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The cloning of glucosidase II (1) permitted the identification of a glyco-code promoting protein folding (2) and another one for dislocation of misfolded glycoproteins to degradation (3). Based on these works, EDEM1 (yeast ortholog Htm1p) was discovered and shown to be a lectin-like glycoprotein directing terminally misfolded glycoproteins for degradation (4,5). Endogenous EDEM1 existed mainly as a soluble glycoprotein and was sequestered in buds forming along rough ER cisternae outside of the transitional ER (6). This resulted in the formation of ~150 nm vesicles in the cytoplasm lacking a recognizable COP I or II coat. Surprisingly, EDEM1 in the cytosol became de-glycosylated and formed detergent-insoluble aggregates that were degraded by autophagy and not be proteasomes (7). EDEM1 vesicles also containedmisfolded Hong Kong variant of -1-antitrypsin (6). Together, these findings demonstrated the existence of a novel vesicular pathway out of the rough ER to remove misfolded luminal glycoproteins for subsequent proteasomal degradation. When the fate of incompletely assembled fibrinogen was studied (8), we made the following observations. Naturally occurring Aa-g assembly intermediates of fibrinogen were substrate for EDEM1 and exited the ER in EDEM1 vesicles. In contrast to the proteasomal degradation of free single chains, surplus Aa-g assembly intermediates of fibrinogen formed detergent-insoluble cytosolic aggregates that were degraded by autophagy. In summary, EDEM1 dislocates not only misfolded glycoproteins but also an assembly intermediate of an oligomeric glycoprotein. This indicates that ER-to-cytosol dislocation of such EDEM1 substrates occurs by a vesicular mechanism rather than by a channel of the ER membrane. Depending on the propensity of the glycoprotein to form aggregates in the cytosol. proteasomal degradation or autophagic elimination ensues.

It has been becoming clear that cell-surface glycans are involved in a variety of important physiological events, such as cell-cell recognition, adhesion, signal transduction, quality control, and circulatory residence of proteins. However, their physiological roles have not been well understood. In this conference, we report two strategies for investigating the physiological role of glycans, i.e., bio-imaging of glycans in a whole body system and synthesis of biologically relevant glycans. Positron emission tomography (PET) is a non-invasive method that quantitatively visualizes the locations and levels of radiotracer accumulation with high imaging contrast. A new DOTA probe 1 was developed for facile labeling of peptides and proteins for PET imaging. The high reactivity of 1 enabled the labeling of lysine residues in peptides and proteins at very low concentration (~10-8 M) within a short reaction time (10~30 min). The labeled glycoproteins and glycoclusters were then subjected to PET imaging in order to examine their in vivo dynamics. MicroPET of the glycoproteins, [68Ga]DOTA-orosomucoid and asialoorosomucoid thus successfully visualized the differences in the circulatory residence of glycoproteins in rabbit, in the presence or absence of the sialic acids in glycans on glycoproteins [1,2]. Synthetic study of glycans including solid-phase synthesis will be also reported.

Chemo-enzymatic glycosylation has become an attractive methodology in the field of synthetic carbohydrate chemistry. The use of glycosidases possesses several merits: Typical glycosidases are stable, easy to handle, and produced on an industrial scale. In principle, glycosidase-catalyzed transglycosylation reactions require activated glycosidic compounds such as glycosyl fluorides and p-nitrophenyl glycosides. However, the preparation of these compounds necessitates the laborious task including the protection and deprotection of the hydroxy groups and the purification of the products. Novel chemo-enzymatic procedures by using 4,6-dimethoxy-1,3,5-triazin-2-yl glycosides1 and sugar oxazolines2 as glycosyl donors have been developed. These glycosyl donors have directly been prepared starting from the corresponding unprotected sugars in aqueous media without the use of any protection and deprotection of hydroxy groups. This is the first example of chemo-enzymatic glycosylation, where both of the glycosyl donor synthesis and the successive glycosylation can be achieved without any protecting and deprotecting steps in water. The new technique of direct activation of the anomeric hydroxyl group3-5 will pave a way to the “one-pot chemo-enzymatic glycosylation” without isolating the glycosyl donors. On the basis of this concept, one-pot syntheses of oligoxyloglucans and chitoheptaose have been demonstrated.

Heterologous expression of different combinations of genes from the 4,6-disubstituted aminoglycoside biosynthetic gene cluster in a non-aminoglycoside producing strain of S. venezuelae caused production of intermediates from the biosynthetic pathway. This provided experimental evidence for assigning the functions of individual gene products and also allowed us to recognize a complete biosynthetic pathway for generation of 4,6-disubstituted aminoglycosides. This work has established an efficient heterologous expression platform based on a genetically amenable streptomycete to dissect the biosynthetic pathways of other aminoglycoside antibiotics produced by less tractable actinomycete.

Glycosylation of plant secondary metabolites is mediated by family 1 glycosyltransferase, called uridine-diphosphate (UDP)-dependent glycosyltransferase (UGT). The N-terminal of UGT recognizes the sugar acceptor and the C-terminal interacts with the sugar-donor. Molecular basis of selection of sugar donor in UGT is not clear. UGT78D2 from Arabidopsis thaliana (AtUGT78D2) transfers a glucose group of UDP-glucose to 3-hydroxyl group of quercetin. On the other hand, the other UGT (ATUGT78D3), which showed 80 % amino acid identity to AtUGT78D2, uses UDP-arabinose as a sugar donor to make quercetin 3-O-arabinodise. Analysis of sugar binding site reveals that five amino acids are different between two UGTs. To find out which amino acid affects to their UDP-sugar donor selectivity, site-directed mutagenesis was performed. One amino acid at the C-terminal of AtUGT78D3 turned out to be critical for the selectionof UDP-sugar. With modeled structures of two AtUGT followed by site-directed mutagenesis, structural basis of UDP-selectivity in UDP-dependent glycosyltransferase is elucidated. Alternation of regioselectivity of UGTs is also challenging area. In order to alter the regioselelctivity, PGT-3 was used. PGTs uses quercetin as substrate and transferrs a glucose to the 4’ hydroxyl group. However, PGT-3 transferred a glucose group to the 3-hydroxyl group of isorhamnetin. Molecular modeling and docking and site directed mutagenesis were carried out to engineer a PGT-3 having a specificity for isorhamnetin but not for quercetin. The engineered PGT-3 that have exclusive activity toward isorhamnetin will be described.

Flavonoids are a group of polyphenolic compounds that have been recognized as important due to their physiological and pharmacological roles and their health benefits. Glycosylation of flavonoids has a wide range of effects on flavonoid solubility, stability, and bioavailability. We previously generated the E. coli BL21 (DE3) Δpgi host by deleting the glucose-phosphate isomerase (Pgi) gene in E. coli BL21 (DE3). This host was further engineered for whole-cell biotransformation by integration of galU from E. coli K12, and expression of calS8 (UDP-glucose dehydrogenase) and calS9 (UDP-glucuronic acid decarboxylase) from Micromonospora echinospora spp. calichensis and arGt-4 (7-O-glycosyltransferase) from Arabidopsis thaliana to form E. coli (US89Gt-4), which is expected to produce glycosylated flavonoids. To test the designed system, the engineered host was fed naringenin as a substrate, and naringenin 7-O-xyloside, a glycosylated naringenin product, was detected. Product formation was verified by HPLC-LC/MS and ESI-MS/MS analyses. The reconstructed host can be applied for the production of various classes of glycosylated flavonoids. A whole-cell biotransformation system was designed in E. coli BL21 (DE3) Δpgi by integrating galU (glucose-1-phosphate uridylyltransferase) from E. coli K12, expressing calS8 (UDP-D-glucose dehydrogenase) and calS9 (UDP-D-glucuronic acid decarboxylase) from Micromonospora echinospora spp. calichensis and arGt-3 (3-O-glycosyltransferase) from Arabidopsis thaliana to form an engineered host, E. coli (US89Gt-3), for the production of glycosylated flavonoids when exogenously fed with flavonoids. To verify the system, quercetin was fed and a glycosylated product, quercetin 3-O-glucuronide, was produced but unable to produce quercetin 3-O-xyloside.

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was cloned and expressed in E scherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.0 and 70ºC in the presence of 1 mM Co2+. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions,such as the D- and L-forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for L-ribulose among all pentoses and hexoses. Thus, L-ribose, as a potential starting material for many L-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300 g/liter L-ribulose by mannose-6-phosphate isomerase at 70ºC for 3 h, with a conversion yield of 71 % and a volumetric productivity of 71 g liter–1 h–1. Two enzymes of L-arabinose isomerase and mannose-6-phosphate isomerase from G. thermodenitrificans produced 118 g/liter L-ribose from 500 g/liter L-arabinose at pH 7.0, 70ºC, and 1 mM Co2+ for 3 h, with a conversion yield of 23.6 % and a volumetric productivity of 39.3 g liter–1 h–1.

The aim of the present study was to analyze the influence of oxidative stress using sodium orthovanadate (SOV) on growth and production of water-soluble exo-polysaccharides in the photosynthetic green alga Haematococcus lacustris. Fourier Transform Infrared (FT-IR) analysis and elemental analysis were performed to determine the primary chemical structure of the polysaccharide, and its monosaccharide composition was characterized by a High Performance Anion-Exchange Chromatography (HPAEC) using Bio-LC (DX-500 Chromatography System, Dionex Co., USA). The molecular weight of the compound was determined by size-fractionation HPLC. Higher production of extracellular polysaccharide from H. lacustris following treatment of SOV was observed at day 2 as compared to untreated controls. Consequently, the total content of crude carbohydrates produced from SOV treated cells (51.2 mg/ml) was approximately 1.7- fold higher than that of the control cells (31.1 mg /ml) at day 2. The molecular mass of the major component of the purified water-soluble polysaccharide was estimated to be approximately 135-kDa by size-fractionation HPLC. In addition, Fourier Transform Infrared (FT-IR) and elemental analysis demonstrated the presence of sulfate groups in this polysaccharide. Taken collectively, our data suggest that oxidative stress by SOV to the cells inhibits cell growth and promotes the expression of water-soluble sulfated polysaccharides from H. lacustris.

Many marine microorganisms produce biopolymers, and some polymers are economical materials in industry. From their wide diversities in structure and physical properties, we have investigated useful biopolymer. The sulfated exopolysaccharide p-KG03, which is produced by the marine microalga Gyrodinium impudicum strain KG03, exhibited impressive antiviral activity in vitro (EC50 = 26.9 μg/ml) against the encephalomyocarditis virus (EMCV), immunostimulating activity by NK cell activation, and bioflocculant activity for the industrial application. P-21653 (Antarctic marine bacterium Pseudoalteromonas arctica KOPRI 21653) is useful cryoprotective agent of red blood cell and animal cell, and also use for enzyme stabilizer. P-m10356 (marine bacterium Hahella chejuensis strain 96CJ10356) is potential release-control matrix for the oral deliverly system and UV-protective agent. And P-11568 (marine bacterium Alteromonas sp. KCTC10324) is excellent emulsifier. Therefore we introduce some results about diversity of marine biopolymer and its applications.

Hyaluronic acid (HA, sodium hyaluronate) is a glycosaminoglycan found abundantly in connective, epithelial, and neural tissues, which was first discovered by Karl Meyer and John Palmer in 1934. HA's high capacity for holding water and high viscoelasticity give it a unique profile among biological materials and make it suitable for various medical and pharmaceutical applications. One of the most successful medical applications of HA is the use of HA hydrogel for the treatment of osteoarthritis, based on the concept of Balazs and Denlinger.1 Also The application of HA in ophthalmology can be cited as another typical medical application. Balazs2 proposed a method using viscoelastic materials (HA) during ophthalmic surgeries. Further applications of HA can be found in artificial tears for dry eye syndrome, drug delivery matrix, wound-healing dressing, and so forth. Recently, chemical modification of HA has been an issue in biomaterials research area. With enhanced chemical and physical characteristics and yet indigenous biocompatibility, HA derivatives have been widely used as a potent biomaterial in variety of fields. The most widely spread application of derivatized HA is injectable dermal filler for soft tissue augmentation. Further applications can be found in post-surgery anti-adhesion barriers and osteoarthritis treatment of single injection regimen. LG Life Sciences Ltd. has been the leading company in Korean HA viscosupplement market, which has been developing variety of HA pharmaceutical products since 1992. In this lecture, the general development procedure and the commercial prospects of HA and HA derivative products are introduced.

Keratins 8 and 18 (K8/K18) are heteropolymeric intermediate filament (IF) phospho-glycoproteins of simple-type epithelia1,2. K8/K18 protect hepatocytes from apoptosis1,3 and their mutations predispose to liver disease4,5. K18 undergoes dynamic single O-linked N-acetylglucosamine (O-GlcNAc)-type glycosylation at Ser30/31/49, the function of which is unknown6. O-GlcNAcylated proteins are modulated on Ser/Thr by O-GlcNAc transferase (OGT) and N-acetyl-D-glucosaminidase (O-GlcNAcase) via GlcNAc addition or removal, respectively7,8. O-GlcNAcylation involves numerous nuclear/cytoplasmic proteins, and regulates several functions including protein turnover, transcription and stress responses7-12. O-GlcNAcylation and phosphorylation frequently occur at adjacent or identical Ser/Thr, and each modification can interfere with the other7-9, 13. Exposure of animal models to streptozotocin (STZ), an O-GlcNAcase inhibitor, results in accumulation of O-GlcNAc-modified proteins, diabetes and neurodegeneration7. Similarly, adipocyte exposure to O-(2-acetamido-2-deoxy-D- glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), another O-GlcNAcase inhibitor, leads to insulin resistance 14 but no reported studies tested the function of site-specific glycosylation in animal models. We addressed the function of K18 glycosylation by generating mice that overexpress K18 S30/31/49A (K18-Gly-), and compared their susceptibility to STZ- or PUGNAc/Fas-mediated injury with nontransgenic and three other control mouse lines that overexpress wild-type K1815, phospho-mutant S53A K182, or R90C K18 which disrupts keratin filaments and predisposes hepatocytes to apoptosis16. In contrast with the other control mouse lines, K18-Gly- mice are significantly more susceptible to STZ- or PUGNAc/Fas-induced liver and pancreatic injury, including prominent hepatocyte apoptosis which is specific to O-GlcNAcase inhibition since Fas-ligand alone induces apoptosis similarly in K18-Gly- and control mice. The enhanced apoptosis in K18-Gly- mice involves Akt1 and protein kinase Cθ inactivation due to site-specific hypophosphorylation. Furthermore, Akt1-T308 plays an important role in reciprocal Akt1 glycosylation and phosphorylation. Therefore, K18 glycosylation provides a unique protective role in epithelial injury by promoting the phosphorylation and activation of cell survival kinases.

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.

It has been shown that membrane microdomains (or rafts) are important as a hot spot for signal transduction. However, little attention has been paid to the fact that glycan chains are enriched in the microdomains. We have recently proposed a hypothesis that the microdomains are involved in the protein- and glycan-mediated cell adhesion and the subsequent signal transduction, and have demonstrated it in sperm-egg interactions at fertilization of sea urchin [1-3] To extend this hypothesis in early embryogenesis, we have studied on microdomains in developing embryos of medaka fish, Oryziaslatipes. Here wes how several lines of evidence supporting our hypothesis: (i) The micro domains prepared from early gastrula embryos were rich in cholesterol and sphingomyelin, and contained E-cadherin and LeX-glycan-containing glycoproteins (LeX-gp) and glycolipids (LeX-gsl), together with transducer proteins like c-Srckinase. The LeX-glycans and E-cadherin are known to be involved in morula compaction (tight cell adhesion) in mouse; (ii) Disruption of the microdomains of early gastrula embryos by treatment with methyl-b-cyclodextrin (MBCD) or C2-ceramide, impaired epiboly, the first cell migration event that occurs during gastrulation process. The most typical phenotype of the microdomain-disrupted embryo was detachment of blastodermal cells. Interestingly, the MBCD-induced disruption of microdomains and the epiboly impairment were restored when cholesterol was added back to the embryos. Thus, microdomain formation is critical in epiboly [4]; (iii) The isolated microdomains could bind to the microdomains in the presence of Ca2+ in LeX-glycan- and E-cadherin-dependent manners. These results suggest that the microdomain formation is important in the protein- and carbohydrate-mediated cell-cell interactions.

The ubiquitin-proteasome system is main degradation pathway of intracellular proteins in eukaryotes. This system is involved in a diverse cellular processes, including cell cycle regulation, stress response, intercellular signaling, transcription regulation, and acquired immunity.It works timely manner and, thus, irreversibly inactivate targeted proteins. However, how activity of 26S proteasome is regulated in each process is not well understood. Here we show that proteasome activity is regulated in EMT via TM4SF5 - slug mediated transcriptional regulation. Transmembrane 4 L6 family member 5(TM4SF5), a tumor antigen, is a member of tetraspanin. The TM4SF5 induces morphological elongation and epithelial-mesenchymal transition (EMT), in hepatocellular carcinoma. In this study, we found that the overexpression of TM4SF5 decreased level of total RNA but kept level of total protein through inhibition of proteasome activity. In TM4SF5-overexpressed hepatocarcinoma, mRNA level of 20s proteasome subunits except that of PSMB4 was decreased. Transcriptional down-regulation of 20s proteasome subunits caused decrease of proteasome activity. Also, when the EMT is induced by HGF, a typical EMT inducing agent, proteasome activity was inhibited. And the blockage of endogenous TM4SF4 by sh-TM4SF5 obstructed the EMT, which is induced by HGF. These results mean that inhibition of proteasome activity by TM4SF5 is a key step in EMT.

Protein O-mannosylation is an evolutionarily conserved protein modification of fundamental importance from bacteria to humans. Protein O-mannosylation in yeast and higher eukaryotes is initiated by protein O-mannosyltransferases (PMTs) that transfer mannose from Dol-P-Man to serine or threonine residues of secretory proteins. We identified and characterized two H. polymorpha PMT1 subfamily genes, HpPMT1-1 and HpPMT1-2, in the thermotolerant methylotrophic yeast Hansenula polymorpha. The HpPMT1-1 and HpPMT1-2 gene encodes two Pmt protein isoforms with an overall protein sequence identity of 52.3 % and 24.6 %, respectively, to Saccharomyces cerevisiae Pmt1p. The analysis of hydropathy profiles predicts the two H. polymorpha Pmt proteins to be integral membrane proteins with multiple transmembrane domains. The promoters of both H. polymorpha genes contain an HpHAC1p binding site, in consistent with their induced expression under UPR condition. Whereas the deletion of HpPMT1-1 resulted in apparent cell growth defects under cell wall stress induction conditions and a temperature sensitive phenotype, any detectable defects were not detected in the HpPMT1-2 deletion. However, the Hppmt1-2 mutant showed a significant sensitivity to the PMT1 inhibitor R3A-1c. Furthermore, analysis of cell wall mannoproteins with lectin blotting indicated significant decrease in O-mannosylation in the Hppmt1-1 mutant, but not in Hppmt1-2, compared to the wild type strain. We constructed a HA epitope-tagged version of HpPmt1-1p and confirmed that this construct was fully functional in vivo and localized at the membrane fraction. Altogether, our data support a major role of HpPMT1-1 in protein O-mannosylation and a redundant function of HpPMT1-2.

The pattern analysis of glycosylation sites is a promising strategy to distinguish the recombination enzyme for the enzyme replacement therapy (ERT) from the native human. Cerezyme is a glycoprotein that has studied for treatment of Gaucher disease. Gaucher disease, the most common lysosomal storage disease, is caused by results of mutations of glucocerebrosidase(GC) which dissolves glucocerebroside. Most patients who suffer from Gaucher disease takes the enzyme replacement therapy (ERT) with imiglucerase, cerezyme(R). The recombinant imiglucocerase must be required as enzyme similar to the native human enzyme sequence. The glycosylation patterns of recombinant imiglucocerase simultaneously should be analogous to those of the native human enzyme. However, the analysis of glycosylation patterns shows that the structure of glycans in recombinant imiglucocerase is similar, but distinct from those of glycans in the native human enzyme. The glycosylation sites of the recombinant enzyme must be consistent with those of the native human enzyme. The patterns of glycopeptides must be characterized in order to identify the glycosylation sites of glycoproteins. Here, we present the glycosylation sites of the imiglucocerease (cerezyme(R)) using MALDI-TOF MS.

Envelope antigen gene of Hepatitis B virus (HBV) consists of successive preS1, preS2, and S regions. Depending on alternative translation starting from each initiation codon of the corresponding regions, three envelop proteins, termed large (L), middle (M) and small (S) proteins, are synthesized. Dobeel Corp. developed improved HBV vaccine as the form of virus-like particle (VLP) comprising all three types of envelope proteins which were expressed from Chinese hamster ovary (CHO) cell. N-glycan profiles of total VLP as well as each L, M, and S protein were analyzed by using normal phase high performance liquid chromatography (NP-HPLC) after labeling of fluorescnece tag and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometry. Sialylated bi-antennary glycans with core fucose (NeuAc1-2 Gal2Man3GlcNAc4Fuc1) were detected as major peaks while bi-antennary neutral glycans terminated with galatose and sialylated tri- and tetra-antennary glycans were also detected in significant amounts. Especially, higher amount of bi-antennary glycan structures were observed in S and M protein whereas the quantities of tri- and tetra-antennary structures were more abundant in L protein. O-glycan, predicted at preS2 region, was also analyzed after the release from unfolded VLP by hydrazinolysis method. Its glycan profile showed that mucin-type sialylated core 1 glycans (NeuAc1-2GalGalNAc) exist in major populations.

This research describes a simple method for direct detection of glycoproteins after affinity-capturing (or enrichment) on the plate using a combination of self-assembled monolayers (SAMs) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI). The terminal hydroxyl groups of the SAMs were activated by N,N‘-disuccinimidyl carbonate (DSC), followed by conjugation with aminophenylboronic acid (APB). The formed APB-functionalized self-assembled monolayers for MALDI (SAMDI) plates were successfully applied to the affinity-capturing of a glycoprotein from the mixture containing non-glycoproteins. The method developed in this work would provide an efficient and convenient analysis approach to proteomics.

Syndecan-4, a cell surface heparan sulfate proteoglycan, is known to regulate the organization of the cytoskeleton and oligomerization is crucial for syndecan-4 function. We therefore explored a possible regulatory role of syndecan-4 oligomerization on the syndecan-4 link to cytoskeleton. Glutathione-S-transferase-syndecan-4 proteins were used to show that syndecan-4 interacted specifically with a-actinin, but not paxillin, talin, and vinculin. Interestingly, only dimeric, and not monomeric, recombinant syndecan-4 interacted with a-actinin in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2), and PIP2 potentiated the interaction of both the cytopasmic domain syndecan-4 peptide and recombinant syndecan-4 proteins with a-actinin, implying that oligomerization of syndecan-4 was importance for this interaction. Consistent with this notion, a-actinin interaction was largely absent in syndecan-4 mutants defective in transmembrane domain-induced oligomerization and a-actinin-associated focal adhesions were decreased in REFs expressing syndecan-4 mutant, compared to that of wild type syndecan-4. Besides, this interaction was consistently lower with the phosphorylation-mimicking syndecan-4 mutant S183E which is known to destabilize the oligomerization of syndecan-4 cytoplasmic domain. Taken together, the data suggest that the oligomeric status of the syndecan-4 plays a crucial role in regulating the interaction of syndecan-4 with a-actinin.

The cell surface heparan sulfate proteoglycan syndecan-2 regulates the activation of matrix metalloproteinase-7 (MMP-7) as a docking receptor. Here, we demonstrate a novel role of MMP-7-mediated syndecan-2 shedding during cancer cell migration. Western blot analysis showed that shed syndecan-2 were found in the culture media from various colon cancer cells and the levels of shed syndecan-2 were elevated in that of HT-29 colon cancer cells overexpressing syndecan-2. MMP-7, but neither MMP-2 nor MMP-9, enhanced shedding of endogenous syndecan-2 in HT-29 cells, suggesting that MMP-7 mediates syndecan-2 shedding. Consistently, MMP-7 cleaved recombinant rat syndecan-2 at the N-terminal of Leu149 residue in the extracellular domain in vitro. The substitution of Asn148-Leu149 with Ser148-Ser149 significantly reduced MMP-7-mediated syndecan-2 shedding and subsequently syndecan-2-induced cancer cell migration. In addition, shed syndecan-2 ectodomain per sei enhanced colon cancer cell migration. Furthermore, overexpression of syndecan-2 enhanced migration and metastatic potential of mouse melanoma cells in mice, but this effect was diminished in non-cleavable syndecan-2 mutant. Taken together, these data suggest that MMP-7 mediates syndecan-2 shedding and shed syndecan-2 plays an important role in the regulation of colon cancer cell migration.

Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into osteoblasts, chondroblasts and adipocytes. The osteoblast is a mononucleate cell that is responsible for bone formation. Gangliosides are key lipid molecules that are required for the regulation of cellular processes such as proliferation and differentiation. The role of gangliosides that play in osteoblastogenesis is not yet clearly understood. Therefore, in this study, we demonstrated the clearly role of gangliosides in the osteoblast differentiation of hDPSCs by shRNA. The results of high-performance thin-layer chromatography (HP-TLC) showed that ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. We showed a localization of ganglioside GD1a with ALP by immunofluorescence staining. Biosynthesis of ganglioside GD1a synthesize with the formation of sialic acid from GM1a by ST2 beta-galactoside alpha-2,3-sialyltransferase 2 (ST3Gal2). We used lentivirus-mediated shRNA expression for knockdown of ST3Gal2 gene expression. Down-regulation of ganglioside GD1a was decreased GD1a expression and ALP activity. Furthermore, these data indicate that the knockdown of ST3Gal2 by shRNA is inhibition of ERK1/2-phosphorylation. Taken together, these results suggest that ganglioside GD1a may play a role in the osteoblast differentiation process of hDPSCs.

In this study, pig ST6GalNAc IV gene was cloned from pig liver tissue and characterized. The pST6GalNAc IV cDNA has the open reading frame (ORF) containing 909 bp encoded 302 amino acids. Entire ORF of pST6GalNAc IV containing sialylmotif, ‘L’- (Large), ‘S’- (Small) and ‘VS’- (Very small) motif has a higher degree of sequence homology with Homo sapiens (90%), Pan troglodytes (91%) and Mus musculus (87%). Expression of pST6GalNAc IV mRNA in various pig tissues was identified by Reverse transcription polymerase chain reaction (RT-PCR) analysis. The pST6GalNAc IV mRNA was highly expressed in tongue, muscle and heart, but not expressed in pancreas.We also established pST6GalNAc IV-transfected PK15 stable cells for observing functional characterization of pST6GalNAc IV gene. Glycosylation pattern of pST6GalNAc IV-transfected PK15 cells was detected by flow cytometry and Immunofluorescence analysis with peanut agglutinin lectin (PNA) and maackia amurensis lectin II (MAL II). These results revealed that Neu5Ac-α2,3Gal-1,3(Neu5Ac α 2-6)GalNAc tetrasaccharide structures found on O-glycan were newly produced by pST6GalNAc IV, whereas Gal-1,3GalNAc disaccharide structures found on O-glycan were decreased by pST6GalNAc IV.

The disialoganglioside GD3 has been considered to be involved in tumor progression or suppression in various tumor cells. However, the significance of the biological functions of GD3 in breast cancer cells is still controversial. This prompted us to study the possible relationship(s) between GD3 expression and the metastatic potential of a breast cancer MDA-MB231 cells. The human GD3 synthase cDNA was stably transfected into MDA-MB231 cells. In vitro invasion potentials of the GD3 synthase overexpressing cells (pc3-GD3s) were significantly suppressed when compared with control cells. Expression of intercellular adhesion molecule-1 (ICAM-1; CD54) was down-regulated in the pc3-GD3s cells and the decrease in ICAM-I expression is directly related to the decrease in invasiveness of the pc3-GD3s cells. Then, we investigated signaling pathways known to control ICAM-1 expression. No difference was observed in the phosphorylation of ERK and p38 between the pc3-GD3s and control cells (pc3), but the activation of AKT was inhibited in pc3-GD3s, and not in the control (pc3). Overexpression of GD3 synthase suppresses the invasive potential of human breast cancer MDA-MB-231 cells through downregulation of ICAM-1.

Cancer is one of serious human diseases that causes angiogenesis, metastasis, and abnormal cell divisions including genetic change. Thus, lots of researchers have been investigated to inhibit cancer. Human 90K (h90K) protein has very crucial role for inhibition of cancer metastasis. h90K is a glycoprotein having potential 7 N-glycosylation sites (4 sites are clear but remaining 3 sites are not yet). In this work, we performed expression of recombinant h90K glycoprotein in insect Drosophila S2 cell system. We constructed stably-transfected S2-h90K cells by antibiotic selection and confirmed h90K expression using Western Blot and in vitro bioactivity analyses. We also identified N-glycan patterns of recombinant h90K glycoprotein using HPLC and MALDI-TOF MS. Based on the identified N-glycan structures, we next engineered N-glycosylation pathway to develop advanced N-glycan patterns using multiple expression platform strategy with recombinant baculoviruses. We found that infection with recombinant baculovirus having galactosyltransferase resulted advanced bi-antennary galactosyl form of N-glycans in h90K glycoprotein.

Posterior capsular opacification (PCO) is caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the human lens epithelial (HLE) cells after cataract surgery. GM3, modulating interactions and migration plays major roles in living organisms. So, we investigated correlation with GM3 and TGFβ receptor, a key regulator during PCO, in the HLE B-3 cells. Our results indicate that TLC data and immuno-fluorescence shows the increase of GM3 and GM2 during EMT induced by TGF-β1 in HLE B-3 cells. The expression of fibronectin, known as an EMT maker, at the mRNA level is controlled by expression of GM3 synthase in evidence of a dose- and time-dependent manner. GD3 synthase, downstream of GM3, and alpha-SMA did not change, however. GM3-depleting agents and siRNA of GM3 synthase specifically inhibited TGF-β–induced activation of fibronectin, TGFβR and Smad-2/3 compare as treatment of TGF-β1 only. Also, GM3 regulated TGF-β–promoted cell migration in HLE B-3 cells. Furthermore, TGF-β1 also increased binding between GM3 and TGFβ receptor detected by immune-precipitation. These results suggest that GM3, which is interacted with TGFβ receptor potentially, regulates the EMT in the human lens epithelial cells.

Plant genetic engineering has led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current antibody expression systems. In this study, the expression levels of mAbP SO57 with or without ER-signal peptides (Lys-Asp-Gly-Leu;KDEL) in transgenic tobacco plants were analysed in transgenic plant. PCR and Reverse Transcription-PCR analyses showed existence of heavy and light chain genes of mAb with or without KEDL and their transcription in plant, respectively. Western blot showed that the expression levels of mAbP SO57 with KDEL were significantly higher than that without KDEL. Flow cytometry analysis showed that the Fc domains of both purified mAbP and mammalian-derived mAb have similar binding activity to the FcγRI receptor. High-performance liquid chromatography analysis showed that the mAbP SO57 with KDEL had glycan profile with both oligomannose and golgi type, whereas the mAbP SO57 without KDEL had only golgi type glycans. Neutralizing analysis with rabies virus CVS-11 showed the similar neutralizing activity between mAbP SO57 with and without KDEL. These results suggest that the potential of mAbP SO57 for rabies immunotherapy is regardless of plant specific glycan structures.

The glycosyl epitope dimeric Lea (Lea-on-Lea), defined by mouse monoclonal antibody NCC-ST-421, was identified previously as tumor-associated antigen, expressed highly in various human cancer tissues and cell lines derived therefrom, but with minimal expression in various normal tissues. In the present study, we observed clearly higher expression of this epitope, defined by ST421, in β-haptoglobin (β-Hap) from sera of patients with colorectal cancer, compared to normal, healthy subjects or patients with chronic inflammatory processes (Crohn\'s disease, ulcerative colitis). We focused, therefore, on biochemical characterization of glycosyl epitope status expressed in β-Hap. We concluded that the dimeric Lea epitope is carried by O-linked but not by N-linked structure, based on the following observations: (i) Strong reactivity of Colo205 supernatant with ST421 was reduced clearly by pre-incubation of cells with benzyl-α -GalNAc. (ii) Treatment of β-Hap with α-L-fucosidase reduced its reactivity with ST421, but did not affect its reactivity with anti-Hap antibody. In contrast, treatment of purified β-Hap with PNGase F, which releases N-linked glycans, had no effect on reactivity with ST421, but changed molecular mass from 40 kDa to 30 kDa.

The mAb CO17-1A recognizes the colorectal antigen GA733-2E, which highly is expressed on the surface membrane of human colorectal carcinoma cells. The higher yield of CO17-1A mAb reflect the use of the ER retrieval motif KDEL(Lys-Asp-Glu-Leu), In this study, the expression level of mAb CO17-1A was compared between mAbs fused with and without KDEL in plant. Western blot showed that the expression levels of mAb CO17-1A with KDEL were significantly higher than that without KDEL Cell ELISA with human colorectal carcinoma cell line confirmed that expression of the mAb with KDEL was significantly higher than that of mAb without KDEL. Flow cytometry analysis showed that the Fc domains of both purified mAbP (with and without KDEL) and mammalian-derived mAb have similar binding activity to the FcγRI receptor. The glycosylation analysis revealed that mAb CO17-1A without KDEL had plant specific glycans structure while mAb CO17-1A with KDEL had mainly oligomannose type glycans. Taken together, these studies suggest that KDEL affects expression of mAb and its glycan structures.

N-glycosylation is a major post-translational protein modification, which alters physicochemical properties of the protein, affecting the folding, distribution, stability and thus biological function and efficiency of protein. β1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans are potentially immunogenic in mammals. In this study, to remove the plant specific sugar residues and humanize the N-glycosylation in plant, we isolated mutants corresponding plant glycosyltransferase genes (N-acetylglucosaminyltransferaseI, N-acetylglucosaminyltransferaseII, α 1,3-fucosyltransferaseI, α1,3-fucosyltransferaseII, β1,2-xylosyltransferase, β 1,3-galactosyltransferase, α1,4-fucosyltransferase). Double, triple, quadruple and quintuple mutants were made by crossing the mutants and two quadruple mutants (fuct1/fuct2/xylt/gntII), (fuct1/fuct2/xylt/galt) are on the construction. The triple (fuct1/fuct2/xylt) and quadruple (fuct1/fuct2/xylt/fuct3), quintuple (fuct1/fuct2/xylt/fuct3/galt) mutants did not show significant developmental defects in a normal growth condition and they did not produce the plant specific sugar residues on the N-glycan. [Supported by BK21 program]

In this study, pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of N-glycolylneuraminic acid was cloned from pig small intestine and characterized. The open reading frame of pcmah was 1,734 bp encoded 577 amino acids and consists of fourteen exons. Organ expression pattern analysis reveals that pig CMAH mRNA is mainly expressed in pig rectum, tongue, spleen, and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when PK15 cells and ECV304 cells were transfected with the cloned pcmah, NeuGc contents of these transfectants were greater in comparison to vector transfectants used as control. In addition, in the functional analysis of NeuGc, human serum-mediated cytotoxicity was elevated in the ectopic NeuGc expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG were slightly increased indicating that the human IgM type was a major anti-NeuGc antibody. From the results, it was concluded that the pig CMAH gene was capable to synthesize the NeuGc acting as a xenoantigen in human, confirming the NeuGc-mediated rejection response in pig-to-human xenotransplantati