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More than 60% of the currently available therapeutic proteins are glycoproteins attached with glycans, which play important roles in the protein folding, therapeutic efficacy, in vivo half-life, and immune reactions. Our group has focused on N-glycosylation pathway engineering of recombinant expression hosts including yeasts and Chinese hamster ovary (CHO) cells for production of therapeutic glycoproteins with improved potency. Different engineering strategies are required depending on the classes of therapeutic proteins because their optimal glycan structures are different. Many therapeutic enzymes used for treatment of lysosomal storage diseases require high contents of mannose-6- phosphate (M-6-P) glycan, which is essential for cellular uptake and targeting to lysosomes. Although yeasts do not have M-6-P glycans, they have some glycans containing mannosylphosphorylated mannose residues which can be converted to M-6-P structure through removal of the outer capping mannose. Recently, we constructed a glyco-engineered Saccharomyces cerevisiae mnn1Δoch1Δ strain overexpressing YlMPO1 gene, which was shown to have very high content of mannosylphosphorylated glycans [1, 2]. As they were efficiently converted to high content of M-6-P glycans through uncapping process, our engineering strategy showed a promise for production of biobetter enzymes with enhanced lysosomal targeting capabilities. For prolonged in vivo half-life of glycoproteins, sialic acids present on non-reducing terminal of glycan are important. We compared several bacterial α(2,6)-sialyltransferases and found that they have inherent siallidase activities, which decreased the amount of sialylated products [3]. These sialidase activities were inhibited by simple treatment of alkaline phosphatase, which desphosphorylates free cytidine monophosphate (CMP) generated from a donor substrate (CMP-N-acetylneuraminic acid) during the sialyltransferase reaction. It greatly enhanced the efficiency of bacterial α(2,6)- sialyltransferase reaction, which resulted in the increased content of bi-sialylated N-glycan up to ~98% without any decrease in prolonged reactions. Our efforts to construct Chinese hamster ovary (CHO) cells for production of glycoproteins with increased content of sialic acids shall be introduced also.

In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle.

Glycans are key functional components in protein biopharmaceuticals, disease biomarkers, viral vaccines, prebiotics, and a host of other myriad applications. Consequently, analytical methods for glycan characterization are in constant demand, as are the skills necessary to carry out such an analysis. The Asia Glycomics Reference Site (AGRS) specializes in mass spectrometric characterization of glycans, glycoproteins, glycolipis, and proteins. The AGRS develops and validates new analytical platforms for glycomic and glycoproteomic-analysis, using cutting-edge facilities and equipment made available through industrial partnerships with leading instrument manufacturers. In addition to providing core glyco-analytical support to biopharmaceutical companies as well as governmental regulatory agencies, the AGRS conducts basic glycobiological research in collaboration with academic labs around the world.

MicroRNAs (miRNAs) are regulatory small RNAs that have important roles in numerous developmental, metabolic, and disease processes of plants and animals. The individual levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis. Thus, innovative new tools for rapid, specific, and sensitive detection of miRNAs are an important field of research. Using the fluorescence properties of DNA-nanosilver clusters (DNA/AgNC), we have designed a DNA/AgNC probe that can detect the presence of target miRNA. Here, we show that the red fluorescence of the DNA/AgNC probe is diminished upon the presence of target miRNA without pre- or postmodification, addition of extra enhancer molecules, and labeling. The DNA/AgNC probe emission was lowest when the complementary miRNA target was present and was significantly higher for four other control miRNA sequences. Also, when adding whole plant endogenous RNA to the DNA/AgNC probe, the emission was significantly higher for the mutant where miRNA was deficient. On the basis of these findings, we suggest that these DNA/AgNC probes could be developed into a new, simple, inexpensive, and instant technique for miRNAs detection.

Cancer is often difficult to achieve early diagnosis, which is, however, a decisive requisite for favorable outcome in cancer treatments. Cancer biomarkers have been sought for several purposes preferably in blood and pinpointing cancer cells-derived aberrant glycoproteins would be a well-grounded approach to cancer biomarker discovery. One of the glycosyltransferases responsible for aberrant glycosylation in cancer is N-acetylglucosaminyltransferase V (GnT-V), which catalyzes an addition of b1,6-N-acetylglucosamine (GlcNAc) to the core N-glycan, and many lines of evidence have demonstrated the role of N-acetylglucosaminyltransferase V (GnT-V) in cancer development. Tissue inhibitor of metalloproteinase-1 (TIMP-1) and protein tyrosine phosphatase kappa (PTPk) were suggested to be involved in cancer malignancy upon aberrantly glycosylation by GnT-V. In addition to GnT-V, several glycosyltransferase co-works to render the altered glycan structures in cancer cells including sialyl Lewis antigen and core-fucosylation, which prompted us to mine serological biomarker candidates in cancer sera. Immunodepleted on an immune-LC column, serological proteins were enriched by carbohydrate beads conjugated with lectins like L-PHA, DSA, E-selectin, AAL, Con-A. The fraction refractive to lectin enrichments was resolved on an SDS-PAGE gel, and fractionated by molecular mass. Both the captured glycoproteins and gel-separated proteins were tryptic-digested for sequence determination in an LTQ-FTICR mass spectrometer. Candidate proteins showing high sensitivity and specificity during the discovery phase were selected and the panel of biomarker candidates is currently under in-depth analyses for validation.

The therapeutic effects of medicinal plants used in traditional medicine were evaluated only on the basis of the empirical practice. Searching for active biopolymers with antitussive and bronchodilatory properties, several plants were investigated in term of chemical structure and their pharmacodynamics properties. The study was focused on the extractable polymer complexes of flowering parts of medicinal plants. Alkaline extractions of flowers from selected herbs afforded the dark-brown glycoconjugates (polysaccharide-protein-phenolic complexes) differing in molecular mass, carbohydrate, phenolic and protein contents. Carbohydrate parts of conjugates were rich mainly in rhamnogalacturonans/galacturonans and arabinogalactans. Antitussive activity tests showed in some conjugates the reduction of the number of cough efforts in the dose-dependent manner, while in some conjugates this dose dependence was not observed. The tests evaluating the influence of different doses of conjugates on airway smooth muscle reactivity revealed more significant effect of some plant conjugates in comparison with a commercial bronchodilator used. Comparative tests showed that antitussive activity of the most effective conjugate was lower than that of codeine, the strongest antitussive drug used in the clinical practice. Some glycoconjugates were shown to be promising candidates for the application in the herbal medicine as antitussive and bronchodilatory agents.

Glucose is a primary carbon source for the most of the life form of earth. Cellluose, starch, and many of polysaccharides exclusively or mainly consist of anhydroglucose. Uses of glucose polymers from maltodextrin to starch include food, paper, textile, plastics, pharmaceutical, and cosmetics industries. Recently, market size of glucose/glucose polymer is expected to show annual growth greater than 8% and expended up to 42.2 billion dollars in 2021. However, producing structurally pure glucose polymer is still a challenging task. Because, glucose polymer with degree of polymerization (DP) 100 has, literally, innumerous structures by anomeric forms (α or β) and linkage positions. To solve these problems, our research uses both in vivo and in vitro approaches. For pure maltodextrin synthesis, a pure but cheap substrate was supplied to an immobilized enzyme reactor. Polysaccharides that had different branched structure but similar DP were produced from amylopectin by purified branching enzymes. But, glycogen-like polysaccarides that are natural dendrimers were synthesized in Escherichia coli cell system and purified. These approaches still need to be improved for industrialization, but provide worthwhile methods as progress in glucose polymer synthesis.

Various native and hydroxyethylated starch granules were reacted as substrates for a debranching enzyme, pullulanase, from Bacillus acidopullulyticus , and the kinetic mechanism was investigated in initial rate studies. The apparent K m values of the native starch hydrolysis reactions decreased in the order of rice, corn, and potato, whereas V max remained constant for all starches. Assuming that starch granules were spherical, the double reciprocal of the initial rates of the hydrolysis reactions along with the surface area of the three native starch granules could be described in a linear function. Therefore, enzymatic hydrolysis of native starches during the initial stage is mainly affected by particle size of starch granules. In case of hydroxyethylated corn starch (HES) and native corn starch, the apparent K m values of the native corn starch and HES hydrolysis reactions showed similar values, but V max of HES was higher than that of native corn starch, which was resulted from reduced activation energy of HES toward a debranching enzyme. From these results, the surface area as well as particle size of granules have important effects on the initial rate at which native starch is digested by debranching enzyme. Also, we could modulate the enzyme activity toward native starch by modifying the surface of native starches.

Genome editing technologies are versatile tools to delete, insert, or correct a certain span of nucleotide sequences under viable conditions. The generation of model animals became quite easier and has been facilitated due to the development of genome editing methods. We aim to generate glycoengineering mice using CRISPR/Cas9 system that have modified glycan structures either systemically or at a specific proteins. One of the engineering mice is a glycan-humanized mouse that mimics glycan structures of human. Another is the development of aglycosylated antibody-producing mice for precision diagnosis. These glycoengineering mice are expected to provide more efficient preclinical model mice for drug development as well as antibody-producing hosts for personalized, precision medicine.

Gangliosides are glycolipid having sialic acid. It is present in the central nervous system and involved in multiple functions of cell membranes and nerve function. However, roles of gangliosides in the differentiation of mesenchymal stem cells (MSCs) is unclear. Therefore, we investigated the effects of ganglioside in neuronal differentiation of adipose-derived MSCs (AD-MSCs). In the differentiated neuronal cells from AD-MSCs. we observed the expression of neuronal markers (MAP-2 and GFAP) and the high levels of mRNA expression of neuron-specific genes. Also, The expression of ganglioside GM2 and GD3 were significant increased in neuronal differentiation of AD-MSCs. In addition, this study showed that differentiation of AD-MSCs into neuronal cells significant decrease in the knockdown of UDP-glucose ceramide glucosyltransferase (UGCG) which well known as first step of ganglioside synthase and beta-1,4-N-acetyl-galactosaminyl transferase 1(B4GALNT1) gangllioside GM2 synthase. The results indicate that ganglioside GM2 and GD3 might be important roles in the neural differentiation of mini-pig AD-MSCs.

Alzheimer's disease(AD), known as a chronic neurodegenerative disease, is the most common type of dementia with clinical symptoms. The pathogenesis of AD is highly believed to have a effect on the production and deposition of β-amyloid(Aβ) peptide. Swedish mutation or Familial Alzheimer’s disease genetic mutation is one of the most well known genetic variation that causes early onset familial AD. Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation and cell signaling. However the mechanism is not unclear between ganglioside and AD. This study was investigated relationship of gangliosides between ganglioside and AD. We made a cell line(WK-H4) using transformed Vector that is forced to gene over-expression of Aβ. We investigate the expression by using high performance thin-layer chromatograph(HPTLC), Western Blot(WB) and Immunofluoresence(IF). According to IF, the results show Aβ significantly increase in WK-H4. Also, HPTLC analysis shows that the expression of ganglioside GD1a is markedly diminished in WK-H4 than H4. Furthermore, β-Catenin at Wnt signal increases on WB analysis. While GSK-3β imperceptibly decrease in WK-H4. In conclusion, This study may reason out a conclusion that ganglioside is associated with AD and the selective control of ganglioside is helpful treatment of AD as a fresh bio-marker.

Mesenchymal stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. The Ganglioside is included in biomarker, that are the major component of cytoplasmic cell membranes complex glycosphingolipids, and play a role in the control of biological processes. However, role of ganglioside in osteogenesis of mini-pig adipose derived mesenchymal stem cells (AD-MSCs) unclear . We have knocked down was ganglioside synthase UDP-glucose ceramide glucosyltransferase (UGCG)using a siRNA differentiation into osteoblast was markedly decreased in UGCG-knocked down AD-MSCs. The results showed significant inhibits alkaline phosphatase, osteocalcin, osteopontin, and Runt-related transcription factor 2 in UGCG knock down AD-MSCs compared to with the control AD-MSCs. Thus we guess, maybe ganglioside is essential factor to stimulate osteogenesis in AD-MSCs.

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-xylose and α 1,3-fucose residues Man3XylFucGlcNAc2. Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan3GlcNAc2 inhibits additions of the core β1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man3XylFucGlcNAc2 and GlcNAc2Man3XylFucGlcNAc2 structures. To investigate the physiological effects of the addition of the 6-arm GlcNAc residue to the N-glycans in plants, phenotypes of Col-0, gnt2, 35S:GnTII and a triple-knockout mutant with mutated alleles of N-acetyl-beta-D-hexosaminidases (Hex1, Hex2 and Hex3) were analysed in the absence or presence of exogenously supplied tunicamycin (TM) or sodium chloride (NaCl). gnt2, compared with Col-0, 35S:GnTII and the triple mutant, displayed increased sensitivity to TM and NaCl during germination and seedling development, indicating that GnTII is important to confer stress tolerance on plants.

Endoplasmic reticulum (ER) stress induced by various stimuli leads to accumulation of unfolded proteins and unfolded protein response (UPR) in plants. P58IPK induced by ER stress is known to inhibit the activity of protein kinase RNA-like ER kinase (PERK) in mammalian cells. Both genetic reduction of PERK expression and pharmacological inhibition of its activity reduces p-eIF2α levels resulting in a reduction in general protein translation in mammalian cells. However, it has been unclear whether a similar mechanism is applicable to the P58IPK in plants. To investigate physiological function and molecular mechanism of the P58IPK in plants, a loss-of-function mutant of P58IPK(p58ipk) and P58IPKoverexpressingplants(CVMVp:P58IPK) have been analysed. We found that p58ipk is more resistant to tunicamycin (TM), an inhibitor of protein N-glycosylation, than the Arabidopsis Columbia-0 (Col-0) ecotype background and CVMVp:P58IPK. Gene expression profiling studies indicate that transcript levels of ER stress marker genes are more higher in p58ipk than those in Col-0 and CVMVp:P58IPK. However, unlike its mammalian counterpart, P58IPK in Arabidopsis do not affect p-eIF2α levels but interacts with ribosome to inhibit protein synthesis. These results indicate that P58IPK is also implicated in UPR in plants but different molecular mechanism accounts for physiological effects of the loss-of-function mutation of P58IPKin plants.

Genetic engineering approaches to improve therapeutic potentials of mesenchymal stem cells (MSCs) have been made by viral or non-viral gene delivery methods. Viral methods have severe limitations in clinical application due to potential oncogenic, pathogenic, and immunogenic risks while non-viral ones have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery. Conventional cationic liposome method was not effective for MSC transfection to result in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles up to 66% in MSCs without significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression, compared to plasmid microporation. When the MSCs transfected to express firefly luciferase by minicircle microporation were subcutaneously injected to mice, their bioluminescence was monitored for more than a week whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By using minicircle microporation as a non-viral gene delivery, the engineered MSCs to overexpress CXCR4 were shown to have greatly increased homing ability toward the injury site through in vivo bioluminescence imaging in mice, which showed a promise to enhance therapeutic potentials of MSCs in clinical applications.

Mannosylphosphorylated glycans, which confers negative charges on the cell surface, are only found in yeasts and fungi groups. Both MNN4 and MNN6 genes are required for mannosylphosphorylation in the traditional yeast Saccharomyces cerevisiae. Mnn4 protein has been known to be a positive regulator of Mnn6p, a real enzyme for mannosylphosphorylation. We constructed the quadruple-disrupted och1Δmnn1 Δmnn4Δmnn6Δ strain by disrupting MNN4 and MNN6 genes from the och1Δmnn1Δ strain in which yeast-specific hyper-mannosylated glycan and immunogenic α(1,3)-mannose structures were already abolished [1]. However, yeast-specific mannosylphosphorylated N-glycan structures were still observed in the och1Δmnn1Δmnn4Δmnn6Δ strain, which suggests that there is another gene involved in mannosylphosphorylation. From the och1Δmnn1Δmnn4Δmnn6Δ strain, we further disrupted the genes (MNN14, YUR1, KTR2, KTR4, KTR5, or KTR7) having homology with MNN4 or MNN6 genes. Analyses of N-glycans obtained from cell wall mannoproteins of six quintuple-disrupted strains indicated that mannosylphosphorylated glycan structure was completely abolished only in the och1Δmnn1Δmnn4Δ mnn6Δmnn14Δ strain. Since MNN14 has the homology with MNN4, the och1Δmnn1Δmnn14Δ and och1 Δmnn1Δmnn4Δmnn14Δ strains were constructed. While mannosylphoshorylated N-glycans were observed in och1Δmnn1Δmnn14Δ strain, there was no mannosylphosphorylation in the och1Δmnn1Δmnn4Δmnn14Δ strain, which clearly shows that disruption of both MNN4 and MNN14 genes was required to remove mannosylphosphorylated glycan structures. Complementation of one gene (either MNN4 or MNN14) restored mannosylphosphorylation in the och1Δmnn1Δmnn4Δmnn14Δ strain, which supports that MNN4 and MNN14 can play a redundant role on the deficiency of one gene. The och1Δmnn1Δmnn4Δmnn14Δ strain without any yeast-specific glycan structure will serve as an useful platform for production of glycoproteins with human-compatible N-glycans.

Sialic acids present on non-reducing terminal of glycan is important for prolonged in vivo half-life of glycoproteins. Many efforts have been made to increase the sialic acid content in the manufacturing of therapeutic glycoproteins because glycoproteins containing non-sialylated glycans do not show effective in vivo efficacies owing to a short half-life. It was reported that knock down of neuraminidase expression using the RNA interference (RNAi) technology enhanced protein sialylation in CHO cells which are the most widely used for therapeutic glycoprotein production [1]. However, knock down strategy did not completely shut down the neuraminidase expression. In this study, we performed knockout of neuraminidase genes in CHO cells by using the CRISPR/Cas9 genome editing technology. Only three neuraminidase (Neu1, Neu2, and Neu3) were actively expressed in CHO cells when analyzing their expressions using real-time polymerase chain reaction (PCR). Neu3 is located in the plasma membrane while Neu1 and Neu2 exist in lysosome and cytosol, respectively. After the transfection of vectors expressing Cas9 and sgRNAs, Neu-disrupted clones were selected by using the T7 endonucleases 1 (T7E1) assay and further confirmed by the sequencing analysis of the corresponding editing sites. All of single mutants (Neu1-, Neu2-, and Neu3-disrupted clones) displayed similar proliferation ability compared to wild-type CHO cells. Two of double Neu-disrupted mutants (Neu1Neu3- and Neu2Neu3-dirupted mutants) were currently constructed. Our goal is to construct the triple knockout CHO cell mutant (Neu1Neu2Neu3-disrupted clone) and investigate its phenotypes and features.

Syndecans are a cell surface single-pass transmembrane protein which is involved in various cellular functions. It is known that syndecans form homo-, hetero-dimer through transmembrane domain and the extent of dimerization affects intracellular signaling. However, little has been known about transmembrane domain of syndecan-3. In this research, it is discovered that syndecan-3 forms SDS-resistant higher-order oligomerization through transmembrane domain and alanine located nearby GxxxG motif has a crucial role to maintain oligomer among 25 hydrophobic transmembrane amino acids. Moreover, the pattern of higher-order oligomerization has an influence to regulate cellular function such as migration in neuroblastoma. In conclusion, syndecan-3 has a distinct feature of higher-order oligomerization through transmembrane domain in constrast with other syndecan family proteins and alanine is a key amino acid which has an ability to form oligomer and also these pattern of oligomerization in syndecan-3 is linked to cell behavior.

O-GlcNAcylation is a dynamic posttranslational modification that occurs in the serine or threonine residues of nuclear and cytosolic proteins. O-GlcNAcylation regulates various cellular events, such as a nutrient sensing, epigenetic regulation, translational regulation, and cell proliferation, as well as disease-related signal pathways. UDP-GlcNAc, a precursor of O-GlcNAcylation, is synthesized from glucose by the hexosamine biosynthetic pathway (HBP). The level of UDP-GlcNAc depends on the cellular glucose condition. For this reason, O-GlcNAcylation is called a nutrient sensor. Although O-GlcNAcylation plays a role in nutrient sensing and metabolism, the relationship between O-GlcNAcylation and autophagy remains unknown. In previous studies, decreased O-GlcNAcylation by OGT siRNA induced autophagy without starvation. In this study, we observed that a unc-51 like autophagy activating kinase 1(ULK1), a regulator of autophagy, is O-GlcNAcylated. Under glucose deprivation, O-GlcNAcylated ULk1 is increased, and increased O-GlcNAcylation induces ULK1 S757 phosphorylation, which leads to ULK1 inactivation. The ULK1 S757A mutant decreases O-GlcNAcylation on ULK1 and binding affinity with OGT compared with ULK1 wild type. These data imply that O-GlcNAcylation on ULK1 decreases ULK1 kinase activity by increasing S757 phosphorylation and reduces autophagy induction.

Nucleocytoplasmic O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues. Although the cellular localisation of OGT is important to regulate a variety of cellular processes, the molecular mechanisms regulating the nuclear localisation of OGT is unclear. Here, we characterised three amino acids (DFP; residues 451–453) as the nuclear localisation signal of OGT and demonstrated that this motif mediated the nuclear import of non-diffusible β-galactosidase. OGT bound the importin α5 protein, and this association was abolished when the DFP motif of OGT was mutated or deleted. We also revealed that O-GlcNAcylation of Ser389, which resides in the tetratricopeptide repeats, plays an important role in the nuclear localisation of OGT. Our findings may explain how OGT, which possesses a NLS, exists in the nucleus and cytosol simultaneously.

Most cancer cells need more glucose. Because they produce energy by fermentation, even though they exist in the presence of oxygen. Cancer cell can escape a poor environment like hypoxia and under nutrition condition. Considering this fact, It is deducible that hypoglycemia affect cancer metastasis. But the research about cancer metastasis has been limited to hypoxia condition until now. Interestingly, it was reported that O-GlcNAc modification on protein was increased under hypoglycemic condition. And it was thought that O-GlcNAcylation is related with EMT. In our study, we want to examine whether hypoglycemic condition can induce cancer metastasis and it is related to increased O-GlcNAc modification or not. So we focused on EMT to visualize cancer metastasis. We found that the amount of several EMT marker were changed under hypoglycemic condition in cancer cell. And it was mitigated by OGT knock down. These findings tell us that hypoglycemic condition can be closely connected with cancer metastasis via O-GlcNAclation. So we suggest possibilities that hypoglycemic condition induced EMT in cancer cell and it related with increased O-GlcNAc modification.

O-GlcNAcylation is a carbohydrate post-translational sugar modification occurs on hydroxyl groups of serine or threonine residues of cytosolic and nuclear proteins. It is known that O-GlcNAc addition and removal regulate many biological events like transcription, protein degradation, proliferation. Epithelial-mesenchymal transition(EMT) is a process that epithelial cells lose its polarity and cell-cell contact, therefore get migratory capabilities to become mesenchymal stem cells. There are several reports that O-GlcNAc is associated with cancer EMT and metastasis. But there are still much to discover the role of O-GlcNAc on TGF-β-induced EMT. TGF-β is a powerful EMT inducer, and is primarily dependent on TGF-β/Smad signaling pathway. Here we found that Smad protein expression is regulated by global O-GlcNAc change, consequentially controls TGF-β-induced EMT. Now we’re planning to verify how O-GlcNAc controls Smad expression.

O-GlcNAc modification is glycosylation that occurs on Serine and Threonine residues of proteins located in nucleus and cytoplasm. O-GlcNAcylation regulates various cellular events including transcription, translation, cell death and cell proliferation as cellular nutrient sensor. It also has important roles in development processes such as neurogenesis, osteogenesis, adipogenesis and myogenesis. Although some roles and molecular mechanism of O-GlcNAcylation in various development process has been studied, the relationship between O-GlcNAcylation and muscle differentiation is not clearly established. We confirm that physiological conditions to regulate cellular O-GlcNAc level as well as O-GlcNAcase inhibitor treatment influenced the myogenin expression and myogenesis. We also confirm that this results was shown in mouse model system. We demonstrate that these phenomenons result from O-GlcNAc of Mef2c. Furthermore, we identify the 3 O-GlcNAc sites in Mef2c and find that one of these site has role regulating the DNA binding affinity with promoter of myogenin. Our results suggest that one specific O-GlcNAc site of Mef2c is closely associated with muscle differentiation through the regulation of myogenin transcription.

The N-glycan profile of a biologics, especially therapeutic antibody is commonly defined as a critical quality attribute (CQA) in bioprocess development. Fc glycosylation is reported to be important in the antibody-dependnet cell-mediated cytotoxicity (ADCC) and for complement-dependent cytotoxicity (CDC) functions through modulating the binding to Fcy receptors and C1q respectively. Removal of core fucose can also increase the ADCC. Therefore the analysis of N-glycan is very important and critical in the function of therapeutic antibodies. Recently, most of the analytical techniques in N-glycan analysis are using PNGase and Fluorescent labeling such as 2-AB. Resultant released glycans are analysed in the HPLC coupled with fluorescent detector and Mass. As the methods take 2~3 day to get the results it cannot be good ones in bioprocess development of therapeutic antibodies that they are requiring rapid analysis. Here, we tested an N-glycan labeling method that could provide rapid and enhanced fluorescence response/Mass sensitivity in the detection of N-glycan. Furthermore, this method has simplicity and high throughput capacity.

Curcumin is polyphenols extracted from the roots of the plant Curcuma longa Linn (Zingiberaceae). Curcumin is the yellow pigment associated with the curry spice, Turmeric, and to a lesser extent Ginger. Potential factors that limit the bioavailability of curcumin include insolubility in water (more soluble in alkaline solutions) and non-absorption. Glycosylation is one of the important tools to diversify and extend such biological functions in natural products and secondary metabolites. In this study, in order to increase the solubility and enhance the biological activity of curcumin we applied a one-pot two different enzyme systems, using the 6 glycosylation enzymes: ACK (Acetate kinase), UMK (Uridine monophosphate kinase), NAGK (N-acetyl-D-glucosamine kinase) / PMM (Phosphomannomutase), GalU (UDP-D-glucose synthase) / YjiC (Glycosyl-transferase). UDP-D-glucose was considered as a sugar donor and a curcumin as acceptor substrate. These methods may be the strategic approach for the production of an important small bioactive molecule by glycosylation in an industrial scale.

In this work, the specificity of glycosyl donors was studied to understand the material balance analysis on the products using a commercial CGTase. 4-hydroxyphenyl α-glucopyranoside (α-arbutin) and 4-hydroxyphenyl β-glucopyranoside (β-arbutin) were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from sp.(Toruzyme®3.0L). The reactions were practiced in an aqueous system containing hydroquinone (HQ) and starch as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutinorβ-arbutin) were 20 mM hydroquinone, starch (3%,w/v) in pH5 and 0.04 mg/ml toruzyme at 75°C for 6 h. The transfer efficiency of hydroquinone glycosylation was 17% respectively, when starch was employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-d-glucopyranoside (arbutin) on the basis of mass spectrometric analysis. The highest molar yield of arbutin (17.0%) was obtained when starch was used as the donor substrate. At room temperature the solubility of arbutin in water was determined to be 12.8 g/100 ml which is approximately 1.8 fold higher than that of hydroquinone.

The enzymatic reaction systems were developed for the efficient synthesis of glycoside products of arbutin, exists in two conformations, and . Beta-arbutin is often referred to as just arbutin. As a natural extract found in bearberry plants, arbutin also can infiltrate into the skin quickly without affecting the concentration of cell multiplication and effectively prevent activity of tyrosinase in the skin and the forming of melanin. Though arbutin is a natural derivative of hydroquinone, it does not possess the same risks or side effects. Arbutin has been shown to be a very safe ingredient and does not break down into hydroquionone very readily. The aim of the present study is to produce beta-arbutin by one-step reaction containing recycle systems, which is an enzyme reaction, not a chemical synthesis method. In an effort to produce UDP-glucose, we used specific enzyme, such as GalU, ACK, UMK, utilizing UMP as the substrates, and then beta-arbutin is synthesized through one-step reaction using YjiC, YdhE as a glycosyltransferase. The synthesized beta-arbutin was confirmed through the high-performance liquid chromatography (HPLC-PDA) and Mass, ¹H and ¹³C nuclear magnetic resonance (NMR). In excess of the substrate, the maximum productions of Hydroquinone glucosides (54% overall conversion of Hydroquinone) was achieved at 3 h incubation period. All the hydroquinone glycosides exhibited improved water solubility, diverse hydroquinone glycosides were produced in significant quantities by recycling UDP-a-D-glucose within one-pot in the glycosylation reactions.

Compound K (CK) is formed by bio-deglycosylation of major protopanaxadiol (PPD)-type ginsenosides (mainly Rb1, Rb2, Rd and Rc). Although it has never been identified in Panax plants, it is the main functional component detected in mammalian blood or organs after oral administration of ginseng or ginsenosides. It possesses remarkable bioactivities like anti-inflammation, hepatoprotection, antidiabetes and anti-cancer activities. It has even been approved by the China Food and Drug Administration to commence clinical trials for arthritis prevention and treatment. Neisseria β (1,4) galactosyltransferase, LgtB introduces galactose into glucose at its 1 position to form lactose. In the present study, we performed the in vitro enzymatic reaction of CK using UDP-D-galactose as a sugar donor and compound K as an acceptor substrate. Reaction products were analyzed by HPLC and TLC and the product was confirmed by high resolution LC-QTOF-ESI/MS, which revealed the addition of one galactose unit into compound K. This is the first study regarding the addition of galactose to protopanaxadiol type ginsenosides.