• Journal of Life Science
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• v.23 no.4
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• pp.586-594
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• 2013

Post-translational O-GlcNAc modification (O-GlcNAcylation) of serine or threonine is a new protein modulation mechanism. In contrast to the classical glycosylation, O-GlcNAcylation occurs in a one-step transfer of O-GlcNAc on both nuclear and cytoplasmic proteins. In contrast to the general consensus that O-GlcNAc is a final modification, a recent paper (J Proteome Res. 2011 10:2725-2733) showed the presence of O-GlcNAc-P on a synaptic assembly protein AP180. This finding raises a fundamental question about its prevalence. To address this question, we used proteomics to identify those proteins that were phospho-signal enriched by GlcNAc kinase (NAGK). Comparison of pDsRed2-$NAGK_{WT}$-transfected HEK293T cell extract with pDsRed2-$NAGK_{D107A}$-transfected control culture revealed 15 phospho-signal increased spots. Excluding those spots that had no detectable amount of protein expression yielded 7 spots, which were selected for ID determination. Among these, two duplicate spots (two $HSP90{\beta}$ and two ENO1 spots) were shown to be O-GlcNAcylated, two (dUTP nucleotidohydrolase mitochondrial isoform 2, glutathione S-transferase P) were not known to be involved in O-GlcNAcylation, and one (heat shock protein gp96 precursor or grp94) was a glycoprotein. The increase in the phospho-levels of O-GlcNAc by NAGK strongly indicates that these proteins are phosphorylated on O-GlcNAc. Our present data support the idea that O-GlcNAc is not a terminal modification.

• Journal of Life Science
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• v.23 no.2
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• pp.324-331
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• 2013

The addition and removal of N-acetylglucosamine (GlcNAc) molecules on serine or threonine residues of a protein is called O-GlcNAcylation. This post-translational modification occurs on both cytoplasmic and nuclear protein, and is fast and reversible as comparable to phosphorylation. In contrast to the phospho-signaling cycles, this emerging moon-lightening signaling is cycled by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The simple machinery is a good evolutionary adaptation of a cell for quick accommodation to continuously fluctuating intra- and extracellular microenvironments. Rather than "switching" on or off a specific proteins - this would be done by phosphorylation where numerous specific kinases and phosphatases are involved - O-GlcNAcylation would play a "rheostat" which would be much more delicately increase or decrease the efficacy of signal transductions in response to cellular nutrient and stress conditions. Interestingly, recent evidence indicates that O-GlcNAc is further modified by phosphorylation. The O-GlcNAc-P will upgrade the modulation efficiency of cellular processes to continuous 'analogue' level. So far, only one protein AP180 was reported to have O-GlcNAc-P on Thr310. But, proteomic data from our laboratory indicate that there are multiple O-GlcNAc-P proteins, constituting "O-GlcNAc-P'om". This will focus on the possibility of existence of "O-GlcNAc-P'om".

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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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.

• Proceedings of the Microbiological Society of Korea Conference
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• 2004.05a
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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.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.147-153
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• 2004

Protein modification by N-acetyl-${\beta}$-D-glucosamine (O-G1cNAc) on the hydroxyl groups of Ser or Thr ubiq-uitously occurs in eukaryotic cells and is involved in many cellular phenomena. The level of O-G1cNAc-mod-ified protein is regulated by OGT and O-GlcNAcase enzymes. We have tried to produce recombinant O-GlcNAcase in E. coli as an effort to establish in vitro screening system for modulators of O-GlcNAcase. The culture conditions for improvement of O-GlcNAcase productivity, were as follows: induction temperature, $30^{\circ}C$; the concentration of L-arabinose, 0.02% and induction time, 5 hr. Under these culture conditions, E. coli cells containing O-GlcNAcase gene had no enzyme activity until up to 3 hr culture. However, O-GlcNAcase activity dramatically increased from 3 to 5 hr culture. It almost maintained the same level after 5 hr culture. Western blot analysis verified the amount of expressed O-GlcNAcase increased with culture time, being con-sistent with activity data. The optimal reaction condition determined in this study was as follows: protein quan-tity, $5{\mu}g$; reaction time, 30 min; reaction temperature, $45^{\circ}C$; substrate concentration, 2 mM; reaction pH, 6.5. Methanol had little effect on O-GlcNAcase activity and 90% of activity were retained at 10%. Only 15% resid-ual activity were detected at 5% of chloroform.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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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.

• Molecules and Cells
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• v.38 no.5
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• pp.402-408
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• 2015

Protein O-GlcNAcylation, dictated by cellular UDP-N-acetylglucosamine (UDP-GlcNAc) levels, plays a crucial role in posttranslational modifications. The enzyme GlcNAc kinase (NAGK, E.C. 2.7.1.59) catalyzes the formation of GlcNAc-6-phosphate, which is a major substrate for the biosynthesis of UDP-GlcNAc. Recent studies have revealed the expression of NAGK in different types of cells especially in neuronal dendrites. Here, by immunocytochemistry (ICC) and immunonucleochemistry (INC) of cultured rat hippocampal neurons, HEK293T and GT1-7 cells, we have showed that NAGK immuno-reactive punctae being present in the nucleoplasm colocalized with small nuclear ribonucleoprotein-associated protein N (snRNPN) and p54NRB, which are speckle and paraspeckle markers, respectively. Furthermore, NAGK IR cluster was also found to be colocalized with GTF2H5 (general transcription factor IIH, polypeptide 5) immuno reactive punctae. In addition, relative localization to the ring of nuclear lamin matrix and to GlcNAc, which is highly enriched in nuclear pore complexes, showed that NAGK surrounds the nucleus at the cytoplasmic face of the nuclear outer membrane. By in situ proximity ligation assay (PLA) we confirmed the colocalization of NAGK with snRNPN in the nucleus and in dendrites, while we also verified the interactions of NAGK with p54NRB, and with GTF2H5 in the nucleus. These associations between NAGK with speckle, paraspeckle and general transcription factor suggest its regulatory roles in gene expression.

• Journal of Life Science
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• v.12 no.1
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• pp.77-86
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• 2002

Chitin, a $\beta$-1,4 polymer of N-acetyl-D-glucosamine, is one of the most abundant organic compounds in nature. Chitinase (EC 3.2.1.14) is an enzyme that degrades chitin to chito-oligosaccharides, diacetyl rhitobiose and N-acetyl-D-glucosamine. An extracellular chitinase-producing bacterial strain was isolated from soil and named to as Bacillus subtilis JK-56. Optimum culture condition of B. subtilis JK-56 for the production of chitinase was 1% chitin, 0.5% polypepton, 0.1% KCl, 0.05% MnS $O_4$.4$H_2O$, 37$^{\circ}C$, initial pH 7.0 and 40 hour culture time. When B. subtilis JK-56 was grown in the optimum medium, one major active band and two minor active bands were detected by native-PAGE and active staining of the gel. Among them, the major band was purified from the culture supernatant by 70% ammonium sulfate precipitation and native-PAGE with BIO-RAD Model 491 Prep-Cell and named as Chi-56A. Its molecular weight was estimated to be 53kDa monomer and the isoelectric point (pI) was pH 4.3. The pH and temperature for the optimum activity of Chi-56A were pH 6.0 and $65^{\circ}C$, respectively. Chi-56A was stable up to $65^{\circ}C$ and in alkaline region. Its $K_{m}$ value for colloidal chitin was 17.33g/L. HPLC analysis of the reaction products confirmed that Chi-56A was an exo type chitinase.e.

• Molecules and Cells
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• v.40 no.7
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• pp.476-484
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• 2017

C-X-C chemokine receptor 4 (CXCR4) stimulates cancer metastasis. NF-${\kappa}B$ regulates CXCR4 expression in cancer cells, and O-GlcNAc modification of NF-${\kappa}B$ promotes its transcriptional activity. Here, we determined whether CXCR4 expression is affected by O-GlcNAcylation of NF-${\kappa}B$ in lung metastasis of cervical cancer. We found elevated levels of O-linked-N-actylglucosamine transferase (OGT) and O-GlcNAcylation in cervical cancer cells compared to those in non-malignant epithelial cells and detected increased expression of NF-${\kappa}B$ p65 (p65) and CXCR4 in cervical cancer cells. Knockdown of OGT inhibited the O-GlcNAcylation of p65 and decreased CXCR4 expression levels in HeLa cells. Thiamet G treatment increased O-GlcNAcylated p65, which subsequently enhanced CXCR4 expression levels. Inhibition of O-GlcNAcylation by 6-Diazo-5-oxo-L-norleucine (DON) treatment decreased p65 activation, eventually inhibiting CXCR4 expression in HeLa cells. Lung tissues from mice engrafted with OGT-knockdown HeLa cells (shOGT) exhibited lower expression of Ki-67 and HPV E6 and E7 oncogenes compared to lung tissues from mice engrafted with control HeLa cells (shCTL). In addition, lung tissues from mice engrafted with shOGT cells exhibited lower p65 and CXCR4 immunoreactivity compared to tissues from mice engrafted with shCTL cells. Taken together, our data suggest that p65 O-GlcNAcylation promotes lung metastasis of cervical cancer cells by activating CXCR4 expression.

• 한국당과학회:학술대회논문집
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• 2006.11a
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• pp.42-43
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• 2006