• BMB Reports
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• 제44권10호
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• pp.686-691
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• 2011

Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.

• KSBB Journal
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• 제32권3호
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.

• Molecules and Cells
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• 제38권6호
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• pp.496-505
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• 2015

A variant peak was detected in the analysis of RP-HPLC of rHu-EPO, which has about 7% relative content. Fractions of the main and the variant peaks were pooled separately and further analyzed to identify the molecular structure of the variant peak. Total mass analysis for each peak fraction using ESI-TOF MS shows differences in molecular mass. The fraction of the main peak tends to result in higher molecular masses than the fraction of the variant. The detected masses for the variant are about 600-1000 Da smaller than those for the main peak. Peptide mapping analysis for each peak fraction using Asp-N and Glu-C shows differences in O-glycopeptide profiles at Ser126. The O-glycopeptides were not detected in the fraction of the variant. It is concluded that the variant peak is non-O-glycosylated rHu-EPO and the main peak is fully O-glycosylated rHu-EPO at Ser126.

• 약학회지
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• 제44권6호
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• pp.588-594
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• 2000

To investigate the effects of iota-carrageenan (CAR) and/or alum on the adjuvancity as well as the structural difference of oligosaccharide on the IgG2b in the adjuvant effect, C57BL/6 mice were immunized twice with fetuin as a model antigen. CAR alone showed no significant effect on induction of antibody except IgG1. In contrast, Alum-CAR (after mixing of antigen-Alum, CAR adjuvant was prepared) and CAR-Alum (after formulation of antigen-CAR, Alum adjuvant was prepared) enhanced production of antibody, especially, IgG2b. After separation of IgG2b, changes of glycosylation were investigated using enzymelinked lectin assay. High affinity of IgG2b to N-acetylneuraminic acid, galactose and mannose-specific lectin were induced by CAR-Alum adjuvant, however, the affinity of IgG2b induced by CAR-Alum to GlcNAc and GalNAc-specific lectin were much less than that induced by Alum-CAR.

• 약학회지
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• 제54권6호
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• pp.449-454
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• 2010

The glycosylation of glycoproteins from mammalian or plants can affect their efficacy, stability, solubility, and half-life. In the present study, we investigated plant glycosylation and their relative intensity (%) in a plant carbohydratebinding protein with the hemagglutination and antiproliferative activities. The hemagglutination activity on the deglycosylated protein was decreased as a 16-fold than that of intact glycoprotein. Using the HPLC with fluorescence detector and mass spectrometer, the major eight bi- or triantennary oligosaccharides containing xylose, fucose, mannose, galactose, and N-acetylglucosamine were identified and structurally characterized. The present results indicate that the oligosaccharides on this plant glycoprotein is necessary for their own property.

• Journal of Microbiology
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• 제40권3호
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• pp.219-223
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• 2002

In order to investigate the function of Soo1p/${\alpha}$-COP during post-translational modification and intra-cellular transport of cell wall proteins in Saccharomyces cerevisiae, cell wall proteins from the soo1-1/ret1-1 mutant cells were analyzed. SDS-PAGE analysis of biotin labeled cell wall proteins suggested that the soo1-1 mutation impairs post-translational modification of cell wall proteins, such as N- and/ or Ο-glycosylation. Analysis of cell wall proteins with antibodies against ${\beta}$-1,3-glucan and ${\beta}$-1,6-glucan revealed alteration of the linkage between cell wall proteins and ${\beta}$-glucans in the soo1-1 mutant cells. Compositional sugar analysis of the cell wall proteins also suggested that the soo1-1 mutation impairs glycosylation of cell wall protein in the ER, which is crucial for the maintenance of cell wall integrity.

• 대한수의학회지
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• 제45권2호
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• pp.207-214
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• 2005

The nonstructural protein 4 (NSP4) of rotavirus encoded by gene 10, plays an important role in rotavirus pathogenicity. In this study, NSP4 gene of avian rotavirus (AvRV-1, AvRV-2) was analyzed and expressed using baculovirus expression system. The sequence data indicated that the NSP4 gene of AvRV-1 and AvRV-2 were 727 bases in length, encoded one open reading frame of 169 amino acids beginning at base 41 and terminating at base 550, and had two glycosylation sites. Nucleotide sequences of NSP4 gene of AvRV-1 and AvRV-2 exhibited a high degree of homology ($88.1{\pm}7.6%$) with avian rotaviruses, namely Ty1, Ty3 and PO-13. Phylogenetic analysis showed that AvRV-1 and AvRV-2 belonged to genotype NSP4[E], which is widely found in group A avian rotaviruses. The baculovirus-expressed NSP4 migrated at 20-28 kDa and reacted with NSP4-specific antiserum by FA and Western blot. Furthermore, it was found to be a glycoprotein by using tunicamycin, which is a specific inhibitor of N-linked glycosylation.

• BMB Reports
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• 제32권3호
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• pp.317-319
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• 1999

A leucine aminopeptidase has been isolated from the culture medium of the soil fungus, Aspergillus flavus. The enzyme was found to be a glycoprotein, as judged by electrophoresis analysis and the subsequent staining by the periodic acid-Schiff's reagent. Carbohydrate moieties could be cleaved by N-glycosidase, but not by O-glycosidase, indicating that the glucans are linked to the asparagine residue in the protein. Removal of N-glucans was observed without prior denaturation of the protein, implying that the N-glycosidic linkage is exposed and accessible to glycosidase. When the activity of native or deglycosylated enzyme was measured in the presence of various metal ions, removal of carbohydrates increased the aminopeptidase activity of the enzyme.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.651-652
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• 2003

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.649-650
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• 2003