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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, bio-nanocapsule (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., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

N-Glycosylation is an important post-translational modification in most secreted and membrane-bound proteins in eukaryotic cells. However, the insect N-glycosylation pathway and the potentials contributing to the N-glycan synthesis are still unclear because most of the studies on these subjects have focused on mammals and plants. Here, we identified Bombyx mori sialyltransferase (BmST), which is a Golgi-localized glycosyltransferase and which can modify N-glycans. BmST was ubiquitously expressed in different organs and in various stages of development and localized at the Golgi. Biochemical analysis using Sf9-expressed BmST revealed that BmST encoded α2,6-sialyltransferase and transferred N-acetylneuraminic acid (NeuAc) to the nonreducing terminus of Galβ1-R, but exhibited the highest activity toward GalNAcβ1,4-GlcNAc-R. Unlike human α2,6-sialyltransferase, BmST required the post-translational modification, especially N-glycosylation, for its full activity. N-Glycoprotein analysis of B. mori fifth instar larvae revealed that high-mannose-type structure was predominant and GlcNAc-linked and fucosylated structures were observed but endogenous galactosyl-, N-acetylgalactosaminyl- and sialyl-N-glycoproteins were undetectable under the standard analytical approach. These results indicate that B. mori genome encodes an α2,6-sialyltransferase, but further investigations of the sialylation potentials are necessary., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human beta1,4-galactosyltransferase. In this study, we analyze the N-glycan structures of glycoproteins secreted from GT6 cells to the spent medium. The N-glycans were liberated by hydrazinolysis, and the resulting oligosaccharides were labeled with 2-aminopyridine (PA). The pyridylaminated glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of 2D PA sugar chain mapping, MS/MS analysis, and exoglycosidase digestion. The distribution of proposed N-glycan structures of GT6-secreted glycoproteins (GalGNM5 [26.8%], GalGNM4 [18.4%], GalGNM3 [19.6%], and GalGNM3X [35.2%]) is different from that found in intracellular glycoproteins (M7A [9.3%], M7B [15.9%], M6B [19.5%], M5 [1.4%], M3X [6.6%], GalGNM5 [35.5%], and GalGNM3 [11.8%]). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggest that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.