In vitro and in silico analyses of amino acid substitution effects at the conserved N-linked glycosylation site in hepatitis B virus surface protein on antigenicity, immunogenicity, HBV replication and secretion.

The "a" determinant, a highly conformational region within the hepatitis B virus large surface protein (LHBs), is crucial for antibody neutralization and diagnostic assays. Mutations in this area can lead to conformational changes, resulting in vaccination failure, diagnostic evasion, and disease progression. The "a" determinant of LHBs contains a conserved N-linked glycosylation site at N320, but the mechanisms of glycosylation in LHBs remain unclear.

Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage.

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.

Production and -glycan engineering of Varlilumab in .

-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two -glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. has become one of the most attractive production systems for therapeutic antibodies.

High accumulation of the ManGlcNAc structure by combining N-acetylglucosaminyltransferase I gene suppression and mannosidase I gene overexpression in Nicotiana tabacum SR1.

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 ManGlcNAc 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.

Effects of various disaccharide adaptations on recombinant IgA1 production in CHO-K1 suspension cells.

Unlabelled: 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.

The specific core fucose-binding lectin Pholiota squarrosa lectin (PhoSL) inhibits hepatitis B virus infection in vitro.

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.

High-level transient production of a protease-resistant mutant form of human basic fibroblast growth factor in leaves.

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.

Production of recombinant β-glucocerebrosidase in wild-type and glycoengineered transgenic Nicotiana benthamiana root cultures with different N-glycan profiles.

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.

Establishment of serum-free adapted Chinese hamster ovary cells with double knockout of GDP-mannose-4,6-dehydratase and GDP-fucose transporter.

Unlabelled: 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.

Analysis of -glycan profile of Arabidopsis cell culture.

-Glycosylation is essential for protein stability, activity and characteristics, and is often needed to deliver pharmaceutical glycoproteins to target cells. A paucimannosidic structure, ManGlcNAc (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.

Improved assay system for acidic peptide: N-glycanase (aPNGase) activity in plant extracts.

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.

Production of Human Acid-Alpha Glucosidase With a Paucimannose Structure by Glycoengineered Cell Culture.

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 -glycans, the inability to produce essential -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 cell culture.

Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type -Glycan Structure in Glycoengineered Plants.

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 -glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor.

Functional characterization and overexpression of Δ12-desaturase in the oleaginous yeast Rhodotorula toruloides for production of linoleic acid-rich lipids.

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.

Bombyx mori β1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis.

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.

Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation.

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.

Transglycosylation toward naringenin-7-O-glucoside using an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase.

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.

Characterization of Bombyx mori N-acetylglucosaminyltransferase II splicing variants.

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.

Ethanol and H2O2 stresses enhance lipid production in an oleaginous Rhodotorula toruloides thermotolerant mutant L1-1.

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.

Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B.

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.

Enhancement of sialylation in rIgG in glyco-engineered Chinese hamster ovary cells.

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.

Transcriptome sequencing and identification of cytochrome P450 monooxygenases involved in the biosynthesis of maslinic acid and corosolic acid in .

Grey mangrove () 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 ; however, the chemical profiles of triterpenoids in and their biosynthetic genes have not been identified.