• Journal of Life Science
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• v.16 no.6
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• pp.955-959
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• 2006

A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. In this work, immunocytochemistry was carried out to investigate the subcellular expression of GlcNAc kinase (NAGK, EC 2.7.1.59) that catalyzes the phosphorylation of GlcNAc to GlcNAc 6-phosphate. Immunostainings of cultured rat hippocampal neurons revealed patchy or punctate distribution of NAGK. When NAGK is doublestained with caveolin-1 or flotillin, markers for caveolae and lipid rafts, respectively, NAGK was co-localized with these markers. These results indicate that most, if not all, of the NAGK immunopunctae represent caveolae and lipid rafts, and suggest NAGK's role in these membrane microdomains.

• 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.

• Journal of Animal Science and Technology
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• v.57 no.3
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• pp.12.1-12.5
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• 2015

Background: The Korean native pig (KNP) is generally thought to have come from northern China to the Korean peninsula approximately 2000 years ago. KNP pigs were at the brink of extinction in the 1980s, since then efforts have been made to restore the breed by bringing together the remaining stocks in South Korea. As a result, KNP was registered as a breed in 2006. To find additional breed-specific markers that are distinct among pig breeds, variations in O-linked N-acetylglucosamine transferase (OGT) were investigated. OGT is located on chromosome X and catalyzes the post-translational addition of a single O-linked-${\beta}$-N-acetylglucosamine to target proteins. Findings: Length polymorphism in the intron 20 of OGT was identified. The intron 20 of OGT from Duroc, Landrace, and Yorkshire breeds was 281-bp longer than that from either KNP or Chinese Meishan pigs. The difference between the Western pig breeds (BB genotype) and KNP or Meishan pigs (AA genotype) was due to an inserted 276-bp element and the 5-bp ACTTG. Conclusions: The polymorphism in OGT identified in this study may be used as an additional marker for determining the breed of origin among Meishan and the Western pig breeds. The length polymorphism suggests that the locus near OGT is not fixed in KNP. This marker would be relevant in determining the breed of origin in crossbred pigs between KNP pigs with known genotypes and the Western pig breeds with BB genotypes, thus confirming the contribution of the X chromosome from each breed.

• BMB Reports
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• v.29 no.3
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• pp.266-271
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• 1996

A recombinant human erythropoietin (EPO), expressed in Chinese hamster ovary (CHO) cells, is glycosylated at Asn 24, Asn 38, Asn 83, and Ser 126. After release of the N-linked carbohydrate chains by $peptide-N^{4}-(N-acetyl-{\beta}-glucosaminyl)$ asparagine amidase F, the oligosaccharides were analyzed by FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). The O-linked carbohydrate chain was separated by hydrazine, and analyzed by FACE. The monosacccharide composition of recombinant EPO showed man nose, fucose, galactose, N-acetylglucosamine, N-acetylneuraminic acid, and a trace of N-acetylgalactosamine, which are typical monosaccharides in the glycoproteins from the CHO cell. Sequences of N-linked and O-linked oligosaccharides were determined. The structure and composition of oligosaccharides attached to recombinant human EPO, expressed in the CHO cell, are identical to the reported oligosaccharide structure in human EPO isolated from urine.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1086-1092
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• 2017

Objective: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of O-GlcNAc and GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling and transcription. Pig OGT is located near the region of chromosome X that affects follicle stimulating hormone level and testes size. The objective of this study was to find the variations of OGT between European and Chinese pigs. Methods: Pigs were tested initially for polymorphism in OGT among European and Chinese pigs by polymerase chain reaction and sequencing at the U.S. Meat Animal Research Center (USMARC). The polymorphism was also determined in an independent population of pigs including European and Chinese Meishan (ME) breeds at the National Institute of Animal Science (NIAS, RDA, Korea). Results: The intron 20 of OGT from European and Chinese pigs was 514 and 233 bp, respectively, in the pigs tested initially. They included 1 White composite (WC) boar and 7 sows ($2Minzu{\times}WC$, $2Duroc\;[DU]{\times}WC$, $2ME{\times}WC$, $1Fengzing{\times}WC$) at USMARC. The 281-bp difference was due to an inserted 276-bp element and GACTT in European pigs. When additional WC and ME boars, the grandparents that were used to generate the $1/2ME{\times}1/2WC$ parents, and the 84 boars of 16 litters from mating of $1/2ME{\times}1/2WC$ parents were analyzed, the breeds of origin of X chromosome quantitative trait locus (QTL) were confirmed. The polymorphism was determined in an independent population of pigs including DU, Landrace, Yorkshire, and ME breeds at NIAS. OGT was placed at position 67 cM on the chromosome X of the USMARC swine linkage map. Conclusion: There was complete concordance with the insertion in European pigs at USMARC and NIAS. This polymorphism could be a useful marker to identify the breed of origin of X chromosome QTL in pigs produced by crossbreeding Chinese and European pigs.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.87-93
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• 2004

Dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) on Ser and Thr residues is ubiquitous in higher eukaryotes. And this modification may serve as a signaling mod-ification analogous to protein phosphorylation. Addition and cleavage of O-GlcNAc are catalyzed by O-linked GlcNAc transferase (OGT) and O-linked N-acety1glucosaminidase (O-GlcNAcase), respectively. Two types of human O-GlcNAcase gene were cloned and expressed as three fusion proteins in Escherichia coli. O-GlcNA-case activity showed in the order of thioredoxin fusion> $6{\times}His$ tag> GST fusion. O-GlcNAcase had enzy-matic activity against only ${\rho}$NP-GlcNAc of seven tested substrate analogs. Blast search revealed that O-GlcNAcase has two conserved domains, amino terminal hyaluronidase-like domain and carboxy terminal N-acetyltransferase domain. Extensive deletion studies were done to define catalytically important domains. The deletions of hyaluronidase-like domain and N-acetyltransferase domain abolished enzyme activity. But, N-ter-minal 55 amino acid deletion and C-terminal truncation showed lower activity. Based on deletion analysis, we suggest that hyaluronidase-like domain is essential for enzyme activity and carboxy terminal N-acetyltrans-ferase domain may be modulatory function.

• Parasites, Hosts and Diseases
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• v.59 no.5
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• pp.501-505
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• 2021

The pathogenic free-living amoeba Naegleria fowleri causes primary amoebic meningoencephalitis, a fatal infection, by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves. N. fowleri can induce host cell death via lytic necrosis. Similar to phosphorylation, O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), respectively. However, the detailed mechanism of host cell death induced by N. fowleri is unknown. In this study, we investigated whether N. fowleri can induce the modulation of O-GlcNAcylated proteins during cell death in Jurkat T cells. Co-incubation with live N. fowleri trophozoites increased DNA fragmentation. In addition, incubation with N. fowleri induced a dramatic reduction in O-GlcNAcylated protein levels in 30 min. Moreover, pretreatment of Jurkat T cells with the OGA inhibitor PUGNAc prevented N. fowleri-induced O-deGlcNAcylation and DNA fragmentation. These results suggest that O-deGlcNAcylation is an important signaling process that occurs during Jurkat T cell death induced by N. fowleri.

• BMB Reports
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• v.47 no.10
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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).

• 한국당과학회:학술대회논문집
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• 2006.11a
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• pp.44-44
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• 2006

• BMB Reports
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• v.40 no.6
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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.