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Flavonoids are generally glycosylated, and the glycan moieties of flavonoid glycosides are known to greatly affect their physicochemical and biological properties. Thus, the development of a variety of tools for glycan remodeling of flavonoid glycosides is highly desired. An endo-β-N-acetylglucosaminidase mutant Endo-CC N180H, which is developed as an excellent chemoenzymatic tool for creating sialylglycoproteins, was employed for the glycosylation of flavonoids. Endo-CC N180H transferred the sialyl biantennary glycans from the sialylglyco peptide to pNP-GlcNAc and narigenin-7-O-glucoside. The kinetic parameters of Endo-CC N180H towards SGP and pNP-GlcNAc were determined. Flavonoid glucosides harboring a 1,3-diol structure in the glucose moieties acted as substrates of Endo-CC N180H. We proposed that the sialyl biantennary glycan transfer to the flavonoid by Endo-CC N180H could pave the way for the improvement of the inherent biological functions of the flavonoids and creation of novel flavonoid glycoside derivatives for future human health benefits including foods and drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

N-Acetylglucosaminyltransferase II (GNTII), which catalyzes the transfer of N-acetylglucosamine to N-glycans, plays an essential role in the biosynthesis of branched and complex-type N-glycans. Some characteristics of the GNTIIs from various species have been identified, but not all features have been revealed because some insects have GNTII redundancies due to the possession of splicing variants. In this study, we focused on four splicing variants of silkworm Bombyx mori GNTII (BmGNTII) that differ only in the absence or presence of Exon 2, Exon 9 or both, and we characterized the spatiotemporal transcript levels and enzymatic properties of each. Two of the splicing variants, BmGNTII-B and BmGNTII-D, lack Exon 9, and were expressed more highly in silk glands than any other organs. With respect to the enzymatic properties, optimal temperature and pH were similar among the recombinant BmGNTIIs, but the specific activities and temperature stabilities differed according to the presence or absence of Exon 9 in the splicing variants. These results demonstrate that the B. mori genome encodes splicing variants of GNTII with different enzymatic properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

Complex glycans at the cell surface play important roles, and their alteration is known to modulate cellular activity. Previously, we found that HBV replication in HepAD38 altered cell-surface sialylated N-glycan through the upregulation of St6gal1, Mgat2, and Mgat4a expression. Here we studied the effects of knocking them down on HBV replication in HepAD38. Our results showed that St6gal1 knockdown (KD) reduced intracellular HBV rcDNA level by 90%, that Mgat2 KD did not change the intracellular HBV rcDNA level, and that Mgat4 KD increased the intracellular HBV rcDNA level by 19 times compared to Tet(-). The changes in intracellular rcDNA level were followed by the alteration of Pol and HBc expression. Our study suggests that St6gal1 KD contributes more to the HBV life cycle than Mgat2 or Mgat4a KD through the modification of intracellular L, Pol, and HBc expression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

l-Galactose (l-Gal) containing N-glycans and cell wall polysaccharides have been detected in the l-Fuc deficient mur1 mutant of Arabidopsis thaliana. The l-Gal residue is thought to be transferred from GDP-l-Gal, which is a structurally related analog of GDP-l-Fuc, but in vitrol-galactosylation activity has never been detected. In this study, we carried out preparative scale GDP-l-Gal synthesis using recombinant A. thaliana GDP-mannose-3',5'-epimerase. We also demonstrated the l-galactosylation assay of mouse α1,6-fucosyltransferase (MmFUT8) and A. thaliana α1,3-fucosyltransferase (AtFucTA). Both fucosyltransferases showed l-galactosylation activity from GDP-l-Gal to asparagine-linked N-acetyl-β-d-glucosamine of asialo-agalacto-bi-antennary N-glycan instead of l-fucosylation. In addition, the apparent K m values of MmFUT8 and AtFucTA suggest that l-Fuc was preferentially transferred to N-glycan compared with l-Gal by fucosyltransferases. Our results clearly demonstrate that MmFUT8 and AtFucTA transfer l-Gal residues from GDP-l-Gal and synthesize l-Gal containing N-glycan in vitro., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50-100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.

After endocytosis, most membrane proteins and lipids return to the plasma membrane (recycling pathway), but some membrane components are delivered to lysosomes (degradation pathway). These two pathways diverge in early endosomes. The recycling pathway involves recycling endosomes and the degradation pathway incorporates late endosomes and lysosomes. In many cell lines, these organelles often are located in the perinuclear region where they visually intermix. The present study, by tracking specific ligands (epidermal growth factor and transferrin) and expression of Rab proteins (Rab5, Rab7, and Rab11), demonstrated that, in COS-1 cells, the two pathways were spatially segregated. Recycling endosomes were mostly confined within the ring-shaped structure of the Golgi complex ("the Golgi ring"), whereas late endosomes and lysosomes were excluded from inside the Golgi ring. Thus, the unique organization of endocytic organelles in COS-1 cells can be utilized to visualize endocytic trafficking pathways in detail.

Previously, we developed a transgenic tobacco BY2 cell line (GT6) in which glycosylation was modified by expressing human beta(1,4)-galactosyltransferase (betaGalT). In this study, we produced a mouse monoclonal antibody in GT6 cells, and determined the sugar chain structures of plant-produced antibodies. Galactose-extended N-linked glycans comprised 16.7%, and high-mannose-type and complex-type glycans comprised 38.5% and 35.0% of the total number of glycans, respectively. N-linked glycans with the plant-specific sugars beta(1,2)-xylose and alpha(1,3)-fucose comprised 9.8%. The introduction of human betaGalT into suspension cultured tobacco cells resulted in the production of recombinant proteins with galactose-extended glycans and decreased contents of beta(1,2)-xylose and alpha(1,3)-fucose.

Plant cells have no beta1,4-galactosylated and sialylated glycan, which plays important roles in biological functions in animal cells. Previously, we generated transgenic tobacco BY2 suspension-cultured cells that produced human beta1,4-galactosyltransferase [N.Q. Palacpac, S. Yoshida, H. Sakai, Y. Kimura, K. Fujiyama, T. Yoshida, T. Seki, Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation pattern, Proc. Natl. Acad. Sci. USA 96 (1999) 4692-4697]. In this study, we introduced two critical genes encoding human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter into tobacco suspension-cultured cell to pave a route for sialic biosynthetic pathway. The recombinant human proteins showed their biological activities. These results show that the plant cell can be a useful bioreactor for the production of mammalian glycoproteins.

The full-length mouse interferon-beta (mIFN-beta) cDNA, including the secretion signal peptide coding region under control of the polyhedrin promoter, was introduced into Bombyx mori nucleopolyhedrovirus (BmNPV). Recombinant mIFN-beta (rmIFN-beta) was accumulated in the haemolymph of infected silkworm larvae. Western blot analysis showed isoforms of rmIFN-beta, suggesting that rmIFN-beta is glycosylated. The glycan structures of purified rmIFN-beta were determined. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by gel filtration, reversed-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains were identified by a combination of two-dimensional PA-sugar chain mapping, MS analysis, and exoglycosidase digestions.