• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2008년도 제49회 학술심포지움 및 국제학술대회
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• pp.61.2-61.2
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• 2008

• 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.

• Molecules and Cells
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• 제37권4호
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• pp.322-329
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• 2014

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the ${\gamma}$-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.

• BMB Reports
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• 제47권10호
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• pp.593-598
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• 2014

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of ${\beta}$-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain insight into the PTM of hSCP1, we used the Western blot, immunoprecipitation, succinylayed wheat germ agglutinin-precipitation, liquid chromatography-mass spectrometry analyses, and site-directed mutagenesis and identified the $Ser^{41}$ residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s).

• Molecules and Cells
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• 제38권5호
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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.

• Applied Microscopy
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• 제31권1호
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• pp.49-57
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• 2001

섬유아세포 표면의 미세구조적 특성과 세포표면에 존재하는 당 단백질 말단 GlcNAc(N-acetylglucosamine)와 NeuNAc(N-acetylneuraminic acid)는 섬유세포의 이동과 세포의 인식에 중요한 역할을 하는 것으로 알려졌다. 따라서 섬유세포의 미세구조적 특성을 전자현미경을 사용하여 관찰하였으며, 당 단백질 말단 GlcNAc와 NeuNAc의 분포를 확인하기 위하여 WGA 황금입자 복합체를 반응시켜 전자현미경으로 관찰하였다. 그 결과 배양섬유세포의 표면에 미세구조적 특성은 세포의 분화 정도와 세포의 부위에 따라 다양한 형태를 형성하며, 세포의 배양시간에 따라 정도의 차이는 있으나 일반적인 미세융모의 분포와 세포질 돌기의 분화는 세포의 주위 환경에 따라 다양한 형태로 분화하는 특성이 있는 것으로 확인되었다. 섬유세포의 세포표면에 분포하는 당 단백질 말단의 일종으로 섬유세포의 이동과 세포인식에 관여하는 lectin WGA 수용체 인 sialic acid (GlcNAc; N-acetylgalactosamine, NeuNAc; N-acetyl neuraminic acid)는 세포질의 조면소포체에서 생성되어 액포상태로 이동되어 섬유세포의 외로 분비되고 분비된 sialic acid는 세포의 표면과 돌기의 표면에 당 단백질 말단으로 분화하여 섬유세포의 이동과 세포인식 등의 섬유세포 기능에 관여하는 것으로 규명되었다.

• Journal of mucopolysaccharidosis and rare diseases
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• 제2권1호
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• pp.13-16
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• 2016

Mucolipidosis (ML) II/III are autosomal recessive diseases caused by deficiency of post-translational modification of lysosomal enzymes. The mannose-6-phosphate (M6P) residue in lysosomal enzymes synthesized by N-acetylglucosamine 1-phosphotransferase (GlcNAc-phosphotransferase) serves as recognition marker for trafficking in lysosomes. GlcNAc-phosphotransferase is encoded by GNPTAB and GNPTG. Mutations in GNPTAB cause severe ML II alpha/beta and the attenuated ML III alpha/beta. Whereas mutations in GNPTG cause the ML III gamma, the attenuated type of ML III variant. For the diagnostic approaches, increased urinary oligosaccharides excretion could be a screening test in clinically suspicious patients. To confirm the diagnosis, instead of measuring the activity of GlcNAc phosphotransferase, measuring the enzymatic activities of different lysosomal hydrolases are useful for diagnosis. The activities of several lysosomal hydrolases are decreased in fibroblasts but increased in serum of the patients. In addition, the sequence analysis of causative gene is warranted. Therefore, the confirmatory diagnosis requires a combination of clinical evaluation, biochemical and molecular genetic testing. ML II/III show complex disease manifestations with lysosomal storage as the prime cellular defect that initiates consequential organic dysfunctions. As there are no specific therapy for ML to date, understanding the molecular pathogenesis can contribute to develop new therapeutic approaches ultimately.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.891-900
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• 2004

UDP-N-Acetylglucosamine(GlcNAc):$\beta$1,4-D-mannoside$\beta$-l ,4N-acetylglucosaminyltransferase-III (GnT-III) and UDP-N-GlcNAc:$\alpha$-6-D-mannosid$\beta$-1,6N-acetylglucosaminyltransferase-V(GnT - V) activities were determined in human hepatoma cell lines and metastatic colon cancer cells, and their activities were compared with those of normal liver cells and fetal hepatocytes. GnT-III activities were higher than those of GnT-V in hepatic carcinoma cells. When the two enzyme activities were assayed in highly metastatic colon cancer cells, GnT - V activities were much higher than those of GnT-III. When GlcN, GlcN-biant-PA and UDP-GlcNAc were used as substrates, the enzymes displayed different kinetic properties between hepatic and colon cancer cells, depending on their metastatic potentials. Normal cells of two origins had characteristically very low levels of GnT-III and -V activities, whereas hepatoma and colon cancer cells contained high levels of activities. These data were supported by RT-PCR and Northern blot analyses, showing that the expression of GnT-III and -V mRNAs were increased in proportion to the enzymatic activities. The increased GnT-III, md -V activities were also correlated with increased glycosylation of the cellular glycoproteins in hepatoma and colon cancer cells, as examined by lectin blotting analysis by using wheat germ glutinin (WGA), erythroagglutinating phytohemagglutinin (E-PHA), leukoagglutinating phytohemagglutinin (L-PHA), and concanavalin A (Con A). Treatment with retinoic acid, a differentiation agent, resulted in decreases of both GnT-III and -V activities of HepG2 and HepG3 cells. In colon carcinoma cells, however, treatment with retinoic acid resulted in a reduction of GnT-V activity, but not with GnT-III activity. Although the mechanism underlying the induction of these mzymes is unclear, oligosaccharides in many glycoproteins have been observed of cancer cells.

• BMB Reports
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• 제45권10호
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• pp.554-559
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• 2012

Oligosaccharide modification by N-acetylglucosaminyltransferase-V (GnT-V), a glycosyltransferase encoded by the Mgat5 gene that catalyzes the formation of ${\beta}1$,6GlcNAc (N-acetylglucosamine) branches on N-glycans, is thought to be associated with cancer growth and metastasis. Overexpression of GnT-V in cancer cells enhances the signaling of growth factors such as epidermal growth factor by increasing galectin-3 binding to polylactosamine structures on receptor N-glycans. In contrast, GnT-V deficient mice are born healthy and lack ${\beta}1$,6GlcNAc branches on N-glycans, but develop immunological disorders due to T-cell dysfunction at 12-20 months of age. We have developed Mgat5 transgenic (Tg) mice (GnT-V Tg mice) using a ${\beta}$-actin promoter and found characteristic phenotypes in skin, liver, and T cells in the mice. Although the GnT-V Tg mice do not develop spontaneous cancers in any organs, there are differences in the response to external stimuli between wild-type and GnT-V Tg mice. These changes are similar to those seen in cancer progression but are unexpected in some aspects. In this review, we summarize what is known about GnT-V functions in skin and liver cells as a means to understand the physiological roles of GnT-V in mice.

• 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.