• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.227-233
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• 2000

Although filamentous fungi are used extensively for protein expression, their use for the production of heterologous glycoproteins is constrained by the types of N-glycan structures produced by filamentous fungi as compared to those naturally found on the glycoproteins. Attempts are underway to engineer the N-glycan synthetic pathways in filamentous fungi in order to produce fungal expression strains which can produce heterologous glycoproteins carrying specific N-glycan structures. To fully realize this goal, a detailed understanding of the genetic components of this pathway in filamentous fungi is required. In this review, we discuss the characterization of the $\alpha$-mannosidase gene family in filamentous fungi and its implications for the elucidation of the N-glycan synthetic pathway.

• Mass Spectrometry Letters
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• 제9권3호
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• pp.67-72
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• 2018

Alkaline phosphatase (AP) is a membrane-bound glycoprotein that is widely distributed in the plasma membrane of cells of various organs and also found in many organisms from bacteria to humans. The complete amino acid sequence and three-dimensional structure of human placental alkaline phosphatase have been reported. Based on the literature data, AP consists of two presumptive glycosylation sites, at Asn-144 and Asn-271. However, it only contains a single occupied N-linked glycosylation site and no occupied O-linked glycosylation sites. Hydrophilic interaction chromatography (HILIC) has been primarily employed for the characterization of the glycan structures derived from glycoproteins. N-glycan structures from human placental alkaline phosphatase (PLAP) were investigated using HILIC-Orbitrap MS, and subsequent data processing and glycan assignment software. 16 structures including 10 sialylated N-glycans were identified from PLAP.

• 약학회지
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• 제57권4호
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• pp.250-257
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• 2013

Biosimilars are important drugs in medicine and contain many glycosylated proteins. Thorough analysis of the glycosylated protein is a prerequisite for evaluation of biosimilar glycan drugs. A method to assess the diversity of N-glycosylation sites and N-glycans from biosimilar glycan drugs has been developed using two separate methods, LC-MS/MS and MALDI-TOF MS, respectively. Development of sensitive, accurate, and efficient methods for evaluation of glycoproteins is still needed. In this study, analysis of both N-glycosylation sites and N-glycans of glycoprotein was performed using the same LC-MS/MS with two different nano-LC columns, nano-C18 and nano-porous graphitized carbon (nano-PGC) columns. N-glycosylated proteins, including RNAse B (one N-glycosylation site), Fetuin (three sites), and ${\alpha}$-1 acid glycoprotein (four sites), were used, and small amounts of each protein were used for identification of N-glycosylation sites. In addition, high mannose N-glycans (one type of typical glycan structure), Mannose 5 and 9, eluted from RNAse B, were successfully identified using nano-PGC-LC MS/MS analysis, and the abundance of each glycan from the glycoprotein was calculated. This study demonstrated an accurate and efficient method for determination of N-glycosylation sites and N-glycans of glycoproteins based on high sensitive LC-MS/MS using two different nano-columns; this method could be applied for evaluation of the quality of various biosimilar drugs containing N-glycosylation groups.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.221-224
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• 2001

Effect of hyperosmotic pressure on growth of recombinant Chinese hamster 。 vary cells and Erythropoietin (EPO) production was investigated. Cells were cultivated in batch modes at various osmolalities. When the osmolality increased from 314 to 463mOsm/Kg, specific EPO productivity (qp) was increased up to 1.6-fold but cell growth was inhibited. EPO has a complex oligosaccharide structure that plays an important role in biological activity in vivo. To investigate the influence of hypoerosmotic pressure on the glycosylation, structural analysis of oligosaccharide was calTied out. Recombinant human EPO was produced by CHO cells grown under various osmotic pressure and purified from culture supernatants by heparin-sepharose affinity column and immunoaffinity column. N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were labeled with fluorescent dye, 2-aminobenzamide and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of GU (glucose unit) value. Glycan analysis by HPLC showed that neutral (asialo) oligosaccharide was increased slightly with an increase in osmolality. In portion of sialylated glycan, total relative amount of mono- and di-sialyated glycan was increased but that of tri- and tetra-sialylated glycan decreased as osmolality was increased.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.859-865
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• 2008

Bacterial glycosyltransferases have drawn growing attention as economical enzymes for oligosaccharide synthesis, with their easy expression and relatively broad substrate specificity. Here, we characterized a glycosyltransferase homolog (Fnu_GT) from a human oral pathogen, Fusobacterium nucleatum. Bioinformatic analysis showed that Fnu_GT belongs to the glycosyltransferases family II. The recombinant Fnu_GT (rFnu_GT) expressed in Escherichia coli displayed the highest glycosylation activity when UDP-galactose (Gal) was used as a donor nucleotide-sugar with heptose or N-acetylglucosamine (GlcNAc) as an acceptor sugar. Interestingly, rFnu_GT transferred the galactose moiety of UDP-Gal to a nonreducing terminal GlcNAc attached to the trimannosyl core glycan, indicating its potential as an enzyme for human-type N-glycan synthesis.

• Mass Spectrometry Letters
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• 제12권3호
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• pp.66-75
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• 2021

Interleukin-4 (IL-4) and IL-13 are cytokines secreted by immune cells. Cytokines induce the proliferation of macrophages or promote the differentiation of secretory cells. The initiation and progression of allergic inflammatory diseases, such as asthma, are dependent on cytokines acting through related receptor complexes. IL-4 and IL-13 are N-glycoproteins. Glycan structures in glycoproteins play important roles in protein folding, protein stability, enzymatic function, inflammation, and cancer development. Therefore, the glycan structure of IL-4 and IL-13 needs to be elucidated in detail for the development of effective therapies. We report the first attempt to characterize the site-specific N-glycosylation of recombinant IL-4 and IL-13 via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The tandem mass spectra of intact N-glycopeptides were identified using the Integrated GlycoProteome Analyzer (I-GPA) platform, which can automatically and rapidly analyze multiple N-glycopeptides, including their glycan composition and amino acid sequences. The recombinant IL-4 and IL-13 were identified with amino acid sequence coverages of 84% and 96%, respectively. For IL-4, 52 glycoforms on one N-glycosylation site were identified and quantified. In IL-13, 232 N-glycopeptides from three N-glycosylation sites were characterized, with the site Asn52 being the most extensively glycosylated (~80%). The complex glycans were the most abundant glycan on IL-4 and IL-13 (~96% and 91%, respectively), and the biantennary glycans were the most abundant in both recombinant IL-4 and IL-13 proteins.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• Bulletin of the Korean Chemical Society
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• 제30권11호
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• pp.2723-2728
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• 2009

The conformational properties of oligosaccharides are important to understand carbohydrate-protein interactions. A trimannoside, methyl 3,6-di-O-($\alpha$-D-Man)-$\alpha$-D-Man (TRIMAN) is a basic unit of N-linked oligosaccharides. This TRIMAN moiety was further modified by GlcNAc (BISECT), which is important to biological activity of N-glycan. To characterize the trimannoside and its bisecting one we performed a molecular dynamics simulation in water. The resulting models show the conformational transition with two major and minor conformations. The major conformational transition results from the $\omega$ angle transition; another minor transition is due to the $\psi$ angle transition of $\alpha$ (1 $\rightarrow$ 6) linkage. The introduction of bisecting GlcNAc on TRIMAN made the different population of the major and minor conformations of the TRIMAN moiety. Omega ($\omega$) angle distribution is largely changed and the population of gt conformation is increased in BISECT oligosaccharide. The inter-residue hydrogen bonds and water bridges via bisecting GlcNAc residue make alterations on the local and overall conformation of TRIMAN moiety. These changes of conformational distribution for TRIMAN moiety can affect the overall conformation of N-glycan and the biological activity of glycoprotein.

• Molecules and Cells
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• 제38권1호
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• pp.65-74
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• 2015

Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after ${\alpha}1,3$-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (${\alpha}$-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

• 약학회지
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• 제53권1호
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• pp.34-40
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• 2009

The glycosylation of therapeutic glycoproteins can affect their efficacy, stability, solubility, and half-life. Analyzing the composition of monosaccharides, such as that of neutral and amino sugars, is the first step for elucidating the structure of glycan attached to glycoproteins. In the present study, neutral and amino sugars of lectin obtained from Maackia fauriei were analyzed using an enzyme-linked lectinsorbent assay (ELLA) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Peroxidase-labeled lectins such as concanavalin A, Ricinus communis agglutinin, and soybean agglutinin were used for ELLA, since they specifically bind to the monosaccharide residue most frequently encountered in a glycan. The hydrosylate of lectin was prepared by treatment with trifluoroacetic acid, which resulted in the lectin mainly possessing the N-glycan consisting of 98.1 pmol Fuc, 342.1 pmol GlcN, 51.9 pmol Gal, 678.9 pmol Man, and 330.7 pmol Xyl. The present results demonstrate that ELLA and HPAEC-PAD are very effective methods for rapidly estimating the types and relative amounts of monosaccharides in intact glycoproteins.