• 대한유전성대사질환학회지
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• 제20권1호
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• pp.1-13
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• 2020

Gaucher disease (GD)는 glucocerebrosidase 유전자(GBA)의 돌연변이에 의하여 발병하는 전세계적으로 가장 유병율이 높은 리소좀 축적질환이다. GD는 신경학적인 증상의 유무에 따라 3가지 임상형으로 구분된다. 신경병증 GD인 2형과 3형의 경우는 대뇌에서 glucosylceramide (GlcCer)와 glucosylsphingosine (GlcSph)의 농도가 증가하면서 신경세포의 심각한 손실이 야기되는 특징을 보인다. 신경교종에서 유래한 H4 세포를 GD에서 증가하는 기질인 GluCer와 GlcSph를 첨가하여 배양하였을 때, 심각한 DNA손상과 더불어 세포의 사멸이 야기되는 것과 이러한 신경세포의 사멸은 GluCer 보다는 GlcSph을 처리하였을 때 더 현저하게 증가하는 것을 관찰하였다. H4 세포에 히스톤 탈아세틸화 효소(HDAC) 6의 저해제인 tubacin과 GlcSph을 함께 처리하였을 경우에는 DNA손상은 물론 GlcSph에 의하여 유도된 세포사멸과 관련된 단백질 인자들의 발현이 모두 감소되었다. 본 연구를 통해 GlcSph이 세포사멸을 통하여 신경병증 GD의 발병에 주요한 역할을 한다는 것을 알 수 있었고, HDAC6 저해제가 신경병증 GD 환자를 위한 치료제 후보물질로 제시될 수 있는 가능성을 확인하였다.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2004년도 International Meeting of the Microbiological Society of Korea
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• pp.65-67
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• 2004

O-GlcNAcylation of p53 has been already identified and reported, but the function of O-GlcNAc on p53 has not been studied well. In this report, the general function of O-GlcNAc modification on p53 has been investigated using mouse fibroblast cell, L929. When streptozotocin (STZ), a non-competitive O-GlcNAcase inhibitor was treated to L929, O-GlcNAc modification level was dramatically increased on nucleocytoplasmic proteins, including p53. Because it has been already reported that $TNF\alpha$ induced the production of p53 in L929, $TNF\alpha$ was treated to obtain more p53. Approximately two times more amount of p53 was found from the cells treated STZ and $TNF\alpha$ simultaneously compared to the cell treated $TNF\alpha$ alone. The p53 increment in the presence of STZ was not caused by the induction of p53 gene expression. When new production of p53 induced by the $TNF\alpha$ was inhibited by the treatment of cycloheximide, O-GlcNAc modification decreased and phosphorylation increased on pre-existing p53 after $TNF\alpha$ treatment. But in the presence of STZ and $TNF\alpha$ at the same time, more O-GlcNAcylation occurred on p53, The level of ubiquitination on p53 was also reduced in the presence of STZ. Approximately three times less amount of Mdm2 bound to this hyperglycosylated p53. From this result it might be concluded that treatment of STZ to inhibit O-GlcNAcase increased O-GlcNAc modification level on p53 and the increment of O-GlcNAc modification stabilized p53 from ubiquitin proteolysis system.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.306-311
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• 2002

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O- GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.

• 생명과학회지
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• 제16권6호
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• pp.955-959
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• 2006

단백질의 serine 및 threonine 잔기에 O-linked N-acetylglucosamine (O-GlcNAc)의 수식은 핵단백질과 세포질 단백질의 주요 조절인자로 부각되고 있다. 본 연구에서는 GlcNAc를 인산화시켜 GlcNAc 6-phosphate로 만드는 GlcNAc kinase (NAGK, EC2.7.1.59)의 세포내 표현을 면역화학적 방법으로 조사하였다. 배양한 해미신경세포에서 NAGK는 가지돌기를 따라 점박이(punctae)를 형성하였으며, 이 점박이들은 caveolin-1 혹은 flotillin 항체에도 염색이 되었다. 이들은 각각 caveolac와 lipid raft의 표지단백질이기 때문에 본 연구결과는 NAGK가 세포막의 이러한 특수 미세부분(microdomain)에 존재함을 의미하며, 이 미세부분에서 아직 알려지지 않은 어떤 기능을 할 것을 시사한다.

• BMB Reports
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• 제40권6호
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• pp.1058-1068
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• 2007

The post-translational modifications of Ser and Thr residues by O-linked $\beta$-N-acetylglucosamine (O-GlcNAc), i.e., O-GlcNAcylation, is considered a key means of regulating signaling, in a manner analogous to protein phosphorylation. Furthermore, it has been suggested that the increased flux of glucose through the hexosamine biosynthetic pathway (HBP) stimulates O-GlcNAcylation, and that this may be responsible for many of the manifestations of type 2 diabetes mellitus. To determine whether excessive O-GlcNAcylation of target proteins results in pancreatic $\beta$ cell dysfunction, we increased nucleocytoplasmic protein O-GlcNAcylation levels in $\beta$ cells by exposing them to streptozotocin and/or glucosamine. Streptozotocin and glucosamine co-treatment increased O-GlcNAcylated proteomic patterns as assessed by immunoblotting, and these increases in nuclear and cytoplasmic protein O-GlcNAcylations were accompanied by impaired insulin secretion and enhanced apoptosis in pancreatic $\beta$ cells. This observed $\beta$cell dysfunction prompted us to examine Akt and Bcl-2 family member proteins to determine which proteins are O-GlcNAcylated under conditions of high HBP throughput, and how these proteins are associated with $\beta$ cell apoptosis. Eventually, we identified ten new O-GlcNAcylated proteins that were expressed during $\beta$ cell apoptosis, and analyzed the functional implications of these proteins in relation to pancreatic $\beta$ cell dysfunction.

• BMB Reports
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• 제57권2호
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• pp.92-97
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• 2024

Elevated blood glucose is associated with an increased risk of atherosclerosis. Data from the current study showed that glucosamine (GlcN), a normal glucose metabolite of the hexosamine biosynthetic pathway (HBP), promoted lipid accumulation in RAW264.7 macrophage cells. Oleic acid- and lipopolysaccharide (LPS)-induced lipid accumulation was further enhanced by GlcN in RAW264.7 cells, although there was no a significant change in the rate of fatty acid uptake. GlcN increased acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), scavenger receptor class A, liver X receptor, and sterol regulatory element-binding protein-1c (SREBP-1c) mRNA expression, and; conversely, suppressed ATP-binding cassette transporter A1 (ABCA-1) and ABCG-1 expression. Additionally, GlcN promoted O-GlcNAcylation of nuclear SREBP-1 but did not affect its DNA binding activity. GlcN stimulated phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Rapamycin, a mTOR-specific inhibitor, suppressed GlcN-induced lipid accumulation in RAW264.7 cells. The GlcN-mediated increase in ACC and FAS mRNA was suppressed, while the decrease in ABCA-1 and ABCG-1 by GlcN was not significantly altered by rapamycin. Together, our results highlight the importance of the mTOR signaling pathway in GlcN-induced macrophage lipid accumulation and further support a potential link between mTOR and HBP signaling in lipogenesis.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.210-214
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• 2001

GlcNAc 2-epimerase gene from human was cloned. However GIcNAc 2-epimerase was expressed in E. coli as inclusion body formation. Several approaches were tried such as expression in low temperature and low concentration of IPTG. With these treatments production of active form of human GIcNAc 2-epimerase ι ,vas enhanced. For the direct synthesis of NeuAc from GlcNAc and pyruvate, NALase and GlcNAc 2-epimerase were characterized in terms of temperature effect on activity. equilibrium and stability, inhibition by pyruvate etc. For cheap and ease preparation of both the NALase and GlcNAc 2-epimerase, pEN24ma vector was made. which express both the NALasc and GIcNAc 2-epimerase simultaneously. In addition, E. coli BL21(DE3) harboring two plasmids was also made. Of the two systems, the latter was better for the expression of both enzymes.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2003년도 제39회 학술심포지움
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• pp.23-37
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• 2003

A novel inhibitor against maltose-producing a-amylase was prepared via stepwise degradation of a high molecular weight acarbose (HMWA) using Thermus maltogenic amylase (ThMA). The structure of the purified inhibitor was determined to be ${\alpha}$-D-glucopyranosyl-${\alpha}$-acarviosinyl-D-glucopyranose (GlcAcvGlc). Progress curves of p-nitrophenyl-${\alpha}$-D-maltoside (PNPG2) hydrolysis by various amylolytic enzymes, including maltogenase (MGase), ThMA, and cyclodextrinase(CDase) I-5, in the presence of acarbose or GlcAcvGlc indicated a slow-binding mode of inhibition. The inhibition potency of GlcAcvGlc for MGase, ThMA, and CDase I-5 was 3 orders of magnitude higher than that of acarbose.

• Bulletin of the Korean Chemical Society
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• 제24권3호
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• pp.339-344
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• 2003

The conformation of synthetic oligosaccharide can be elucidated by employing molecular modeling and highfield proton NMR (nuclear magnetic resonance) spectroscopy. Information with respect to the composition and configuration of saccharide residues and the sequence and linkage positions of the oligosaccharide can be obtained by employing a variety of one- and two-dimensional NMR techniques and molecular modeling. These techniques are also useful in establishing the solution conformation of the oligosaccharide moiety. This study is focused on the elucidation of linkage positions of synthetic trisaccharides, Gal(β1-4)Glc(β1-3)Glc, Gal(β1-4)Glc(β1-4)Glc and Gal(β1-4)Glc(β1-6)Glc.

• Journal of Microbiology and Biotechnology
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• 제28권11호
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• pp.1850-1858
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• 2018

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase ($Dac_{ph}$) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature $40^{\circ}C$, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.