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Herein, we report two cases of erratic coil migration from the bronchial artery to the bronchus after bronchial artery embolization (BAE). Neither patient exhibited haemoptysis recurrence, but chest radiographs revealed that part of the coil had disappeared. In Case 1, the patient coughed up the coil 4.5 years after BAE. We performed repeat BAE to minimize the possibility of haemoptysis considering bronchoscopic and angiographic findings. In Case 2, the patient had severe dry cough 2 years after BAE. Chest radiography showed migrated coils in the trachea; bronchoscopy revealed a migrated fragment of the coil protruding from the elevated mucosa. We used a loop cutter to split the coil and then removed it using forceps. Coil migration to the bronchus is an infrequent late‐stage complication of super‐selective bronchial artery coil embolization, and only one other case has been reported. Accordingly, we propose treatment strategies and speculate on the mechanism of fistula formation.

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Tepjanta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

N -glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two N -glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. Nicotiana benthamiana has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown N. benthamiana , resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type N. benthamiana lacked the terminal galactose residues in its N -glycan. Therefore, N -glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine β1,4-galactosyltransferase (β1,4-GALT) to modify plant N -glycan with β1,4-linked Gal residue(s) and Arabidopsis thaliana β1,3-galactosylatransferase (β1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of N -glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine β1,4-GALT in soil-grown N. benthamiana had 42.5% of N -glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown N. benthamiana plants. Concomitantly, N- glycan profile analysis of IgG co-expressed with β1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary β1,4-galactosylated N -glycan structures., Competing Interests: Authors RK and TY was employed by the company GreenLand-Kidaya group Co Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nguyen, Kajiura, Kamiya, Yoshida, Misaki and Fujiyama.)

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man 5 GlcNAc 2 structure by combining N-acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N-glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man 6-9 GlcNAc 2 structure (2.8%, 7.1%) and high abundance of the Man 5 GlcNAc 2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man 5 GlcNAc 2 structure, and Man I overexpression enhanced the conversion of Man 6-9 GlcNAc 2 structures to the Man 5 GlcNAc 2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins., (Copyright © 2023 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Immunoglobulin A (IgA) has been showing potential as a new therapeutic antibody. However, recombinant IgA suffers from low yield. Supplementation of the medium is an effective approach to improving the production and quality of recombinant proteins. In this study, we adapted IgA1-producing CHO-K1 suspension cells to a high concentration (150 mM) of different disaccharides, namely sucrose, maltose, lactose, and trehalose, to improve the production and quality of recombinant IgA1. The disaccharide-adapted cell lines had slower cell growth rates, but their cell viability was extended compared to the nonadapted IgA1-producing cell line. Glucose consumption was exhausted in all cell lines except for the maltose-adapted one, which still contained glucose even after the 9th day of culturing. Lactate production was higher among the disaccharide-adapted cell lines. The specific productivity of the maltose-adapted IgA1-producing line was 4.5-fold that of the nonadapted line. In addition, this specific productivity was higher than in previous productions of recombinant IgA1 with a lambda chain. Lastly, secreted IgA1 aggregated in all cell lines, which may have been caused by self-aggregation. This aggregation was also found to begin inside the cells for maltose-adapted cell line. These results suggest that a high concentration of disaccharide-supplemented induced hyperosmolarity in the IgA1-producing CHO-K1 cell lines. In addition, the maltose-adapted CHO-K1 cell line benefited from having an additional source of carbohydrate., Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00571-5., Competing Interests: Conflict of interestThe authors have no conflict of interest to declare., (© The Author(s), under exclusive licence to Springer Nature B.V. 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents., (© 2023. The Author(s).)

The human basic fibroblast growth factor (bFGF) is a protein that plays a pivotal role in cellular processes like cell proliferation and development. As a result, it has become an important component in cell culture systems, with applications in biomedical engineering, cosmetics, and research. Alternative production techniques, such as transient production in plants, are becoming a feasible option as the demand continues to grow. High-level bFGF production was achieved in this study employing an optimized Agrobacterium -mediated transient expression system, which yielded about a 3-fold increase in production over a conventional system. This yield was further doubled at about 185 µg g -1 FW using a mutant protease-resistant version that degraded/aggregated at a three-fold slower rate in leaf crude extracts. To achieve a pure product, a two-step purification technique was applied. The capacity of the pure protease-resistant bFGF (PRbFGF) to stimulate cell proliferation was tested and was found to be comparable to that of E. coli -produced bFGF in HepG2 and CHO-K1 cells. Overall, this study demonstrates a high-level transient production system of functional PRbFGF in N. benthamiana leaves as well as an efficient tag-less purification technique of leaf crude extracts., Competing Interests: Conflict of interestThe authors declare no conflict of interest., (© 2022 Japanese Society for Plant Biotechnology.)

Gaucher disease is an inherited lysosomal storage disorder caused by an insufficiency of active β-glucocerebrosidase (GCase). Exogenous recombinant GCase via enzyme replacement therapy is considered the most practical treatment for Gaucher disease. Mannose receptors mediate the efficient uptake of exogenous GCase into macrophages. Thus, terminal mannose residues on N-glycans are essential for the delivery of exogenous GCase. In this study, recombinant GCase was produced in root cultures of wild-type (WT) and glycoengineered transgenic Nicotiana benthamiana with downregulated N-acetylglucosaminyltransferase I expression. Root cultures of WT and glycoengineered transgenic N. benthamiana plants were successfully generated by the induction of plant hormones. Recombinant GCases produced in both root cultures possessed GCase enzyme activity. Purified GCases derived from both root cultures revealed different N-glycan profiles. The WT-derived GCase possessed the predominant plant-type N-glycans, which contain plant-specific sugars-linkages, specifically β1,2-xylose and α1,3-fucose residues. Notably, the mannosidic-type N-glycans with terminal mannose residues were abundant in the purified GCase derived from glycoengineered N. benthamiana root culture. This research provides a promising plant-based system for the production of recombinant GCase with terminal mannose residues on N-glycans., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Although antibodies have attracted attention as next-generation biopharmaceuticals, the costs of purifying the products and of arranging the environment for cell cultivation are high. Therefore, there is a need to increase antibody efficacy and improve product quality as much as possible. Since antibodies are glycoproteins, their glycan structures have been found to affect the function of antibodies. Especially, afucosylation of the N -linked glycan in the Fc region is known to significantly increase antibody-dependent cellular cytotoxicity. In this study, we established a double-mutant ΔGMDΔGFT in which GDP-mannose 4,6-dehydratase and GDP-fucose transporter were knocked out in Chinese hamster ovary cells, a platform for biopharmaceutical protein production. By adapting ΔGMDΔGFT cells to serum-free medium and constructing suspension-cultured cells, we established host CHO cells with no detected fucosylated glycans and succeeded in production of afucosylated antibodies. We also demonstrated that, in culture in the presence of serum, fucosylation occurs due to contamination from serum components. Furthermore, we found that afucosylation of glycans does not affect cell growth after adaptation to serum-free medium as compared to wild-type CHO cells growth and does not significantly affect the expression levels of other endogenous fucose metabolism-related enzyme genes., Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-021-00501-3., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

Polygalacturonases (PGs) hydrolyze α-1,4-linked d-galacturonic acid (GalUA) in polygalacturonic acid. Previously, PG activity in pea seedlings was found in the Golgi apparatus, where pectin biosynthesis occurs. However, the corresponding genes encoding Golgi-localized PG proteins have never been identified in the higher plants. In this study, we cloned the 5 Arabidopsis genes encoding putative membrane-bound PGs from clade F PGs (AtPGFs) as the first step for the discovery of the Golgi-localized PGs. Five AtPGF proteins (AtPGF3, AtPGF6, AtPGF10, AtPGF14 and AtPGF16) were heterologously produced in Schizosaccharomyces pombe. Among these, only the AtPGF10 protein showed in vitro exo-type PG activity toward fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as a substrate. The optimum PG activity was observed at pH 5.5 and 60°C. The recombinant AtPGF10 protein showed the maximum PG activities toward PA-OGA with 10 degrees of polymerization. The apparent K m values for the PA-OGAs with 7, 11 and 14 degrees of polymerization were 8.0, 22, and 5.9 μM, respectively. This is the first report of the identification and enzymatic characterization of AtPGF10 as PG carrying putative membrane-bound domain., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

N -Glycosylation is essential for protein stability, activity and characteristics, and is often needed to deliver pharmaceutical glycoproteins to target cells. A paucimannosidic structure, Man 3 GlcNAc 2 (M3), has been reported to enable cellular uptake of glycoproteins through the mannose receptor (MR) in humans, and such uptake has been exploited for the treatment of certain diseases. However, M3 is generally produced at a very low level in plants. In this study, a cell culture was established from an Arabidopsis alg3 mutant plant lacking asparagine-linked glycosylation 3 (ALG3) enzyme activity. Arabidopsis alg3 cell culture produced glycoproteins with predominantly M3 and GlcNAc-terminal structures, while the amount of plant-specific N -glycans was very low. Pharmaceutical glycoproteins with these characteristics would be valuable for cellular delivery through the MR, and safe for human therapy., (© 2021 Japanese Society for Plant Biotechnology.)

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Plant cell cultures have emerged as a promising platform for the production of biopharmaceutics due to their cost-effectiveness, safety, ability to control the cultivation, and secrete products into culture medium. However, the use of this platform is hindered by the generation of plant-specific N -glycans, the inability to produce essential N -glycans for cellular delivery of biopharmaceutics, and low productivity. In this study, an alternative acid-alpha glucosidase (GAA) for enzyme replacement therapy of Pompe disease was produced in a glycoengineered Arabidopsis alg3 cell culture. The N -glycan composition of the GAA consisted of a predominantly paucimannosidic structure, Man 3 GlcNAc 2 (M3), without the plant-specific N -glycans. Supplementing the culture medium with NaCl to a final concentration of 50 mM successfully increased GAA production by 3.8-fold. GAA from an NaCl-supplemented culture showed a similar N -glycan profile, indicating that the NaCl supplementation did not affect N -glycosylation. The results of this study highlight the feasibility of using a glycoengineered plant cell culture to produce recombinant proteins for which M3 or mannose receptor-mediated delivery is desired., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sariyatun, Florence, Kajiura, Ohashi, Misaki and Fujiyama.)

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme β-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N -glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium -mediated transient expression in both wild-type (WT) and N -acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N -glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N -glycan analysis revealed that a mannosidic-type N -glycan structure lacking plant-specific N -glycans (β1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N -glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Uthailak, Kajiura, Misaki and Fujiyama.)

Linoleic acid (LA) has garnered much attention due to its potential applications in the oleochemical and nutraceutical industries. The oleaginous yeast Rhodotorula toruloides has outstanding lipogenecity, and is considered a potential alternative to the current plant-based platforms for LA production. Δ12-fatty acid desaturases (Δ12-Fads) are involved in LA synthesis in various fungi and yeasts, but their functions in R. toruloides remain poorly understood. To achieve the production of LA-rich lipids in R. toruloides, we investigated the function of the native Δ12-FAD (RtFAD2). First, the overexpression of RtFAD2 and its co-overexpression with RtFAD1 (encoding R. toruloides Δ9-Fad) and their effects on LA production in R. toruloides were investigated. The function of RtFad2 was confirmed by heterologous expression in Saccharomyces cerevisiae. Overexpression of RtFAD2 significantly elevated the LA contents and titers in the wild-type strain R. toruloides DMKU3-TK16 (TK16) and in a thermotolerant derivative of TK16 (L1-1). Additionally, overexpression of RtFAD2 in R. toruloides strains also increased the lipid titer and content. Overexpression of RtFAD1 was down-regulated in the RtFAD1 and RtFAD2 co-overexpressing strains, suggesting that the elevated LA content may function as a key regulator of RtFAD1 expression to control C18 fatty-acid synthesis in R. toruloides. We characterized the function of RtFAD2 and showed that its overexpression in R. toruloides increased the lipid and LA production. These findings may assist in the rational design of metabolic engineering related to LA or polyunsaturated fatty acid production in R. toruloides., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Cytosolic peptide:N-glycanase (cPNGase), which occurs ubiquitously in eukaryotic cells, is involved in the de-N-glycosylation of misfolded glycoproteins in the protein quality control system. In this study, we aimed to provide direct evidence of plant cPNGase activity against a denatured glycoprotein using a crude extract prepared from a mutant line of Arabidopsis thaliana lacking 2 acidic PNGase genes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcβ1,2-R with β1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of β1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

Glycosylation, the most common posttranslational modification of proteins, is a stepwise process that relies on tight regulation of subcellular glycosyltransferase location to control the addition of each monosaccharide. Glycosyltransferases primarily reside and function in the endoplasmic reticulum (ER) and the Golgi apparatus; whether and how they traffic beyond the Golgi, how this trafficking is controlled, and how it impacts glycosylation remain unclear. Our previous work identified a connection between N-glycosylation and Rab11, a key player in the post-Golgi transport that connects recycling endosomes and other compartments. To learn more about the specific role of Rab11, we knocked down Rab11 in HeLa cells. Our findings indicate that Rab11 knockdown results in a dramatic enhancement in the sialylation of N-glycans. Structural analyses of glycans using lectins and LC-MS revealed that α2,3-sialylation is selectively enhanced, suggesting that an α2,3-sialyltransferase that catalyzes the sialyation of glycoproteins is activated or upregulated as the result of Rab11 knockdown. ST3GAL4 is the major α2,3-sialyltransferase that acts on N-glycans; we demonstrated that the localization of ST3GAL4, but not the levels of its mRNA, protein, or donor substrate, was altered by Rab11 depletion. In knockdown cells, ST3GAL4 is densely distributed in the trans-Golgi network, compared with the wider distribution in the Golgi and in other peripheral puncta in control cells, whereas the α2,6-sialyltransferase ST6GAL1 is predominantly localized to the Golgi regardless of Rab11 knockdown. This indicates that Rab11 may negatively regulate α2,3-sialylation by transporting ST3GAL4 to post-Golgi compartments (PGCs), which is a novel mechanism of glycosyltransferase regulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Since about 70% of commercial biopharmaceutical products have been produced in Chinese hamster ovary (CHO) cells, this cell line is undeniably a workhorse for biopharmaceuticals production. Meanwhile, sialic acid terminals were reported to affect anti-inflammatory activity, antibody-dependent cellular cytotoxicity efficacy of IgG antibodies. Taking these findings together, we aimed to establish CHO cell lines that highly produce sialic acid terminals by overexpressing two N-acetylneuraminic acid-based key enzymes, α(2,6)-sialyltransferase and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase using dihydrofolate reductase/methotrexate gene amplification method. Indeed, the number of total sialic acid terminal glycan structures increased tremendously, by 12-fold compared to the wild type in total protein extracts. With the methotrexate supplementation, a targeted cell line, CHOmt17-100, showed up to 1.4 times more sialylated structures of glycoforms in total proteins. Interestingly, immunoglobulin G, used as the model protein in CHOmt17-100, showed about 53% sialylated structures in its glycoforms. These resultant sialylated glycans exhibited more than approximately 14.5 times increase as compared to that of the wild type. Moreover, the resultant glycan structures mostly had N-acetylneuraminic acid terminals, while N-glycolylneuraminic acid terminal composition remained less than 5% as compared to the wild type. Engineered antibodies derived from CHO cell lines that produce high levels of sialic acid will contribute to the examination of glycoforms' efficacy and usefulness toward bio-better products.

Stress tolerance is a desired characteristic of yeast strains for industrial applications. Stress tolerance has been well described in Saccharomyces yeasts but has not yet been characterized in oleaginous Rhodotorula yeasts even though they are considered promising platforms for lipid production owing to their outstanding lipogenicity. In a previous study, the thermotolerant strain L1-1 was isolated from R. toruloides DMKU3-TK16 (formerly Rhodosporidium toruloides). In this study, we aimed to further examine the ability of this strain to tolerate other stresses and its lipid productivity under various stress conditions. We found that the L1-1 strain could tolerate not only thermal stress but also oxidative stress (ethanol and H2O2), osmotic stress (glucose) and a cell membrane disturbing reagent (DMSO). Our results also showed that the L1-1 strain exhibited enhanced ability to maintain ROS homeostasis, stronger cell wall strength and increased levels of unsaturated membrane lipids under various stresses. Moreover, we also demonstrated that ethanol-induced stress significantly increased the lipid productivity of the thermotolerant L1-1. The thermotolerant L1-1 was also found to produce a higher lipid titer under the dual ethanol-H2O2 stress than under non-stress conditions. This is the first report to indicate that ethanol stress can induce lipid production in an R. toruloides thermotolerant strain., (© FEMS 2020.)

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine-human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately 10 i.p. LD 50 /100 µg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of two HuMAbs, M2 (1.25 µg) and M4 (1.25 µg), was able to completely neutralize BoNT/B1 (80 i.p. LD 50 ) with a potency greater than 80 i.p. LD 50 /2.5 µg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.

The oil plants provide a sufficient source of renewable lipid production for alternative fuel and chemical supplies as an alternative to the depleting fossil source, but the environmental effect from these plants' cropping is a concern. The high oleic acid (OA; C18:1) content in plant-derived products provide advantages of multiple uses with improved oxidative stability and a wide range of applicable temperature. Here we used a promising lipid producer, the oleaginous yeast Rhodosporidium toruloides, to attempt to obtain an OA-enriched lipid. Saccharomyces cerevisiae OLE1 (ScOLE1) gene encodes Δ9 fatty acid desaturase (Δ9FAD), which is generally known to synthesize palmitoleic acid (POA; C16:1) and OA, but the functions of putative R. toruloides Δ9FAD gene are not well understood. In a complementary test, the RtΔ9FAD gene rescued the survival of an OA-deficient Scole1Δ mutant, and we introduced the RtΔ9FAD gene into R. toruloides strains for the production of OA-enriched lipid. Increasing lipid production was observed in ScOLE1 and genomic RtΔ9FAD gene-overexpressing R. toruloides strains. The ScOLE1 transformant output fivefold more OA content in total amount, with >70% of total lipid. Different enhancing effects from the protein coding sequence and genomic sequence of RtΔ9FAD genes were also observed. Overall, this study resulted in ScOLE1 and RtΔ9FAD gene overexpression in R. toruloides to obtain OA-enriched lipid as a candidate source of designed biodiesel and lipid-related chemicals., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

l-Galactose (l-Gal) is one of the components of plant cell wall polysaccharides. In the GDP-l-fucose-deficient Arabidopsis thaliana mutant mur1, l-fucose (l-Fuc) residues in xyloglucan are substituted by l-Gal residues. l-Gal only differs from l-Fuc by the presence of an oxygen at C-6. Thus, we hypothesized that the A. thaliana xyloglucan α1,2-l-fucosyltransferase (AtFUT1) is also responsible for the l-galactosyl transfer to d-galactose residues in xyloglucan. In this study, we heterologously produced AtFUT1 in fission yeast and carried out an in vitro assay for the activities of AtFUT1 on GDP-l-Gal and xyloglucan oligosaccharide. We show that the recombinant AtFUT1 catalyzes l-Gal transfer to xyloglucan oligosaccharides although the initial velocity of l-Gal transfer is 3.1 times lower than that of l-Fuc transfer., (© 2018 Federation of European Biochemical Societies.)

Grey mangrove ( Avicennia marina ) is a traditional medicine used for the treatment of various diseases, including rheumatism and ulcers; however, the compounds responsible for its curative effects remain largely unknown. Triterpenoids are a diverse group of plant-specialized metabolites derived from a common precursor, 2,3-oxidosqualene. Triterpenoids are potentially responsible for the beneficial effects of A. marina ; however, the chemical profiles of triterpenoids in A. marina and their biosynthetic genes have not been identified. Cytochrome P450 monooxygenases (P450s) have key roles in the structural diversification of plant triterpenoids by catalyzing site-specific oxidation of triterpene scaffolds. Recent studies have revealed that the CYP716 family represents the most common clade of P450s involved in triterpenoid biosynthesis. In this study, we performed triterpenoid profiling and RNA sequencing of A. marina leaves. Mining of CYP716 family genes and enzymatic activity assays of encoded proteins revealed that CYP716A259 catalyzed oxidation at the C-28 position of the pentacyclic triterpene skeletons of β-amyrin, α-amyrin, and lupeol to produce oleanolic acid, ursolic acid, and betulinic acid, respectively. The other functionally defined P450, CYP716C53, catalyzed the C-2α hydroxylation of oleanolic acid and ursolic acid to produce maslinic acid and corosolic acid, respectively. The possible involvement of CYP716A259 and CYP716C53 in the biosynthesis of these health-benefiting compounds in A. marina leaves, and the possible contribution of the resulting compounds to the reported bioactivities of A. marina leaf extract, are discussed., (© 2018 The Japanese Society for Plant Cell and Molecular Biology.)

Oleaginous yeast Rhodosporidium toruloides DMKU3-TK16 (TK16), which was isolated from Thailand, is considered a promising lipid producer for biodiesel production. For future industrial applications of this strain, thermotolerant traits are highly desired for their potential to reduce cooling costs in a commercial fermenter. Here, by using an adaptive breeding strategy, we isolated a thermotolerant R. toruloides mutant, L1-1. The isolated L1-1 strain exhibited better growth and higher lipid production at 37°C, and it was found to have significantly higher oleic acid (C18:1) content and yield compared with the wild-type TK16 when cultivated at 37°C. This is the first study to isolate a thermotolerant strain from the oleaginous yeast R. toruloides. The information gained herein will provide new clues for engineering lipid production and for studying the thermotolerance of R. toruloides.

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.)

Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase (GCase). Currently, enzyme-replacement therapy using recombinant GCase produced in mammalian cells is considered the most effective treatment. Plants are an attractive alternative host for recombinant protein production due to the low cost of large-scale production and lack of risk of contamination by human pathogens. Compared to whole plants, root cultures can grow faster. Therefore, this study aimed to produce recombinant GCase in a Nicotiana benthamiana root culture. Root culture of a GCase-producing transgenic plant was induced by indole-3-acetic acid at the concentration of 1 mg/L. Recombinant GCase was successfully produced in roots as a functional protein with an enzyme activity equal to 81.40 ± 17.99 units/mg total protein. Crude proteins were extracted from the roots. Recombinant GCase could be purified by concanavalin A and phenyl 650C chromatography. The productivity of GCase was approximately 1 µg/g of the root. A N -glycan analysis of purified GCase was performed using nano LC/MS. The Man₃XylFucGlcNAc₂ structure was predominant in purified GCase with two plant-specific glycan residues. This study presents evidence for a new, safe and efficient system of recombinant GCase production that might be applied to other recombinant proteins.

Sialic acid plays important roles in stabilization and modulation of the interaction of molecules and membranes in organisms. Due to its high electronegativity, sialic acid can promote binding effects of molecules and support the transportation of drugs and ions in cells. This also strengthens cells against degradation from glycosidases and proteases. Hence sialic acid helps glycoproteins extend their half-lives and bioactivity. On the other hand, Chinese hamster ovary (CHO) cells have been widely used as a workhorse in biopharmaceutical fields in part due to the similarity between their glycan properties and those in humans. Thus, a high sialylation produced by CHO host cell line is strongly desired. In this study, we simultaneously overexpressed two key sialylated-based enzymes human β-galactoside α(2,6) sialyltransferase I and UDP-GlcNAc 2-epimerase/ManNAc kinase to achieve greater sialylation pattern produced host cells. The single-cell line thus-generated produced an approximately 41.6% higher level of total free sialic acid, and the glycan profiles showed a significant increase of more than 7-fold in the relative amount of total sialylated N-glycan as compared to the wild-type. These results demonstrated that co-expression of these two sialylated-based key enzymes yielded a cell line that effectively produced glycoproteins with superior sialylation and achievable human-like glycoforms., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) is the smallest partially double-stranded DNA virus known to infect humans. Worldwide, more than 50% of hepatocellular carcinoma (HCC) cases are related to chronic Hepatitis B. Development of HCC from normal liver cells is characterized by changes in cell surface N-glycans, which can promote the invasive behavior of tumor cells, leading ultimately to the progression of cancer. However, little is understood about the cell surface N-glycans of HBV-infected liver cells. We try to address this by taking advantage of the HepAD38 cell line, which can replicate HBV in the absence of tetracycline [tet(-)] in growth medium. In the presence of tetracycline [tet(+)], this cell line is free from the virus due to the repression of pregenomic (pg) RNA synthesis. In culture medium without tetracycline, cells express viral pgRNA and start to secrete virions into the supernatant. Here we studied the expression of glycosyltransferases and the cell surface N-glycan composition of tet(+) and tet(-) HepAD38. Among the glycosyltransferases upregulated by the expression of HBV were GnT-II, GnT-IVa, ST6Gal1, and GnT-V, whereas GnT-I, GnT-III, β4GalT1, and FUT8 were downregulated. About one-third of the total cell surface N-glycans found on tet(-)HepAD38 were sialylated. As for tet(+)HepAD38, sialylation was 6% lower compared to the tet(-) cells. Neither treatment changed the cell surface N-glycans expression of the total complex type or the total fucosylated type, which were about 50% or 60%, respectively. Our results showed that the expression of HBV triggers higher sialylation in HepAD38 cells. Altogether, the results show that HBV expression triggered the alteration of the cell surface N-glycosylation pattern and the expression levels of glycosyltransferases of HepAD38 cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Rhodosporidium toruloides DMKU3-TK16 (TK16), a basidiomycetous yeast isolated in Thailand, can produce a large amount of oil corresponding to approximately 70 % of its dry cell weight. However, lack of a sufficient and efficient transformation method makes further genetic manipulation of this organism difficult. We here developed a new transformation system for R. toruloides using a lithium acetate method with the Sh ble gene as a selective marker under the control of the R. toruloides ATCC 10657 GPD1 promoter. A linear DNA fragment containing the Sh ble gene expression cassette was integrated into the genome, and its integration was confirmed by colony PCR and Southern blot. Then, we further optimized the parameters affecting the transformation efficiency, such as the amount of linear DNA, the growth phase, the incubation time in the transformation mixture, the heat shock treatment temperature, the addition of DMSO and carrier DNA, and the recovery incubation time. With the developed method, the transformation efficiency of approximately 25 transformants/μg DNA was achieved. Compared with the initial trial, transformation efficiency was enhanced 417-fold. We further demonstrated the heterologous production of EGFP in TK16 by microscopic observation and immunoblot analysis, and use the technique to disrupt the endogenous URA3 gene. The newly developed method is thus simple and time saving, making it useful for efficient introduction of an exogenous gene into R. toruloides strains. Accordingly, this new practical approach should facilitate the molecular manipulation, such as target gene introduction and deletion, of TK16 and other R. toruloides strains as a major source of biodiesel.

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.)

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.)

In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

For the production of therapeutic proteins in plants, the presence of β1,2-xylose and core α1,3-fucose on plants' N-glycan structures has been debated for their antigenic activity. In this study, RNA interference (RNAi) technology was used to down-regulate the endogenous N-acetylglucosaminyltransferase I (GNTI) expression in Nicotiana benthamiana. One glyco-engineered line (NbGNTI-RNAi) showed a strong reduction of plant-specific N-glycans, with the result that as much as 90.9% of the total N-glycans were of high-mannose type. Therefore, this NbGNTI-RNAi would be a promising system for the production of therapeutic glycoproteins in plants. The NbGNTI-RNAi plant was cross-pollinated with transgenic N. benthamiana expressing human glucocerebrosidase (GC). The recombinant GC, which has been used for enzyme replacement therapy in patients with Gaucher's disease, requires terminal mannose for its therapeutic efficacy. The N-glycan structures that were presented on all of the four occupied N-glycosylation sites of recombinant GC in NbGNTI-RNAi plants (GC(gnt1) ) showed that the majority (ranging from 73.3% up to 85.5%) of the N-glycans had mannose-type structures lacking potential immunogenic β1,2-xylose and α1,3-fucose epitopes. Moreover, GC(gnt1) could be taken up into the macrophage cells via mannose receptors, and distributed and taken up into the liver and spleen, the target organs in the treatment of Gaucher's disease. Notably, the NbGNTI-RNAi line, producing GC, was stable and the NbGNTI-RNAi plants were viable and did not show any obvious phenotype. Therefore, it would provide a robust tool for the production of GC with customized N-glycan structures., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst.

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.)

Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

N-Glycosylation is a ubiquitous protein post-translational modification mechanism in eukaryotes. In this work, a synthetic pathway containing glycosyltransferases from Saccharomyces cerevisiae was introduced to Escherichia coli to synthesize lipid-linked mannosyl-chitobiose (Man-GlcNAc2) and trimannosyl-chitobiose (Man3-GlcNAc2). Transfer of Man3-GlcNAc2 onto a model periplasmic protein occurred in the engineered E. coli cell using oligosaccharyltransferase PglB from Campylobacter jejuni. Mass spectrometric analysis of the fluorescently labeled N-glycan indicated a glycan signal composed of 2 HexNAc and 3 Hex residues. The reversed-phase HPLC analysis suggested that the Hex residues were α1,3-, α1,6- and β1,4-linked mannoses. These results indicated that the constructed system synthesizes a Man3-GlcNAc2, identical to that observed in an early eukaryotic dolichol pathway. Finally, glycopeptide mass spectrometry confirmed the transfer of the assembled glycan moiety onto an engineered glycosylation motif of recombinant maltose binding protein. Surprisingly, the Man3-GlcNAc2 structure but not Man-GlcNAc2 was transferred onto maltose binding protein. This work showed that PglB protein might be able to accommodate the transfer of the further engineered glycan with greater complexity., (Copyright © 2014. Published by Elsevier B.V.)

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.)

Gly m Bd 28K (Gm28K), a soybean allergen, is formed as a preproprotein consisting of a predicted signal peptide, Gm28K, and the 23-kDa peptide (Gm23K). Gm28K and Gm23K are found in the protein-storage vacuoles (PSVs) of developing soybean seeds. However, the complete structure of Gm28K has not yet been identified and its processing and transport to the vacuoles has never been clarified. In the present study, we elucidated the 5'-nucleotide sequence of cDNA encoding the Gm28K precursor and identified a putative signal peptide (SP) with 24 N-terminal amino acid residues. We expressed peptides from the Gm28K precursor as fusion proteins with enhanced green fluorescent protein (EGFP) in tobacco BY2 suspension-cultured cells. BY2 cells transformed by an expression vector for SP-EGFP-Gm28-Gm23K (SP-EGFP-Gm28-Gm23K/BY2 cells) and SP-Gm28-Gm23K-EGFP/BY2 cells produced the EGFP fused-Gm28K precursor, and the EGFP-fluorescence in their vacuoles were recorded. In the experiments with SP-EGFP/BY2 and SP-EGFP-Gm28K/BY2 cells, large amounts of the EGFP segments were secreted into the medium. On the other hand, the fluorescence of EGFP in SP-EGFP-Gm23K/BY2 cells was shown to accumulate only in the endoplasmic reticulum without secretion into the medium. These findings show that the SP signals the precursor to enter the lumen of the endoplasmic reticulum and that both the Gm28K and Gm23K components may be involved in the transport from the endoplasmic reticulum (ER) lumen via the Golgi to the vacuoles in a proprotein form.

The swine-origin pandemic A(H1N1)2009 virus, A(H1N1)pdm09, is still circulating in parts of the human population. To monitor variants that may escape from vaccination specificity, antigenic characterization of circulating viruses is important. In this study, a hybridoma clone producing human monoclonal antibody against A(H1N1)pdm09, designated 5E4, was prepared using peripheral lymphocytes from a vaccinated volunteer. The 5E4 showed viral neutralization activity and inhibited hemagglutination. 5E4 escape mutants harbored amino acid substitutions (A189T and D190E) in the hemagglutinin (HA) protein, suggesting that 5E4 recognized the antigenic site Sb in the HA protein. To study the diversity of Sb in A(H1N1)pdm09, 58 viral isolates were obtained during the 2009/10 and 2010/11 winter seasons in Osaka, Japan. Hemagglutination-inhibition titers were significantly reduced against 5E4 in the 2010/11 compared with the 2009/10 samples. Viral neutralizing titers were also significantly decreased in the 2010/11 samples. By contrast, isolated samples reacted well to ferret anti-A(H1N1)pdm09 serum from both seasons. Nonsynonymous substitution rates revealed that the variant Sb and Ca2 sequences were being positively selected between 2009/10 and 2010/11. In 7,415 HA protein sequences derived from GenBank, variants in the antigenic sites Sa and Sb increased significantly worldwide from 2009 to 2013. These results indicate that the antigenic variants in Sb are likely to be in global circulation currently.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.

β1,2-Xylosyltransferase (XYLT) is a plant-specific glycosyltransferase that contributes to the biosynthesis of N-glycoproteins in plants. However, the specificity of XYLT for N-glycans has not yet been completely clarified. To gain insights into the function of XYLT in the plant N-glycosylation pathway, we examined the acceptor substrate specificity of recombinant Arabidopsis XYLT (AtXYLT) using 2-aminopyridine-labeled N-glycans as the substrates and confirmed the N-glycans of Arabidopsis xylt mutant. Recombinant AtXYLT expressed in insect cells required the β1,2-linked N-acetylglucosamine (GlcNAc) residue at the nonreducing terminus of the α1,3-branched mannose (Man) residue (GlcNAcβ1,2-Manα1,3-Man; GNM3B) for activity. However, AtXYLT showed decreased activity with substrates that contained α1,3-fucose at the chitobiose core-GlcNAc or a terminal GlcNAc at the α1,6-branched Man residue of GlcNAcβ1,2-Man (GlcNAcβ1,2-Manα1,6-Man; GNM3A), whose ratios were 10% and 50% of the optimal substrate, GNM3B, respectively. Moreover, AtXYLT did not show any activity in the transfer of the Xyl residue to N-glycans that contained a mammalian-type β1,4-linked galactose (Gal) residue at the nonreducing terminus of GlcNAcβ1,2-Man. These results indicate that a β1,2-linked GlcNAc residue at the nonreducing terminus of an α1,3-branched Man residue is necessary for AtXYLT activity and that mammalian-type β1,4-linked Gal residue(s) on the same branch completely inhibit(s) the activity. Furthermore, N-glycan analysis showed that approximately 30% of the N-glycans carry the Xyl residue in the wild type. These findings suggest that AtXYLT acts on protein-bound N-glycans prior to α1,3-fucosyltransferase and mannosidase II in planta., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)