• Asian Pacific Journal of Cancer Prevention
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• 제14권4호
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• pp.2447-2451
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• 2013

Objective: To study changes of tumor associated carbohydrate antigen (TACAs) expression and mRNA levels for tumor associated glycosyltransferases, and assess subcellular localizations of N-acetyl galactosyltransferases (GalNAc-Ts) in the K562 leukemia cell line after imatinib treatment. Methods: RT-PCR was performed to analyze the expression of glycosyltransferases which synthesize O-glycan in tumor-associated carbohydrate antigens (TCTAs). The expression of Tn antigen, T antigen and sialyl T antigen on K562 cell membranes was measured by flow cytometry after treatment with different concentrations of imatinib. Co-localization of GalNAc-Ts and ER (endoplasmic reticulum) was determined by confocal laser scanning microcopy. Results: Transcript expression levels of several glycosyltransferases related to TCTAs were decreased after imatinib ($0-0.3{\mu}M$) treatment. Expression of Tn antigen and T antigen was increased while that of sialyl T antigen was decreased. Co-localization of GalNAc-Ts and ER was reduced by $0.2{\mu}M$ of imatinib. Conclusion: Imatinib inhibited the expression of O-glycan related TACAs and several related glycosyltransferases, while decreasing the co-localization of GalNAc-Ts and ER and normalizing O-glycosylation in the K562 human leukemia cell.

• Journal of Animal Science and Technology
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• 제47권2호
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• pp.167-176
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• 2005

PIGK(phosphatidylinositol glycan, class K) is a subunit of GPI transamidase that cleaves the signal peptide in proproteins and replaces it with GPI. In addition, the structure and synthesis of GPI are critically involved in some of the cellular actions of insulin. Therefore, PIGK would be essential for mammalian development and many specific cellular functions as well as for metabolic activity of insulin associated with GPI. Two types of" full-length cDNAs of porcine PIGK were cloned through RT-PCR and RACE experiments. One is thought to be a normal form(consist of 395 amino acids) and the other is considered as an alternative spliced form(consist of 371 amino acids) which contains additional 63 bps in intron 7. Since a stop codon was contained within the insertion, the spliced form has a shorter coding sequence than that of normal form. A missense mutation (T314I) in exon 6 was detected and used for genotyping to estimate association with the growth and fat deposition traits for 545 $F_2$ animals(Korean native boars ${\times}$ Landrace). From the PCR-RFLP analysis using HpyCH4III, CT genotype showed highly significant relationship(P< 0.01) with carcass fat contents.

• 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.

• Bulletin of the Korean Chemical Society
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• 제34권9호
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• pp.2663-2670
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• 2013

The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature ($25^{\circ}C$ and $40^{\circ}C$) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at $40^{\circ}C$ and pH 9, respectively.

• BMB Reports
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• 제45권11호
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• pp.623-628
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• 2012

Tissue inhibitor of metalloproteinases (TIMPs; TIMP-1, -2, -3 and -4) are endogenous inhibitor for matrix metalloproteinases (MMPs) that are responsible for remodeling the extracellular matrix (ECM) and involved in migration, invasion and metastasis of tumor cells. Unlike under normal conditions, the imbalance between MMPs and TIMPs is associated with various diseased states. Among TIMPs, TIMP-1, a 184-residue protein, is the only N-linked glycoprotein with glycosylation sites at N30 and N78. The structural analysis of the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1 suggests new possibilities of the role of TIMP-1 glycan moieties as a tuner for the proteolytic activities by MMPs. Because the TIMP-1 glycosylation participate in the interaction, aberrant glycosylation of TIMP-1 presumably affects the interaction, thereby leading to pathogenic dysfunction in cancer cells. TIMP-1 has not only the cell proliferation activities but also anti-oncogenic properties. Cancer cells appear to utilize these bilateral aspects of TIMP-1 for cancer progression; an elevated TIMP-1 level exerts to cancer development via MMP-independent pathway during the early phase of tumor formation, whereas it is the aberrant glycosylation of TIMP-1 that overcome the high anti-proteolytic burden. The aberrant glycosylation of TIMP-1 can thus be used as staging and/or prognostic biomarker in colon cancer.

• Journal of Plant Biotechnology
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• 제37권3호
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• pp.292-304
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• 2010

High value-added therapeutic proteins have been leading the biologics industry and occupied major portion of the market. More than 60% of the currently available protein therapeutics are glycoproteins attached with glycans which play crucial roles for the protein folding, therapeutic efficacy, in vivo half-life and immunogenecity. This review introduces the process of glycosylation and the impacts of glycans in the aspects of therapeutics. The important glycan structures in therapeutic performances were also summarized focusing on three representative categories of glycoproteins, cytokines, therapeutic antibody and enzyme. Currently, mammalian expression systems such as Chinese hamster ovary cells are preferred for the production of therapeutic glycoproteins due to their ability to synthesize glycans having similar structures with human type glycans. However, recent advances of plant glycoengineering to overcome the limitation originating from different glycan structures will soon allow to develop more efficient and economic plant-based production systems for therapeutic glycoproteins.

• Parasites, Hosts and Diseases
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• 제42권2호
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• pp.57-60
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• 2004

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

• 한국정보과학회논문지:시스템및이론
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• 제33권7호
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• pp.380-389
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• 2006

본 논문에서는, MGrid 시스템과 이를 통한 응용 어플리케이션으로서 Glycoconjugates 시뮬레이션 데이타베이스 구축에 대한 연구를 소개한다. MGrid 시스템은 분자 시뮬레이션 계산 및 분석에 대한 그리드 서비스를 상호 운용 가능한 방법으로 제공하는 그리드 시스템이다. e-Glycoconjugates는 당접합체류의 분자 시뮬레이션을 수행하는 그리드 포털이다. 이 프로젝트는 MGrid 시스템을 통해 PDB와 같은 단백질 구조 데이타베이스 상에서 지금까지 알려진 2000 여개의 glycan chain들과 100 여개의 당접합체류에 대한 분자 시뮬레이션 데이타베이스 구축을 목표로 하고 있다. 본 논문에서는, MGrid 시스템의 목표와 시스템 아키텍처, 현재 시스템의 구현과 e-Glycoconjugates의 초기 결과를 기술하고자 한다.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1087-1092
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• 2001

The effect of Sodium Butyrate (NaBu) on the N-linked oligosaccharide structure of Erythropoietin (EPO) was investigated. Recombinant human EPO was produced by CHO cells grown in an $MEM{\alpha}$ medium with or without 5 mM NaBu, and purified from the culture supernatants using a heparin-sepharose affinity column and immunoaffinity column. The N-linked oligosaccharides were released enzymatically and isolated by paper chromatography. The isolated oligosaccharides were then labeled with a fluorescent dye, 2-aminobenzamide, and analyzed with MonoQ anion exchange chromatography and GlycosepN amide chromatography for the assignment of a GU (glucose unit) vague. A glycan analysis by HPLC showed that the most significant characteristic effect of NaBu was a reduction in the proportion of glycans with Sri-and tetrasialylated oligodaccharides from $21.30\%$ (tri-) and $14.86\%$ (tetra-) in the control cultures (without NaBu) to $8.72\%$ (tri-) and $1.25\%$ (tetra-) in the NaBu-treated cultures, respectively. It was also found that the proportion of asialo-glycan increased from $12.54\%\;to\;23.6\%$ when treated with NaBu.

• International Journal of Industrial Entomology and Biomaterials
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• 제36권1호
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• pp.15-24
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• 2018

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (${\alpha}1AGP$) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the ${\alpha}1AGP$ to be a better model for studying glycosylation. The modified ${\alpha}1AGP$ (${\alpha}1AGP{\Delta}$) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the ${\alpha}1AGP$. Subsequently, we confirmed the detailed profile of N-glycan on the ${\alpha}1AGP{\Delta}$ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant ${\alpha}1AGP{\Delta}$ could be usable as a better model glycoprotein of N-glycosylation research in BES.