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The functions of cell surface proteins, such as growth factor receptors and virus/bacteria-entry receptors, can be dynamically regulated by oligosaccharide modifications. In the present study, we investigated the involvement of glycosylation in hepatitis B virus (HBV) entry into hepatoma cells. Infection of oligosaccharide-remodeling hepatoma cells with a pseudo virus of HBV, bionanocapsule (BNC), was evaluated by flow cytometry and confocal microscopy. Among various experiments using several hepatoma cells, marked difference was observed between Huh6 cells and HB611 cells, which were established by HBV gene transfection into hepatoma cells. Comprehensive oligosaccharide analysis showed dramatic increases of core fucosylation in HB611 cells, compared with Huh6 cells. Knock down of fucosyltransferase 8 (FUT8) reduced BNC entry into HB611 cells. In contrast, overexpression of FUT8 in Huh6 cells increased BNC entry. Although expression of sodium taurocholate cotransporting polypeptide (NTCP), which is one of HBV receptors was very similar between Huh6 and HB611 cells, proteins coprecipitated with NTCP were dependent on levels of core-fucosylation, suggesting that core-fucosylation regulates BNC entry into hepatoma cells. Our findings demonstrate that core-fucosylation is an important glycosylation for HBV infection of hepatoma cells through HBV-receptor-mediated endocytosis. Down-regulation of core-fucosylation may be a novel target for HBV therapy. [ABSTRACT FROM AUTHOR]

The usefulness of radiolabeled 3′-fluoro-3′-deoxythymidine (FLT), a thymidine derivative with affinity to cytoplasmic thymidine kinase 1 (TK1), as a tumor proliferation marker was evaluated using [3H]FLT and 22 cultured tumor cell lines. Asynchronously growing tumor cells were used for studies to mimic in vivo status of tumors. FLT uptake in each cell line was compared with [3H]thymidine ([3H]Thd) uptake and %S-phase fraction, both known as acceptable markers of proliferation. Uptake of the mitochondrial TK2 specific substrate [3H]arabinothymidine ([3H]AraT) was studied as a reference. Metabolic fate of FLT in tumor cells was also analyzed to elucidate the retention mechanism of FLT. [3H]FLT uptake was mildly correlated with the %S-phase fraction (r=0.76, p<0.0001]Thd uptake (r=0.88, p<0.0001 specific substrate, [3H]AraT, was not significantly correlated with the %S-phase fraction (r=0.19, p=0.39), although it showed some correlation with the [3H]Thd uptake (r=0.47, p<0.0001]Thd was found in the DNA fraction after 60 minutes incubation. In contrast, most of the radioactivity of [3H]FLT was found in the acid-soluble fraction (95%). [3H]FLT incorporation into the DNA fraction was negligible (0.2%). The [3H]AraT was mainly distributed in the acid-soluble fraction (70%) and the DNA fraction (20%). From our results, we concluded that FLT uptake in tumor cells reflects tumor cell proliferation. However, much more convincing validation is needed to clarify the difference between FLT and true substrates for DNA synthesis, like thymidine. [Copyright &y& Elsevier]

Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing 1-6 branching in asparagine- linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg vs. wild-type (WT) mice. Hepatic expression of apolipoprotein AI (ApoAI) and ATP-binding cassette subfamily A member 1 (ABCA1), two important factors in HDL assembly, were higher in Tg mice compared with WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large-size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis. [ABSTRACT FROM AUTHOR]

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes β1-6 branching in asparagine-linked oligosaccharides and is one of the most important glycosyltransferases involved in carcinogenesis, cancer metastasis and immunity. To investigate the biological functions of GnT-V, the present study developed GnT-V transgenic (Tg) mice and the role of GnT-V in experimental immune-mediated hepatitis, induced by concanavalin A (ConA), were investigated. It was found that the aberrant expression of GnT-V exacerbated ConA-induced hepatitis in the Tg mice compared with the wild-type (WT) mice. The survival rate of the ConA-induced hepatitis at a high-dose of ConA was significantly lower in the Tg mice. Intravenously injected ConA is known to initially bind predominantly to the mannose gland of the liver sinusoidal endothelial cell (LSEC) surface and to leads to the activation of various immune cells. In the present study, the binding affinity of ConA to the LSECs did not differ between the WT and Tg mice. In addition, T cell receptor stimulation by anti-cluster of differentiation (CD)3/CD28 antibodies produced lower levels of T helper (Th)1 cytokine (interferon-γ) and higher levels of Th2 cytokine (interleukin-10) in the Tg mouse splenic lymphocytes compared with WT mice. The composition of the hepatic mononuclear cells revealed that CD11b-positive cells were significantly increased in the GnT-V Tg mice. In addition, F4/80-positive cells were significantly increased in the Tg mouse liver and the depletion of macrophages reduced the difference in the severity of ConA-induced hepatitis between the WT and Tg mice. In conclusion, the present findings indicated that the aberrant expression of GnT-V led to an increase in hepatic macrophage infiltration and enhanced ConA-induced hepatitis. Modulation of glycosylation may be a novel therapeutic target for immunity-associated acute hepatitis. [ABSTRACT FROM AUTHOR]

Our previous studies on a β1,6-JV-acetylglucosaminyltrans-ferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward -linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodies-terase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular gly-cosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions. [ABSTRACT FROM AUTHOR]