Earticle

The JST/NBDC integrated database project has kicked off last year. JCGGDB was selected as a promotion program of DB integration, aiming to integrate all the glycan-related databases in Japan and build user- friendly search systems. As part of the project, the construction of ACGG-DB (an integrated database for the ACGG: Asian Communications for Glycobiology and Glycotechnology) is also planned in cooperation with Asian countries. As of now we have consolidated data from various Japanese institutes into JCGGDB and developed a cross-search function by keyword entry and integrated search functions by glycan stcurctures. These functions enabled users to easily access various glycan-related databases with a single search. Cheminformatics technologies using chemical structural formula for glycan has been also adopted to provide a search for glycan structures, glycan synthetic products by organic chemistry and recombinant enzymes, glycogene inhibitors, glycosides, and commercial glycans. This Summer, we have released AIST GlycoProtDB, which stores the data of experimentally-proven glycosylation sites on each mouse tissue. We are continuously accumulating experimental results of glycosylation sites, while collecting more information from scientific journals, toward the release of ACGG Glycoprotein Database in autumn. For the future, we will keep developing base technologies for DB integration and linking with databases related to glycoscience as well as other study areas. Some more bioinformatics tools are also being developed to support experimental study. Our aim is to create contents which could be easily and intuitively understood by every user.

The Attana Cell 200 biosensor measures label-free, full kinetics in real time with the target in its biological context. In our study, a novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed, where an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.

O-GlcNAc(O-linked-N-acetylglucosamine) modification is one of the protein post- translational modifications that is involved in many cellular processes, including signal transduction, transcription, translation, cell proliferation and apoptosis. It is known that, on some proteins, this modification can affect the O-phosphorylation state of the proteins. UDP-GlcNAc, the precursor of O-GlcNAc, is mainly synthesized from glucose by HBP(Hexosamine Biosynthetic Pathway), which is sensitive to glucose concentration. Here, we confirmed that there is an inevitable decrease in the glucose concentration in the media during cell culture by the cell metabolism. Because O-GlcNAc is sensitive to the glucose concentration, the decrease is supposed to affect the cellular processes. In this study, the decrease in glucose concentration during cell culture was assessed in many cell lines. In some cell lines, the concentration decreased to a much lower level compared to the original. With this change, the O-GlcNAc level also appeared to decrease.A comparison was made between a glucose- concentration-maintained sample and a non-maintained one, where a difference in insulin signal transduction was observed. The phoshporylation level of Akt was likely to decrease with the maintainence of glucose while the O-GlcNAcylation level of it increased. This means decreased level of acitvated Akt. This was confirmed by checking the downstream protein, GSK(Glycogen synthase kinase), which was found to be less phophorylated under glucose-maintained condition.These findings provide us an evidence of the crosstalk between O-GlcNAcylation and O-phosphorylation on Akt. It corresponds with the insulin insensitivity of type II diabetes. These findings also suggest the importance of glucose condition during cell culturing.

Saponins, water soluble compounds composed of sugars and steroid or triterpenoid moieties, have attracted considerable interests due to the increased understanding of their wide spectrum of biological and pharmacological activities.Saponins have been found only in marine phylum Echinodermata and particularly in species of the classes Holothuroidea (sea cucumbers) and Asteroidea (starfishes) in the animal kingdom. Starfish extracts and also purified saponins have shown a broad spectrum of physiological and pharmacological activities. In 1981, Sepositoside A, the major saponin, was isolated from the hydrolysate of the Mediterranean starfish Echinaster sepositussaponins mixture(Figure 1). It has a number of unusual features when compared to the more common asterosaponins: it is devoid of the sulphate group and the Δ7, 3β,6β-dioxygenated steroidal nucleus is unprecedented, and, the most remarkable feature is that the trisaccharide chain is cyclized between C-3 and C-6 of the aglycon, giving rise to a highly strain 16-membered macrocyclic ring reminiscent of a crown ether. Sepositoside A had been shown to display antifungal activity, cytotoxic acitivity toward bovine turbinate cells up to a level of 1 μg ml-1 and inhibition of cell division of fertilized sea urchin eggs (ca. 30% inhibition at 10-5 M).Fascinated by the unprecedented highly strain 16-membered macrocyclic molecular structure of Sepositoside A, we contemplated its total synthesis, which might require new synthetic strategies and technologies to close the challenging 16-membered macrocyclic ring by virtue of its expected high strain and lability. Herein, we report our synthetic studies for the construction of 16-membered macrocyclic ring of Sepositoside A model systems.

To investigate density-dependent binding of glycans by lectins using carbohydrate microarrays, a number of C-terminal hydrazide-conjugated neoglycopeptides with various valences and different spatial arrangements of the sugar ligands were prepared on a solid support. The synthetic strategy includes (1) assembly of alkyne-linked peptides possessing C-terminal hydrazide on a solid support, (2) coupling of azide-linked, unprotected sugars to the alkyne-linked peptides on the solid support utilizing click chemistry, and (3) release of the neoglycopeptides from the solid support. By using this synthetic methodology, sixty five neoglycopeptides with a valency ranging from 1 to 4 and different spatial arrangements of the carbohydrate ligands were generated. Carbohydrate microarrays were constructed by immobilizing the prepared neoglycopeptides on the epoxide-derivatized glass slide and were used to analyze density-dependent binding of glycans by lectins. The results of binding property determinations show that lectin binding is highly dependent on the surface glycan density.

Hyaluronan (also called hyaluronic acid or hyaluronate) is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissue. Hyaluronan, a core component of the extracellular matrix, comprises a repeating oligosaccharides of N-acetylglucosamine and D-glucuronic acid. The purpose of this study was to investigate the anti-adipogenic effect of low-molecular weight hyaluronan (LMW-HA, 43,500 Da) on 3T3-L1 preadipocyte cells. The LMW-HA was prepared by enzyme oligosaccharide (Vibrio splindidus BST398) of high-molecular weight hyaluronan (>3x106 Da) produced from Streptococcus zooepidemicus. LMW-HA did not cytotoxicity on 3T3-L1 preadipocyte cells at up to 1000 μg/ml. Treatment at 3T3-L1 cells maintained in adipocyte-induction media with 25, 50, 100, and 200 μg/ml of LMW-HA resulted in significant decrease in intracellular lipid drops by 75.5, 65.4, 58.0, and 53.5 %, respectively, suggesting that LMW-HA has anti-adipogenic effect on 3T3-L1 adipocyte differentiation. Flow cytometry analysis by Annexin V/FITC and PI staining showed that LMW-HA does not affect apoptotic (or necrotic) cell death. However, incubation of cells with LWM-HA 25, 50, 100, and 200 μg/ml) significantly reduced the expression of PPAR-γ by 81, 69, 58, and 56 %, respectively, and also aP2 by 81, 67, 56, and 55 %, respectively, compared to untreated control group. PPAR-γ and aP2 are known as the major adipocyte-specific genes. Therefore, these results suggested that LMW-HA has anti-obesity effect by inhibiting adipocyte differentiation through down-regulation of PPAR-γ and aP2 expression.

Alzheimer’s disease (AD) is a major public health problem, and there is currently no clinically accepted treatment to cure or stop its progression. Fibrillar aggregates of the β-amyloid peptide (Aβ) are the major constituents of the senile plaques found in the brains of AD patients and have been related to AD neurotoxicity. A formation from amyloid precursor protein (APP) is caused mainly byβ-secretase. Phellinus linteus (PL) has been recognized as a major source of natural antioxidants that could decrease reactive oxygen species (ROS). However, its role in protection of Aβ-induced cytotoxicity and apoptosis in neuronal cells remains unclear. The present study aims to investigate such an effect of PL extracts on neuroprotection by evaluating the inhibition of β-secretase activity. From the hot-water extract (PL-WE) of PL fruiting body, a high-molecular weight fraction (Et-P) and a low-molecular weight fraction (Et-S) were obtained by 75% ethanol (EtOH) precipitation. The latter was further extracted with ethyl acetate (EA). Three major water-soluble polysaccharides (Et-P1, Et-P2, Et-P3) were purified from the Et-P fraction by DEAE-cellulose column chromatography. Molecular masses were estimated to be ~1,629, 1,294, and 21kDa, respectively, by size-exclusion chromatography (SEC). These polysaccharides were shown to be composed of glucose, galactose, and mannose as the major monosaccharides and fucose, xylose as minor ones. The FT-IR analysis suggested that they are typical polysaccharides having at least partially -linkages and possibly existing as complex with phenolic compounds. Laminarinase digestion and HPLC analysis suggested that these polysaccharides are a variant of β-(1,3)-glucan. Theses polysaccharides significantly inhibited the β-secreatase activity, suggesting that they may inhibit the formation of Aβ. Further characterizations of this extract may lead to identification of a potent therapeutic compounds for the prevention and/or decreasing the severity of AD.

In vivo medical research relies primarily on the common laboratory mouse, who exhibits an inherent similarity to its fellow mammal, the human, yet is much more amenable to experimentation (for ethical, economic, and life cycle -related reasons). Numerous studies have elucidated the genome, proteome, and metabolome of the various mouse models available, but few if any have paid any attention to the mouse glycome. However, glycosylation is one of the most common eukaryotic post-translational modifications, and variations in glycosylation have been linked to cancer and other important human diseases by over fifty years of glycobiological research. Elucidation of the mouse glycome would aid glycomic and glycoproteomic biomarker studies based on the mouse model and advance our understanding of mice as models for humans. We have used retrosynthetic analysis to create a library of all possible mouse serum N-glycan compositions, based on our knowledge of the N-glycan biosynthesis pathway. Highly sensitive nano-LC/MS profiling of mouse serum N-glycans allowed us to annotate the theoretical library with experimental data, while isomer-specific nano-LC/MS/MS provided structural information. The annotated mouse serum N-glycan library was applied to automated data analysis of large sample sets for the purposes of biomarker discovery.

A fucoidan-degrading enzyme activity has been produced from Sphingomonas paucimobilis PF-1 (KCTC 11130BP) using Miyeokgui fucoidan, isolated from Korean Undaria pinnatifida sporophyll, as s sole carbon source of culture medium. The enzyme was purified to an apparent homogeneity mainly by sonic disruption of cells, ammonium sulfate precipitation, DEAE-Sepharose column chromatography and chromatofocusing, with an yield of 3.2% and a specific activity of 2.143 U/mg protein. The purified enzyme (tentatively named FNase S) appeared as a single band on Native PAGE gels with a molecular mass of approximately 130 kDa. However, the SDS-PAGE gels gave 3 separate proteins with molecular masses of approx. 130 (designated as S1), 70 (S2) and 60 (S3) kDa, respectively, suggesting a multi-protein complex nature of this enzyme. The first 10 N-terminal amino acid sequences of S1 (SXPEAASLPG), S2 (SPQFDVVXIG), and S3 (SLQFDVVVIG) showed high homology (over 80-90 identity) with the N-terminal regions of ATPase, core domain (EGF 27867), carbohydrate kinase, PfkB family protein (YP003854323) and dihydrolipoyl dehydrogenase (ZP08017938), suggesting that these three proteins may delineate a new family of glycoside hydrolases. The optimum conditions for enzyme activity on Miyeokgui fucoidan were pH 6.0-7.0 and 40-45℃. The activity was stable within pH 5.5-8.0 and most stable at 40-45℃ for 1 day at pH 6.0. The enzyme was activated by Mn2+ and Na+ at the concentration of 1 mM. The enzyme was specific (almost equally active) towards Miyeokgui fucoidan, commercial fucoidan (Sigma) and alginate, but exhibited very low activity towards heparin, starch, laminarin and dextran. Apparent Km, Vmax and Kcat values for Miyeokgui fucoidan were 1.7 mM, 0.62 μmol/mlㆍmin, and 0.38 S-1, respectively. The purified FNase S depolymerized Miyeokgui fucoidan into at least more than 7 distinct low-molecular weight fucose-containing oligosaccharides, ranging from 318 to 3,312 Da, with no production of monosaccharides, suggesting that this enzyme is an endo-acting fucoidanase and may be an attractive material for not only industrial applications utilizing low-molecular weight fucooligosaccharides but also structural analysis of fucose-containing marine polysaccharides.

The glycosaminoglycans (GAGs) from Melibe viridis were extracted and optimized using Multifactorial design. Four factors (Temperature, pH, incubation period, and enzyme ratio) were varied in the optimization experiment. The design used three enzymes namely; Alcalase, Flavourzyme, and Protamex. The optimum results were obtained using Flavourzyme at pH 8.0, incubation temperature of 450C, for 15 hours, and an enzyme ratio of 1.5% relative to sample weight. The extracted Melibe GAGs (prepared at different concentrations) were tested for DPPH scavenging effect, it showed that, it has a comparable activity with standard, the Ascorbic acid. It also showed a reductive capability in a concentration-dependent manner. For the hydroxyl radical scavenging effect, the results showed that the activity is very near with that of the standard too, butylated hydroxyanisole (BHA). From the data obtained in this study, it can be inferred that, Melibe GAGs has a potential in the field of medicine as well as well as they can be used as dietary supplements with specific functional properties

The polysaccharides extracted from Liparis tesellatus eggs (LEP) and Pronase-digested LEP (LEP-PD) were tested for their anti-inflammatory, anti-cancer, and cytotoxic activities. The anti-inflammatory activities were determined using NO production by macrophage RAW 264.7 cell lines induced by lipopolysaccharide (LPS). Results showed that LEP-PD has an activity on all concentrations (0.01, 0.1, 1.0, 10.0, 100.0, and 200.0 ug/ml), whereas, no activity on all concentrations prepared for LEP. The anti-cancer activities of LEP and LEP-PD were evaluated using Human colon adenocarcinoma (HT-29) and Human stomach adenocarcinoma (AGS) cell lines. Results showed that both the LEP and LEP-PD have anti-carcinogenic effects on these specific cell lines. For the cytotoxic effects of LEP and LEP-PD, RAW 264.7 cell line was also used treated with the polysaccharides. The results showed that LEP and LEP-PD are non-toxic to the cells. From these data obtained, polysaccharides from Liparis tesellatus eggs can be used in the field of medicine upon further purification and elucidation of the structures of the glycans responsible for these promising activities.

We present here a high-throughput strategy to discover serological biomarkers for early-detection of hepatocellular carcinoma (HCC). Our strategy is also applicable to assess the progressed liver fibrosis that is associated with virus hepatitis. The glycan structure on glycoproteins derived from cancerous cells is known to be different from that derived from normal cells, specifically, the increased aberrant glycosylation appears in patient serum with virus hepatitis along with either or both the initiation and progression. Based on the above perceptions, in order to identify glycoproteins carrying aberrant glycosylation in serum of liver disease patients, we analyzed lectin-captured glycopeptides by the IGOT method. Many glycoproteins carrying altered glycans were successfully identified. The increased amount of these glycoproteins was clinically relevant to the progression of the liver diseases. We are now selecting appropriate molecules depending on the feasibility to detect an abnormality in the liver, such as the occurrence of liver cell neoplasm.

Recent advances in mass spectrometry make our view of glycosphingolipids (GSLs) com- pletely different from the previous one. LC-MS is applicable to GSL mixture in a smallamount, and provides structural information of carbohydrate chains and ceramides andpossibly quantitative results. However, several subjects remain to be carefully clarified.Critical subjects are matrices used for enhancing ionization of GSLs and the prepara- tion of standard or isotope-labeled GSLs for quantitation. We have tested several matrices for neutral GSLs and acidic GSLs (gangliosides) in the analysis of negative ion mode. Negative ion mode provides more fragment ions derived from carbohydrate chains and ceramides. Matrices used for LC analysis with a C30 reversed phase column of neutral GSLs, such as ammonium formate, formic acid, ammonium acetate, and acetic acid, give adduct ions and the ratios of molecularions [M - H]- and adduct ions are different among molecular species of GlcCer as the simplest neutral GSL. Ammonium bicarbonate containing solvents give lesser amount of adducts and can be used for comparing changes of molecular species of neutral GSLs produced by the different conditions of cultured cells or comparing neutral GSLs of different subsets of cells isolated from living organisms. In the analysis of gangliosides, we detected GM1(NeuAc) as a single charged ion [M – H]-,and GM1(NeuGc) as a double charged ion [M - 2H]-2. Other ganglisoides such as GD1, GT1, and GQ1 molecules were detected as double charged ions, therefore, GM1(NeuAc) gives the highest m/z values among these gangliosides, thus detection effi- ciency becomes very low. We have successfully applied LC-MS analysis to gangliosides of mouse thymocytes and CD4+ and CD8+ T cells (Nagafuku et al. 2012). Fortunately, these gangliosides contain N-glycolylneuraminic acid and give double charged ions, even in the case of GM1. When they are activated, the down regulation of CMP- NeuAc hydroxylase which is the rate liming step for the expression of NeuGc containing ganglisodies (Naito et al. 2007), is possibly triggered. Then, the sensitive detection of GM1(NeuAc) must be assured. This is also required for the analysis of microdomains of neuronal cell mem-brane of neural degenerative diseases such as Alzheimer disease and Parkinson disease.

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17-1A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti-colorectal cancer large single chain (lsc) mAb based on mAb CO17-1A in the baculovirus-insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17-1A were fused together by an interchain linker. The only difference between two mAbs is depending of fusion of an ER retention signal (KDEL) at its C-terminus of HC. PCR analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus-infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti-colorectal lsc mAb recognizing human colorectal cancer cells.

Entamoeba histolytica is anenteric tissue-invasion protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. E. histolytica can induce host cell apoptosis by the induction of various intracellular signal mechanisms. These modulations triggered by E. histolytica are closely associated with tissue pathogenesis and parasitic immune evasion mechanism. O-GlcNAcyation, similar to phosphorylation, has been thought to contribute the various cellular signal processes including apoptosis and proliferation. However, it is unknown whether E. histolytica can affect the O-GlcNAc level in host cells during Entamoeba-induced cell death. In this study, we investigated whether modulation of O-GlcNAcylated protein levels in host cells is involved in HepG2 cell death induced by E. histolytica using virulent strain HM-1 and non-virulent Rahman. Co-incubation of HepG2 cells with HM-1 strain remarkably increased DNA fragmentation and LDH release compared to cells incubated with Rahman strain or medium alone. In addition, Entamoeba HM-1induced tyrosine dephosphorylation, and activation of caspases-3 and calpain in HepG2 cells. Gal-lectin-mediated amoebic adherence of live HM-1 trophozoites to the HepG2 cells decreased of O-GlcNAcylated protein levels in HepG2 cells within 2 min, while non-virulent Rahman strain did not. Indeed, E. histolytica-induced deGlcNAcylation in HepG2 cells was prevented by O-GlcNAcase inhibitors, PUGNAc or Thi-Met G. In addition, DNA fragmentation and LDH release triggered by E. histolytica HM-1 were strongly inhibited by pretreatment of host cells with OGA inhibitor PUGNAc. Our results suggest that decreased O-glycosylation is required for cell death in hepatocytes induced by adherence of E. histolytica.

Effects of the degree of deacetylation (DDA) and the molecular mass of chitosan oligosaccharides (CTS-OS), obtained from the enzymatic hydrolysis of high molecular weight chitosan (HMWC), on antitumor activity was explored. The DDA and molecular weights of CTS-OS were determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF MS) analysis. The CTS-OS were found to be a mixture of mainly dimers (18.8%), trimers (24.8%), tetramers (24.9%), pentamers (17.7%), hexamers (7.1%), heptamers (3.3%), and octamers (3.4%). The CTS-OS were further fractionated by gel-filtration chromatography into two major fractions: (1) COS, consisting of glucosamine (GlcN)

Tunicamyin (TM) blocks the synthesis of all N-linked glycoproteins and causes ER stress. It has been reported that TM inhibits LPS-induced NF-kB activation via suppression of MD-2 N-glycosylations. In this study, we investigated that the inhibitory effect of tunicamycin (TM) on lipopolysaccharide (LPS)-induced inflammation is independent of N-linked glycosylations of MD-2 in RAW264.7 macrophage. TM significantly suppressed nitiric oxide (NO) production, inducible nitric oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines, interleukin 1 beta (IL-1β) and tumor necrosis factor alpa (TNF-α) expression in response to LPS. TM also inhibited the inflammatory reactions in response to other TLR ligands, Polyinosinic:polycytidylic acid (Poly:IC) or Lipoteichoic acid (LTA), which do not require N-glycosylated MD-2 protein for macrophage activation. This indicates that the anti-inflammatory effect of TM may not associated with MD-2 N-linked glycosylations. Other well-known ER stress inducers, A23187 (A23) and thapsigargin (TG) had no effect on LPS-induced inflammation, suggesting that ER stress induction and UPR pathway is not necessarily stimulatory factors for LPS-induced inflammation. We showed that TM inhibited LPS-induced nuclear translocation of p50 and DNA binding of p50 and p65 to NF-κB binding sequence of iNOS promoter. Therefore we conclude that TM repressed LPS-induced inflammation through suppression of NF-kB binding activity and independent of inhibition of N-linked glycosylations of MD-2.

Most cancer cells produce energy by glycolysis followed by fermentation in the cytosol, even though they exist in the presence of oxygen. It means that glucose is probably important to cancer growth and survival more than oxygen. But the research about cancer metastasis has been limited to hypoxia condition until now. Interestingly it was reported that overall O-GlcNAc modification on protein was significantly increased under hypoglycemic condition. O-GlcNAcylation is one of the post-translational protein modification and it appears to be involved in many different cellular activitiesregulating in transcription, translation, protein degradation, protein localization and protein-protein interaction. And O-GlcNAcylation on snail1, p120 and beta-catenin induces the decrease of E-cadherin on plasma membrane, so it was thought that O-GlcNAcylation is also related with EMT (epithelial mesenchymal transition). In our study, we want to examine whether hypoglycemic condition can induce cancer metastasis and it is related to increased O-GlcNAc modification induced by hypoglycemic condition or not. So we focused on EMT to visualize cancer metastasis. We found that vimentin which is mesenchymal cell marker was increased and it was O-GlcNAcylated under hypoglycemic condition in cancer cell. Also, we confirmed that epithelial cell markers were also decreased under hypoglycemic condition. 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.

A porous graphitized carbon (PGC) is mainly employed for effective isomer-specific separation and enrichment of the glycans based on size, polarity, and three-dimensional structure. However, PGC separates α and β isoform in an aldehyde which is produced by PNGase F treatment to release N- glycans, thus by making effective isomer separation difficult. Here, we have optimized glycan reducing conditions to get rid of α and β isoform by reducing aldehyde using a sodium borohydride. Reduced reducing N-glycans released from Immunoglobulin G (IgG) have been analyzed by nano LCchip/QTOF MS and the number and quantity of isomers before and after reducing were examined to optimize the reducing conditions. . After releasing N-glycans using PNGase F, released glycans were purified by SPE (solid phase extraction) and reduced by sodium borohydride. And then reduced glycans were purified and enriched by SPE. Reduced N-glycans were separated and quantified using nanoflow liquid chromatography on PGC chip- based device. Indeed, we found less peaks in chromatogram after reducing N-glycans which suggest the number of glycans isomers were decreased because of the removal of α and β isoform. For example, the ion at m/z 1462.5445 corresponding to [Hex]3[HexNAc]4[Fuc] which is the most abundant glycan in IgG has single peak in chromatogram after reducing glycans. Indeed, this glycan does not have any isomer, which is already reported in the literature. Current reducing condition works for neutral glycans but we should optimize reducing conditions for highly branched and sialylated glycans. This method will be successfully applied to isomer-specific separation of the glycans on a PGC in biopharmaceutical glycoanalysis and glycosylation study.

IgA Nephropathy and thin basement membrane nephropathy (TBMN) have identical hematuria symptom but different seriousness. In general, the biopsy of the kidney is performed to distinguish between IgAN and TBMN. There are a lot of needs for clinician to have a sensitive and specific biomarker for diseases diagnosis. Glycosylation is highly sensitive to the biological environment changes and considerably affected by disease states. Glycan is known as playing key roles in cancer metastasis and intracellular recognition. Therefore, the examination of glycosylation change in urinary exosome may be a new way for potential biomarkers to differentiate TBMN and IgAN. In this study, we have analyzed 18 individuals exosomes secreted into urine from renal epithelial cells. Samples were consisted of three different groups, mainly TBMN, IgAN patients, and healthy volunteers. Briefly, N-glycans from urinary exosomes were released by PNGase F digestion and then enriched by solid phase extraction using a porous graphitized carbon cartridge. N-glycans were eluted with three different solution (10% ACN, 20% ACN, 40% ACN with 0.05% TFA in H2O) based on the glycan size and polarity. Enriched glycans were analyzed using MALDI-TOF/TOF mass spectrometry and nanoLCchip/QTOF mass spectrometry for qualitative and quantitative profiling, respectively. We found that high mannose glycans are high in abundance in neutral fractions while complex type glycans consisting of [Hex]n=4-6 [HexNAc]n=4-5[Fuc]n=0-1[NeuAc]1 are high in abundance in acidic fractions.

O-GlcNAcylation is the addition of β-N-acetylglucosamine(O-GlcNAc) to serine or threonine residues of nuclear and cytoplasmic proteins. It is highly abundant on myriad proteins, and cycles on proteins with a timescale similar to protein phosphorylation, and has surprisingly extensive cross talk with phosphorylation, where is serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Mitochondrial membrane ATP synthase(F1F0 ATP synthase complex) produces ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain. Subunits α and β form the catalytic core in F

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17-1A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti-colorectal cancer large single chain (lsc) mAb based on mAb CO17-1A in the baculovirus-insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17-1A were fused together by an interchain linker. The only difference between two mAbs is depending of fusion of an ER retention signal (KDEL) at its C-terminus of HC. PCR analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus-infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti-colorectal lsc mAb recognizing human colorectal cancer cells.

Anticancer and antioxidative activities of corn silks methanol extracts (CS-Me) containing glycoside flavonoids were examined. A corn variety was grown at the field of National Institute of Crop Science (NICS), Suwon, Korea. Before the silk-stage, ears of corn were covered with paper-bag to prevent pollination, and after 3-5 days unpollinated corn silks (female inflorescence) were collected and air-dried. The dried corn silks were extracted with MeOH and washed several times with water and then freeze-dried. The crude extract obtained was analyzed by reverse phase HPLC using C18 column and showed that it contains several phenolic compounds with glycoside flavonoids as major ones. This crude extract (CS- Me) was tested for anticancer and antioxidative activities. The anticancer activity of the corn silk extract toward PC-3 (human prostate cancer cell, ATCC No. CRL-1435™), A549 (carcinomic human lung alveolar basal epithelial cell, ATCC No. CCL-185™) and HepG2 (human hepatocellular carcinoma cell, ATCC No. HB-8065™), HT-29 (human colon cancer cell, HTB-38), and MKN45 (human gastric cancer cell, 80103) cell lines was examined by determining the inhibitory effect of cell growth. Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FBS and 1% penicillin–streptomycin. The cells were maintained at 37oC under 5% CO2 and subcultured twice a week. For anticancer activity, cells were subcultured in 96-well plates at a density of 5 x 104 cells per well. After monolayer cultivation for 24 h, the medium was removed and replaced with 100 ul of the maintenance medium (MM) containing 2% FBS. Cells were then incubated for 24 h with different concentrations (0 – 200 ug/ml) of CS-Me dissolved in PBS or water. The cell viability was then determined by MTT assay at 570 nm. It was noticeable that the CS-Me showed the most significant inhibition of the cell growth against MKN45 cells while its effect was not significant toward other cell lines tested. The CS-Me inhibited MKN45 cell growth by about 40% and 60% at the concentrations of 50 and 100 ug/ml, respectively. Although it is quite preliminary, this result suggested that the crude methanol extract of corn silk may have a potent anticancer effect against human gastric cancer. Further characterizations of this extract may lead to identification of a potent therapeutic compounds for the prevention and/or decreasing the severity of gastric cancer. On the other hand, pretreatment of HepG2 cells with CS-Me dose dependently reduced the cytotoxicity of H2O2 and the intracellular ROS level. CS-Me also a significant DPPH radical scavenging activity and stimulated the expression of hemeoxygenase-1 (HO-1) mRNA in HepG2 cells. These results suggest that CS-Me has antioxidative activity.

Lectin has been played a key innate immune parameter in invertebrate host defense. Previously, we reported a novel sialic acid specific lectin (MC-sialec) cloned from EST sequence of Manila clam hemocytes infected with Perkinsus olseni. The lectin was expressed in E. coli M 15 and purified by Ni-NTA His binding resin matrix for antibody production. Lectin specific antibody was raised in mice. The presence of this lectin in different tissues of Perkinsus infected manila clam was studied by both Real Time-PCR and immunohistochemical localization assay. The results showed that upon infection, expression of MC-sialec was induced and expressed in foot, gill, adductor, mantle, hemocyte, stomach, intestine and digestive gland, siphon, palp and female gonad. Vibrio Tapetis infection induced expression of MC-sialec in hemocytes.

Anoikis is an anchorage-dependent cell death in which an epithelial cell shows the apoptotic behaviors upon loss of adhesion with cells and extracellular matrix. Anoikis resistance is known to be a crucial step for cancer metastasis, and it is demanding that understanding of the mechanism underlying the process is required to control cancer metastasis at a molecular level. We have previously identified N- Acetylglucosaminyltransferase-V (GnT-V), a glycosyltransferase that catalyzes the addition of N- acetylglucosamine (GlcNAc) to the core moiety of N-glycan of glycoproteins, producing β1,6-linkages, is a key protein inducing metastasis in human colon cancer cells. And the secretion of galectin-3 binding protein (LGALS3BP) was lowered in GnT-V overexpressing WiDr cells. In this study, we hypothesized that GnT-V has an inhibitory effect on anoikis of cancer cells, while LGALS3BP stimulates the apoptotic process. To prove this, we established the stable transfectants (WiDr:GnT-V), the control cells (WiDr:mock). In addition, we investigated the molecular signatures in various colon cancer cell lines: WiDr, HCT116, SW480 and LoVo. To induce anoikis process, cells were cultured atop 1% soft agar-coated plate and their cellular and molecular responses were monitored. As a result, GnT-V was observed to mitigate anoikis and promote cell aggregation in soft- agar plate system. Among human colon cancer cells tested, HCT116 cells formed aggregates poorly and thus showed low cell viability. Of note, the secretion of LGALS3BP was higher than any other colon cancer cellline, which is in line with the result that anoikis resistance was observed in LGALS3BP-suppressed WiDr cells. Taken together, these results may suggest that GnT-V induces anoikis resistance of cancer cells either alone or in cooperation with the target molecules, such as LGALS3BP.

Autoantibodies, which are generated by immune system recognizing the presence of the abnormal tumor-associated antigens, are promising biomarkers for the early detection of tumors. Recently, we established a B cell hybridoma pool derived from HBx transgenic mouse, as a source of tumor-associated autoantibodies without using any extracellular antigens, and have characterized the specific target antigens against them. XC154 autoantibody, one of them, has been investigated in this study and its target antigen was identified by mass spectrometric analysis as aminoimidazole carboxamide ribonucleotide transformylase / inosine monophosphate cyclohydrolase (ATIC) that catalyzes the last two steps of de novo purine biosynthesis. A specific mimotope against XC154 autoantibody was screened from the cyclic random hepta-peptide phage library and, using mimotope display M13 phage as a coating antigen for ELISA, we could distinguish patients with hepatocellular carcinoma (HCC) vs. normal subjects with 86.96% sensitivity and 88.24% specificity. These results imply that anti-ATIC autoantibody is induced in patients with HCC and detection of anti-ATIC autoantibody can be used for the diagnosis of HCC. Now, several attempts have been performed to establish a more reliable and convenient assay to detect tumor-assocoated autoantibodies using recombinant proteins displaying cycling peptide instead of mimotope display M13 phage and the usefulness of improved assay will be discussed.

The development of cancer biomarker is of a great promise to conquer cancer since it leads to an early detection and provide an opportunity for better cancer treatment. Validation is a time-consuming step for biomarker developments requiring investigation of biomarker candidates using thousands or more of biosamples, which is why a sensitive, multiplexing validation method is necessary. To address this challenge, we are developing as antibody-based validation method, in which antibody with oxidized N-glycans was covalently linked to an identifiable DNA tag for use of the template in the transcription by T7 polymerase. The transcripts will be quantified and used for an indicator of the amounts of the antigen bound to the DNA-tagged antibody. This strategy will be multiplexed for an establishment of a novel validation method.

Genome editing is one of the most demanding tools in mammalian cells to unravel the role of genes and to implement gene therapy of genetic diseases. Zinc finger nuclease is an engineered restriction enzyme which is constructed by combining an array of zinc finger DNA-binding motifs in the N-terminus and the FokΙ endonuclease in C-terminus. Almost every ZFN recognizes three consecutive nucleotides starting with guanine in a context-dependent manner, thereby limiting the use of ZFN in genome editing. To address this challenge, we replaced ZFNs with a gene-specific nucleotides, which were connected to FokΙ nuclease by using (his)6-tag and GS linkers. We tested in vitro whether this engineered FokΙ shows a site-specific nuclease activity. For this, the engineered FokI and nucleotide complex was incubated with template DNA which was produced from 70-mer and 100-mer nucleotides base-paired each other. The results indicate that the complex showed a site-specific nuclease activity, which suggests that the nucleotide-based FokΙ can be used for the universal genome editing.