous microanalysis of N-linked oligosaccharides in a glycoprotein using microbore graphitized carbon column liquid chromatography–mass spectrometry

We previously reported that graphitized carbon column liquid chromatography-mass spectrometry (GCC-LC-MS) is very useful for the structural analysis of carbohydrates in a glycoprotein. In this study, GCC-LC-MS was adapted for the simultaneous microanalysis of oligosaccharides. A variety of oligosaccharide alditols prepared from fetuin, ribonuclease B, and recombinant human erythropoietin were used as model oligosaccharides. The use of microbore GCC-LC-MS was found to be successful for rapid, sensitive, and simultaneous analysis of high-mannose-type, desialylated fucosyl complex-type, sialylated complex-type, and sialylated fucosyl complex-type oligosaccharide alditols. Furthermore, we demonstrate that this method is applicable to the analysis of carbohydrate heterogeneity in a glycoprotein that possesses diverse oligosaccharides. Microbore GCC-LC-MS was able to characterize high-mannose-type, hybrid-type, and complex-type oligosaccharides in tissue plasminogen activator produced from human melanoma cells in a single analysis.

Selective glycopeptide mapping of erythropoietin by on-line high-performance liquid chromatography–electrospray ionization mass spectrometry

Selective glycopeptide mapping of recombinant human erythropoietin (rhEPO) used as a model glycoprotein was successfully carried out by on-line high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using a Vydac C18 column eluted in acetonitrile-1 mM ammonium acetate, pH 6.8. rhEPO expressed in a Chinese hamster ovary clone was exhaustively digested into four glycopeptides and nine peptides with endoproteinase Glu-C. Both glycopeptides and peptides were eluted with trifluoroacetic acid as the eluent, whereas only glycopeptides were eluted selectively with ammonium acetate in the following order: N38, N24, 0126, and N83. Furthermore, many glycoforms included in each glycopeptide were found to be separated by differences in the numbers of sialic acid and N-acetyllactosaminyl repeats. Twenty, 16 and 22 different N-linked oligosaccharides were determined at Asn24, 38, and 83, respectively, and two different O-linked oligosaccharides were observed at Ser126. Our method is simple, rapid, and useful for determining the carbohydrate structures at each glycosylation site and for elucidating the site-specific carbohydrate heterogeneity.

Suppression by ganglioside GD1A of migration capability, adhesion to vitronectin and metastatic potential of highly metastatic FBJ-LL cells.

Ganglioside GD1a, which is highly expressed in poorly metastatic FBJ‐S1 cells, has been shown to inhibit the serum‐induced migration capability of highly metastatic FBJ‐LL cells. In the present study, the capacity of FBJ‐S1 cells to adhere to vitronectin was found to be about half that of FBJ‐LL cells. Pre‐treatment of FBJ‐LL cells with GD1a decreased this capacity by 30% that of the control, whereas GM1‐pre‐treatment caused only a 10% decrease, indicating that GD1a specifically inhibits FBJ‐LL cell adhesion to vitronectin. Since FBJ‐LL cells contain almost no GD1a, transfectants capable of expressing GD1a to varying degrees were produced in this study by transfection of FBJ‐LL cells with GM2/GD2‐synthase cDNA. Decrease in the serum‐induced migration capacity of these transfectants was accompanied by an increment in GD1a expression. Adhesion of the transfectants to vitronectin decreased by 30% as compared with mock‐transfected cells. Within 4 to 5 weeks after GD1a‐expressing transfectant and mock‐transfected cells were transplanted into mice, metastatic nodules were observed in liver, lung, kidney and adrenal glands of mock‐transplanted mice, but not in those with GD1a‐expressing transfectants, indicating that GD1a suppresses the metastasis of FBJ‐osteosarcoma cells, possibly by inhibiting cell migration and cell adhesion. The involvement of the ganglioside in the suppression of metastasis is clearly demonstrated in the present study. Int. J. Cancer 83:685–691, 1999.

Annexin A3 Expression Increases in Hepatocytes and is Regulated by Hepatocyte Growth Factor in Rat Liver Regeneration

Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl4) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl4 administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.

Ganglioside GD1a inhibits HGF-induced motility and scattering of cancer cells through suppression of tyrosine phosphorylation of c-Met

We previously reported that ganglioside GD1a, which is highly expressed in poorly metastatic FBJ‐S1 cells, inhibits the serum‐induced motility of FBJ‐LL cells and that the metastatic potential of FBJ‐LL cells is completely suppressed by enforced GD1a expression (Hyuga et al., Int J Cancer 1999;83:685–91). We recently discovered that hepatocyte growth factor (HGF) induces FBJ‐LL cell motility. In the present study, the HGF‐induced motility of FBJ‐S1 cells was found to be one‐thirtieth that of FBJ‐LL cells. This motility of GD1a‐expressing transfectants, which were produced by transfection of FBJ‐LL cells with GM2/GD2 synthase cDNA, decreased with increases in their GD1a expression and HGF induced almost no motility in GD1a‐pretreated FBJ‐LL cells, indicating that GD1a inhibits the HGF‐induced motility of FBJ‐LL cells. The expression of the HGF receptor c‐Met on FBJ‐S1 cells, FBJ‐LL cells, transfectants and a mock‐transfectant was almost the same. The level of tyrosine phosphorylation of c‐Met after HGF stimulation in FBJ‐S1 cells, GD1a‐pretreated FBJ‐LL cells and a GD1a‐expressing transfectant was significantly lower than in FBJ‐LL cells and a mock‐transfectant. These findings suggested that GD1a inhibits the HGF‐induced motility of FBJ‐LL cells through suppression of tyrosine phosphorylation of c‐Met. HepG2 cells, a human hepatoma cell line, were used to investigate whether GD1a interferes with other cancer cells expressing c‐Met. HepG2 cells did not express GD1a. HGF induced cell scattering of HepG2 cells and the scattering was inhibited by pretreating the cells with GD1a. The c‐Met in the cells was autophosphorylated by stimulation with HGF, but after treating the cells with GD1a, the HGF‐induced autophosphorylation of c‐Met was suppressed. These results suggest that GD1a acts as a negative regulator of c‐Met in cancer cells.

Ephedrine alkaloids-free Ephedra Herb extract: a safer alternative to ephedra with comparable analgesic, anticancer, and anti-influenza activities

It is generally accepted that the primary pharmacological activities and adverse effects of Ephedra Herb are caused by ephedrine alkaloids. Interestingly, our research shows that Ephedra Herb also has ephedrine alkaloid-independent pharmacological actions, such as c-MET inhibitory activity. This study describes the preparation of an ephedrine alkaloids-free Ephedra Herb extract (EFE) by ion-exchange column chromatography, as well as in vitro and in vivo evaluation of its pharmacological actions and toxicity. We confirmed that EFE suppressed hepatocyte growth factor (HGF)-induced cancer cell motility by preventing both HGF-induced phosphorylation of c-Met and its tyrosine kinase activity. We also investigated the analgesic effect of EFE. Although the analgesic effect of Ephedra Herb has traditionally been attributed to pseudoephedrine, oral administration of EFE reduced formalin-induced pain in a dose-dependent manner in mice. Furthermore, we confirmed the anti-influenza virus activity of EFE by showing inhibition of MDCK cell infection in a concentration-dependent manner. All assessments of toxicity, even after repeated oral administration, suggest that EFE would be a safer alternative to Ephedra Herb. The findings described here suggest that EFE has c-Met inhibitory action, analgesic effect, and anti-influenza activity, and that it is safer than Ephedra Herb extract itself. Therefore, EFE could be a useful pharmacological agent.

Herbacetin, A Constituent of Ephedrae herba, Suppresses the HGF-Induced Motility of Human Breast Cancer MDA-MB-231 Cells by Inhibiting c-Met and Akt Phosphorylation

Ephedrae herba suppresses hepatocyte growth factor-induced cancer cell motility by inhibiting tyrosine phosphorylation of the hepatocyte growth factor receptor, c-Met, and the PI3K/Akt pathway. Moreover, Ephedrae herba directly inhibits the tyrosine-kinase activity of c-Met. Ephedrine-type alkaloids, which are the active component of Ephedrae herba, do not affect hepatocyte growth factor-c-Met-Akt signalling, prompting us to study other active molecules in the herb. We recently discovered herbacetin glycosides and found that their aglycon, herbacetin, inhibits hepatocyte growth factor-c-Met-Akt signalling. This study revealed a novel biological activity of herbacetin. Herbacetin suppressed hepatocyte growth factor-induced motility in human breast cancer MDA-MB-231 cells by inhibiting c-Met and Akt phosphorylation and directly inhibiting c-Met tyrosine kinase activity. The effects of herbacetin were compared to those of kaempferol, apigenin, and isoscutellarein, all of which have similar structures. Herbacetin inhibition of hepatocyte growth factor-induced motility was the strongest of those for the tested flavonols, and only herbacetin inhibited the hepatocyte growth factor-induced phosphorylation of c-Met. These data suggest that herbacetin is a novel Met inhibitor with a potential utility in cancer therapeutics.

Possible role of hepatocyte growth factor/scatter factor and activin A produced by the target organ in liver metastasis

The molecular mechanism of organ-specific metastasis to the liver remains largely unknown. However, it is conceivable that paracrine growth factors produced by a target organ induce migration and proliferation of malignant cells to that organ, and this is the cause of organ-specific metastasis. In this study, we investigated the effect of hepatocyte growth factor/scatter factor (HGF/SF) and activin A, which are known to be produced by the liver, on the motility and growth of liver-metastatic cell line FBJ-LL. HGF/SF and activin A induced motility synergistically, but they did not affect the proliferation of FBJ-LL cells. Expression of the HGF/SF receptor, the c-met gene, and the activin-receptor type IA, type IB, and type IIA genes in FBJ-LL cells was detected by reverse transcription polymerase chain reaction. These findings suggest that both HGF/SF and activin A promote organ-specific metastasis to the liver by induction of migration through their specific receptors on liver-metastatic cells.

Recent Topics of Research in the Characterization and Quality Control of Biopharmaceuticals in Japan

The research and development of next-generation innovative medicines is a prominent interest of both the government and industries in Japan. On June 29, 2017, a kickoff meeting of a new research group focused on the quality issues of biopharmaceuticals was held in Tokyo with Prof. John Carpenter as an invited guest. The groups research focuses mainly on the evaluation and control of protein aggregates/subvisible particles in drug products, but the research topics also include glycan analysis, host-cell protein evaluation, bioassay validation, and analytical quality by design. The purpose of the groups activities is to resolve the critical and fundamental quality issues important to pharmaceutical companies through the collaboration of industries, academia, and regulatory agencies. In this commentary, our current plan to address these issues and the discussion at the kickoff meeting are described.

Two flavone C -glycosides as quality control markers for the manufacturing process of ephedrine alkaloids-free Ephedra Herb extract (EFE) as a crude drug preparation

As part of our continuing study of ephedrine alkaloids-free Ephedra Herb extract (EFE) in pursuit of its approval as a crude drug preparation, we identified two quantitative markers for the quality control of the manufacturing process of EFE and sought to establish cost-effective and simple methods for quantitative analyses. We analysed Ephedra Herb extracts grown in different habitats and collection years by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and detected two notable peaks common to each extract. These peaks were identified as vicenin-2 (1) and isovitexin 2″-O-rhamnoside (2). Quantitative analyses using the isocratic condition of LC/MS showed that the content percentages of 1 and 2 in EFE were 0.140–0.146% and 0.350–0.411%, respectively. We concluded that 1 and 2 were adequate quality control markers for quantitative analysis of EFE. Furthermore, we quantitatively analysed apigenin (3), an aglycon common to 1 and 2, and found that the conversion factors of 1 to 3 and 2 to 3 were 1.3 and 1.5, respectively. Therefore, we concluded that 3 was a secondary standard for quantifying the contents of 1 and 2 in EFE. A series of results obtained from this study will be valuable for the quality control of EFE.