Earticle

Poly-N-acetyllactosamine (poly-LacNAc, (3Gal1-4GlcNAc1-)n) is a linear carbohydrate polymer composed by repeatingN-acetylglucosamine (GlcNAc) and galactose (Gal) residues. These polysaccharides are found either on N- / O-linked glycoproteins or glycolipids and involve in diverse cellular functions such as differentiation, apoptosis, and metastasis. Poly-LacNAc can be further modified by various glycosyltransferases to create branched structures and display terminal epitopes. Sialylated and fucosylated derivatives of poly-LacNAc have been characterized as specific ligands for different lectins such as selectins and galectins as well as being tumor-associated antigens.It is demanded to have various lengths of oligo-LacNAc for studying LacNAc associated biology. However, the traditional organic synthesis requires tedious multiple protection and de-protection steps which are heavily relied on labor-intensive and time-consuming synthetic routes. To efficiently and quickly produce oligo-LacNAc, we expressed the recombinant bacterial enzymes, □ - 1,3-N-acetyl-glucosaminyltransferase of Helicobacter pylori (HpGnT) and □ -1,4-galactosyltransferase of Neisseria meningitides (NmGalT), from E. coli. Defined lengths of oligo-LacNAcs were synthesized by using the expressed enzymes in the presence of sugar donors, uridine 5’-diphosphate galactose (UDP- Gal) and uridine 5’-diphosphate N-acetylglucosamine (UDP-GlcNAc). To reduce the cost of NDP-sugars used in the oligo-LacNAc synthesis, we set up an enzymatic system for one-pot synthesis of NDP- sugars by using a wild-type bacterial thymidylyltransferase (RmlA). In this study, we applied RmlA to synthesize both UDP-Gal and UDP-GlcNAc. In combination with the use of corresponding kinases, UDP- GlcNAc and UDP-Gal were obtained from cheap starting materials, GlcNAc and Gal. Take the advantage of the great thermal stability of RmlA, the sugar nucleotides were prepared with quantitative yield at 55 oC within two hours. By the alternative addition of HpGnT and NmGalT, different lengths of oligo-LacNAc were synthesized. Compare to previous report, we successfully achieve the synthesis of oligo-LacNAc with a more economical way.

O-GlcNAcase (OGA) is the enzyme which catalyzes the removal of O-GlcNAc moiety on its substrates. This enzyme has two isoforms, full OGA and variant OGA that has alternative stop codon within intron10 which is not spliced-out. As a result, variant OGA lacks C-terminal HAT (histone acetyl transferase) domain and it contains additional 15 amino acids. People believed that full OGA exists both in cytosol and nucleus, primarily to the cytoplasm. There are two opposite reports about the subcellular localization of these two OGA isoforms by two different research groups. The first group figured out that full OGA localizes in cytoplasm, whereas variant OGA exists within nucleus in 2001. However, recently the other group reported that full OGA localized diffusely throughout the nucleus and cytoplasm, whereas variant OGA resides with lipid droplets in cytosol. So these two finding motivates our laboratory to commence the study as to nucleocytoplasmic shuttling mechanism of two OGA isoforms. And we also focus on the various post-translational modifications on OGAs. Full OGA should weigh 103 kDa, however, it weighs approximately 130kDa. So we speculate that various modifications might be related to OGA’s subcellular distribution. To verify and get the answers to several issues about OGAs, we first overexpressed two FLAG tagged-OGA isoforms in HeLa cells to trace the subcellular localization of them. Then we made three C-terminal domain deletion mutants to know the critical domain for its localization. And we also check modification occurred on OGAs by using immune-precipitation and mass spectrometry. Consequentially we can get schematic drawing as to OGA’s nucleocytoplasmic shuttling, and we expect that this research makes it easy to understand and approach O-GlcNAc modification.

Protein O-mannosylation is an essential protein modification that is evolutionarily conserved from bacteria to humans, which is initiated by protein O-mannosyltransferases (Pmts). In the endoplasmic reticulum, Pmt proteins catalyze the transfer of mannose residue to Ser/The residues of secretory proteins. The whole genome information of the thermotolerant methylotrophic yeast Hansenula polymorpha reveals the presence of five PMT homologs, HpPMT1, HpPMT2, HpPMT4, HpPMT5 and HpPMT6. Here we characterized H. polymorpha PMT genes, particularly focusing on a PMT1 subfamily gene, HpPMT5, and a PMT2 subfamily gene, HpPMT6. The promoters of all H. polymorpha PMT genes contain an HpHAC1p binding site, in consistent with their induced expression under UPR condition.Although a single deletion of either HpPMT5 or HpPMT6 did not generate any significant defects, the Hppmt1pmt5 and Hppmt1pmt6 double mutants became more susceptible to cell wall disturbing reagents than the Hppmt1 single deletion mutant. Furthermore, the analysis of HpWsc1 and HpMid2, cell surface sensors of cell wall integrity signaling, and H. polymorpha chitinase, an endogenous O-modified glycoprotein, indicated significant decrease in O-mannosylation in the both Hppmt1pmt5 and Hppmt1pmt6 double mutants. All the fully functional epitope-tagged HpPmt proteins were shown to localize at the ER/Golgi membrane, except for the soluble localization of HpPmt6.The co-IP experiments revealed the complex formation between HpPmt1 and HpPmt2, but no interaction between HpPmt5 and HpPmt2 even in the absence of HpPmt1. Whereas, interestingly, phosphorylation of the mitogen-activated protein kinase Mpk1, which resulted in the stimulation of the cell wall integrity pathway, was markedly activated both in Hppmt1 and Hppmt1pmt5 mutant strains,but not in the Hppmt1pmt6 strain compared to wild type under normal condition. Altogether, these results indicate that HpPmt5 and HpPmt6 have redundant functions to significantly compensate the loss of HpPmt1 in protein O-mannosylation that affects cell growth, cell wall integrity, and stress resistance of H. polymorpha.

N-acetylglucosamine (GlcNAc) kinase (NAGK; EC 2.7.1.59) converts GlcNAc into GlcNAc-6-phosphate. Based on the proposed 3D structure, we produced 3 point mutant NAGKs that were expected to retain differential capacities for substrate binding and reaction velocity. The proteins were expressed in Escherichia coli, affinity-purified to homogeneity, and used for functional analysis. Among the mutants, conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc to Ser (i.e., C143S) had the least affecton enzymatic activity. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc to Ala (i.e., N36A) mildly reduced the enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and acts as a proton acceptor to Ala (i.e., D107A), caused a total loss in the enzyme activity. The eGFP- or RFP (DsRed)-tagged mutant NAGKs increased the complexity of dendritic architecture when overexpressed in rat hippocampal neurons (DIV 5-9) with no statistical difference with wild-type NAGK. These results indicate that the upregulation of dendritic complex by NAGK is the enzyme’s non-canonical function.

EDEM1 is an endoplasmic reticulum-associated protein degradation (ERAD) component. ERAD is a cellular pathway that targets misfolded and misassembled glycoproteins for degradation. EDEM1 is involved in the recognition of misfolded luminal glycoproteins and in routing them for dislocation to the cytosol, followed by their degradation. EDEM1 interacts with substrate glycoproteins after their exit from the calnexin/calreticulin cycle and after processing by ER-mannosidase I. Although EDEM1 was proposed to be lectin–like and to react with Man8GlcNAc2 oligosaccharides, itsmechanism of action and its fate are still largely unknown. In a previous report, we found that EDEM1 becomes rapidly degraded and that this occurs by basal autophagy. Here, we show that EDEM1 forms complexes with the selective autophagy receptor p62, NBR1 and Alfy. This was demonstrated in HepG2 cells by double immunogold electron microscopy. Furthermore, we show the interaction between p62 and EDEM1 by immunoprecipitation- Western blot experiments. By serial section analysis, the origin of the phagophore for selective autophagy of EDEM1 could be identified as modified parts of rough ER cisternae. Hence, we provide new insight into the details of EDEM1 degradation process.

Trichomonas vaginalis is a flagellated protozoan parasite causing vaginal trichomoniasis in women, and can secrete chemotactic lipid mediator LTB4 in their secretory products. However, signaling mechanisms of tissue inflammatory responses by infection with T. vaginalis are not fully understood. Recently, we have recently demonstrated that human mast cells and neutrophils are activated to produce chemokine IL-8 via LTB4 receptor BLT1 in response to T. vaginalis-derived secretory products (TvSP). Here, we report that O-glycosylation is important in BLT1-mediated IL-8 secretion in human mast cells (HMC-1 cell line) induced by TvSP. Incubation of HMC-1 cells with TvSP resulted in marked increase of O-GlcNacylated proteins and up-regulated IL-8 protein secretion. TvSP-induced IL-8 production and O-glycosylation in HMC-1 cells was inhibited by pretreatment with OGT inhibitor or OGT siRNA. Pretreatment of HMC-1 cells with BLT1 antagonist or BLT1 siRNA strongly abolished the stimulatory effects of TvSP on O-GlcNacylation and IL-8 production. Moreover, TvSP-induced phosphorylation of transcription factors NF-B and CREB for IL-8 production was reduced by pretreatment of HMC-1 cells with OGT inhibitor or OGT siRNA. These results suggest that BLT1-mediated O-glycosylation may play an important role in activation of transcription factors CREB and NF-B for IL-8 production in mast cells stimulated with TvSP, which can contribute to mast cell-mediated tissue inflammatory responses in T. vaginalis-infected lesion during human trichomoniasis.

Degranulation in human mast cells contributes to provocation of allergic inflammation and innate immunity to parasites. Trichomonas vaginalis is transmitted by sexual intercourse and causes the acute and chronic allergic inflammation. Although a recent report has shown that mast cells are degranulated in response to T. vaginalis-derived secretory products (TvSP), detailed signaling mechanisms of TvSP- induced mast cell degranulation are not fully understood. We report that N-glycosylation of NOX2 play an important role in surface trafficking of NOX2, which can regulate ROS-dependent degranulation in TvSP- stimulated human mast cells. Stimulation with TvSPinduced NOX2 activation such as p47phox phosphorylation and NOX2 glycosylation, production of intracellular ROS, and release of granular proteins such as histamine and -hexosaminidase via BLT1. Inhibition of ROS generation with NOX2 inhibitors also reduced TvSP-triggered degranulation. Moreover, TvSP promoted trafficking of intracellular NOX2 to the cell surface in a BLT1-dependent manner. Such BLT1-mediated surface migration of NOX2 was dependent upon N-glycosyation status of NOX2. Inhibiting N-glycosylation of NOX2 by N-glycosylation inhibitor tunicamycin reduced surface trafficking of NOX2, ROS generation, and degranulation induced by TvSP. PKC inhibitor prevented TvSP-triggered glycosylation of NOX2, its surface trafficking, ROS generation, degranulation. Finally, disruption of lipid rafts with M □CD prevented glycosylation-dependent surface trafficking of NOX2, ROS generation, and degranulation. These results suggest that PKC-mediated N- glycosylation regulates its trafficking of NOX2 to the lipid rafts, which is required for ROS-dependent degranulation in mast cells induced by T. vaginalis-derived secretory products.

Microdomains are constructed by cholesterol, sphingomyelin, and gangliosides in plasma membrane. The microdomains, which are dynamic structure in the cellular membrane, controls lateral diffusion and signaling of membrane molecules such as growth factor receptors (GFR). However, we still could not figure out the generalized mechanisms. We hope to demonstrate that an alteration of lipid component in microdomain affects lateral diffusion of membrane receptor. Therefore, we established experimental system in which monitored the membrane organization of receptors by analyzing its lateral diffusion parameters in the plasma membrane of living cells using fluorescence recovery after photobleaching (FRAP). In the FRAP analysis, we used CHO-K1 cells stably expressed the insulin receptor fused green fluorescence protein (IR-EGFP). Then, fluorescence recovery curves of IR-EGFP in the bleach area was detected and performed nonlinear curve fitting. Next, we performed FRAP analysis in these cells treated with Methyl-β-cyclodextrin (MβCD) to change the lipid component. In this study, we indicated that the ratio of mobile fraction of IR-EGFP in control and MβCD treated cells were about 87.5% and 78.5%, respectively. However, there was little to distinguish diffusion coefficients of IR-EGFP in the both cells. In this condition, we checked insulin signal transduction pathway by co-expression of IR-EGFP and IRS1-ECFP. Interestingly, we have confirmed that phosphorylation of IRS1 was decreased though the phosphorylation of IR was slightly increased. At present, we are trying measurement of lateral diffusion of IR molecules at the change of membrane environment, such as treatment with inhibitor of glycosylceramide synthase (D-PDMP).

CD22 (siglec-2) is an accessory molecule of the B-cell receptor complex (BCR) that exertsnegative effects on receptor signaling. It is also well-documented that CD22 is a regulatoryprotein that sets a threshold for immune responses. The carbohydrate ligand recognized byCD22 is the sequence Neuα(2,6)Galβ(1,4)GlcNAc found on both neighboringglycoconjugate of the same cell (cis ligand) and on other cells that interact with B cells (transligands). Recently, we have reported that the C-9 amido derivative of sialic acid (GSC718;9-(4’-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gc-OBn) show a potent affinity andselectivity for CD22 than other siglecs such as MAG [1]. Moreover, the compoundpromoted the proliferation of B cells in vitro. As next step of our investigation, we intend toreinforce the promoting activity of GSC718 for B cell growth by chemical modification.Herein, we report the efficient synthesis of GSC718 analogs which have varied aglyconmoieties. To achieve the comprehensive synthesis of GSC718 analogs having varied aglycons, wereexamined every synthetic process to obtain a fine target compound. In case of thesialoside synthesis, the most time-consuming and troublesome process is thechromatographic separation of α-sialoside from other byproducts such as β−isomer, 2,3-enederivative etc. after glycosylation reaction with aglycon part. To improve this process, weemployed 1,5-lactam formation as the key step for separation because the lactam formation isknown to proceed only in α-sialoside [2]. At the beginning of the synthesis of targetmolecules, we synthesized a suitably modified sialic acid donor in good yields. Then, thesialyl donor was reacted with various 2-substituted-ethanols to give the mixtures of α- and β-glycosides and 2,3-ene derivative, which were subsequently advanced to 1,5-lactamformation. As we anticipated, 1,5-lactamized α-sialosides became isolable from themixtures due to its different polarity from the other byproducts. Finally, the obtained 1,5-lactamized silaosides were successfully converted into target structures via reaction sequenceincluding C9-midification with biphenyl amide group, lactam opening and globaldeprotection. The synthesized analogs were advanced to biological assay using B cells. In this poster presentation, we will also discuss the structure-activity relationships of thesynthesized analogs. [1] H. H. M. Abdu-Allah et al, Bioorg. Med. Chem. Lett. 2011, 19, 1966-1971. [2] H. Tanaka et al, Tetrahedron Lett. 2009, 50, 4478-4481.

Stage-specific embryonic antigens1 (SSEA’s) have been identified as glycosphingolipids which play diverse roles in embryonic development, such as regulation of cell growth, recognition and differentiation. It’s well-documented that human ES and iPS cell express predominantly glycosphingolipids of the globo- series2 such as SSEA-3 and SSEA-4, which are also hallmarks of human embryonal carcinoma cells. While, SSEA-1 represents a marker for murine pluripotent stem cells, in which it plays an important role in adhesion and migration of the cells in the preimplantation embryo. We are intended to integrate their biological functions into biocompatible nanomaterials for developing ES and iPS cell culturing system. To conjugate with nanomaterials these molecules have been suitably functionalized at the terminal position. In the present study, a convergent chemical synthesis of SSEA-1 SSEA-3 and SSEA-4 will be discussed.

Benzyl ethers are frequently used ether protecting groups in carbohydrate chemistry. Together with ester groups, they already form the selective protection and deprotection of saccharide building blocks prior to and after the glycosidic linkage step for the synthesis of oligosaccharides. The most general used highly regioselective benzylation means involves the utilization of organotin. The regioselectivity was provided by introduction of dibutylstannylene acetals, which derived from treatment of diols and polyols with organotin. Benzyl bromide was herein used as electrophiles, which was normally performed in the presence of halide anions for the improvement in terms of speed, yield and general convenience. However, the mechanistic origin of the improvement for the reaction by halides has not been fully clarified. In present study, the mechanistic origin as to the promoted organotin-mediated carbohydrate benzylation by halides was explored by the comparison of the activation ability of halides on benzylation of methyl β- D-galactoside. It was demonstrated that the improvement of benzylation in the presence of halides was due to the formation of the activated oxide species by the coordination of the halide anions to the tetracoordinate tin atoms. The halide, being able to form stronger bond with tin, showed stronger activation ability. The results were further applied to more carbohydrate structures and multiple carbohydrate benzylations. A range of prototype carbohydrate structures were efficiently prepared with the guidance of the principle.

Polysialic acids, linearly fused N-acetylneuraminic acid (Neu5Ac), play a central role in numerous biological recognition processes such as lymphocyte homing, tumor metathesis, meningitis, urinary tract infections and pathogenic bacteria infections. Three types of polysialic acids, which are different by the internal linkages, have been isolated and identified from the microorganisms. The α (2→8) polysialic acid is a component of neuroinvasive E. coli K1and N. meningitida serogroup B, while the α (2→9) polysialic acid is a component of N. meningitida serogroup C. The alternating α (2→8)/α (2→9) polysialic acid is isolated from E. coli K92 and Bos-12 strains and not found in mammalian systems, making it a potentially carbohydrate vaccine for medicinal treatment. The glycoconjugates of isolated α (2→9) polysialic acid and a carrier protein (ex: diphtheria or tentanus toxoid) are the current vaccines against Meningococcal group C diseases; however, these vaccines are often heterogeneous or contaminated with other antigenic components due to the difficulty in purifying polysialic acids from natural sources. Recently, our lab has successfully and efficiently synthesized α (2→9) polysialic acids up to dodecamers with high yield using 5 -N,4-O-carbonyl protected phosphate-based donors via the convergent block synthesis strategy.

Heparanase, preferentially expressed in many tumor cells, is an endoβDglucuronidase that cleaves specific sites of heparan sulfate. Elevated levels of heparanase expression correlate with tumor vasculatrity, metastatic potiential, and reduced postoperative survival of cancer patients. Consequencely, heparanase detection is considered a tool for cancer discovery. A library of 20 welldefined Tetra1 analogs were prepared in a convergent “2+2” glycosidation manner utilizing a relatively small number of protected disaccharide donors and acceptors. Among three different donors prepared for the fullprotected tetrasaccharide, trifluoroacetimidate donor was most suitable for the preservation and glycosidation reaction, and in each glycosylation, the yield was good and the major anomer was obtained. After coupling these 12 glycosydic reactions, the observation indicate that the coupling ratio is not only relating to the structure and exact nature of acceptors, but also correlated to the structure and size of donors. Key features of the approach include the use of Bz esters or PMB ethers for those hydroxyls that need sulfation, regioselective opening of the 4,6-O-benzylidene acetal for 4OBn that don’t need sulfation and 6OH that need oxidation, an anomeric TBS group for glycosyl donor synthesis, and benzyl ethers just as permanent protecting groups. The HS oligosaccharides will be employed to probe structural features of HS for cleavage of heparanase.

The human colorectal carcinoma-associated GA733 antigen is an epithelial cell adhesion molecule (EpCAM), initially described as a cell surface protein selectively expressed in some myeloid cancers. We demonstrated that treatment with plant-derived anti-EpCAM mAb (mAbp CO17-1A) and RAW264.7 cells significantly inhibited cell growth in SW620 cells. Expression of p53 and p21 increased, whereas the expression of G1 phase-related proteins, cyclin D1, CDK4, cyclin E, and CDK, decreased. In addition, mAbp CO17-1A and RAW264.7 cell treatment decreased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-α, caspase-8, caspase-9, caspase-3 and caspase-6 increased. HPTLC analysis showed that the cells treated with mAbp CO17-1A and RAW264.7 cells newly expresses GM1 and GD1a. These results suggest that mAbp CO17-1A could have an anticancer effect. Further clinical investigation should be conducted on mAbp CO17-1A to determine its possible chemopreventive and/or therapeutic efficacy for the treatment of human colon cancer.

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1,6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core and Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2,3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2,6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.

Glycans, as attached to proteins and lipids on the cell surface, play crucial roles in various physiological phenomena such as cell-cell communication, differentiation and development. Recently, they have been highlighted as stem cell markers used to assess the pluripotency status. However, analysis of cell surface glycans requires difficult and laborious processes for the isolation of plasma membrane from other cellular components. Especially, efficient removal of endoplasmic reticulum (ER) and Golgi membranes are required since the high amount of high-mannose type glycans in ER has hampered the analysis of complex type glycans on the cell surface. In the present study, we employed the simple adhesion-based method for the isolation of plasma membrane to analyze the glycans coating on the cell surface efficiently. First, cells adhered to an adsorbed layer of polylysine on glass plates and then were lysed by hypotonic pressure method using ice-cold distilled water. After the cell lysis and successive washing steps, plasma membrane fractions containing glycopeptides could be collected from the plate by trypsin digestions. The purity of the isolated plasma membranes was evaluated by fluorescence imaging using organelle-specific probes and optimal isolation conditions were established to minimize the contamination of ER and Golgi fractions. Using this method, N-glycans obtained from the plasma membranes of embryonic carcinoma NTERA-2 and Chinese hamster ovary (CHO) cells were analyzed and compared to N-glycan profiles derived from the corresponding total cell fractions. Complex-type glycans capped with terminal sialic acids were observed as major populations in plasma membrane fractions whereas high-mannose type glycans mainly detected in the profiles of total cells. Taken together, easy and rapid method to isolate plasma membrane was successfully employed and optimized for the analysis of the cell surface N-glycans, which will contribute to elucidating the biological implications of cell surface glycan changes.

Isoflavonoids, polyphenols are biologically active natural products which are abundantly present in soyabean seeds during development. Clinical studies have suggested positive effects of isoflavonoids in human health and nutrition, such as reduction in the risks of hormonally dependent cancers, menopausal symptoms, osteoporosis, and cardiovascular disease. However, the insolubility and instability of those polyphenols prevent the wide applications of these medicinally important compounds. Therefore, the generation of isoflavonoid derivatives by glycosylation gained much attention and interest. In this study, we used E.coli BL21 (DE3) as an expression host for gmIF7Gt, a Glycine max derived glycosyl transferase, cell biocatalyst for the production of glycosylated derivatives of genistein. The supplementation of 0.2mM of genistein in the growing induced culture of E. coli BL21 (DE3) harboring PET15b-GmIF7GT resulted novel spots in TLC. These spots were further analyzed by HPLC and LC-TOF ESI/MS. The exact molecular mass analysis confirmed the production of Genistein glucoside. Further, structural elucidation and product enhancement is necessary for large scale production of this compound. However, this study reveals a method that might be useful for the biosynthesis of glycosylated isoflavonoids and related compounds by in-vivo glycosylation.

TDP-L-rhamnose–producing E. coli host containing Lamiales-derived UGT88D7 glycosyltransferase was employed to generate flavone rhamnosides and glucosides from apigenin, baicalein and luteolin. In comparison with the published literatures, this gene has showed markely flexible activity because it is able to transfer glucose, glucoronic acid and rhamnose moieties to aglycones. Although apigenin was manifested as the most favourite substrate baicalein and luteolin seemed to be good candidates to yield glycosylated products. The productivities of bioconversion of apigenin, baicalein and luteolin were 75.4, 36.1 and 32.4%, respectively, in the presence of methylated cyclodextrin as mediator. The formation of product was confirmed by TLC, HPLC and ESI-MS/MS measurement. The structural identification of products is being continued by NMR method. Data revealed the use of NDP-sugars flexible glycosyltransferase as an effective tool to produce various kinds of flavonoid glycosides by combinatorial biosynthesis in E. coli.

Cancer seems like unsolvable disease to human beings. Since there are so many types of the disease, it’s harder to figure out full cure for it. We had special interest in curing colorectal cancer. Curing and preventing colorectal cancer, we can use mAb CO17-1A. GA733-2 is a glycoprotein highly expressed in colorectal carcinoma cells. mAb CO17-1A recognizes GA733-2 of carcinoma cells. Since our body is unable to produce mAb CO17-1A, we have to get the mAb from other sources, such as plants or insect cells. In this study, we are going to find out the differences between two insect cells, SF9 and SWT-4, in producing mAb CO17-1A. By performing purification and SDS PAGE, we figured out that there were more mAb productions in SWT-4. Also cells with KDEL showed different types of glycan structures to cells without KDEL. KDEL is a signal peptide that retains proteins in ER. KDEL stands for Lysine, Aspartic Acid, Glutamic Acid, and Leucine. High-performance liquid chromatography (HPLC) verified that cells with KDEL showed high-mannose type glycan while cells without KDEL gave us insect cell specified glycan.

A novel GT1 family glycosyltransferase, YjiC from Bacillus licheniformis ATCC 14580 has been PCR amplified, cloned, and expressed in E. coli BL21 (DE3) expression host. The expression of 396 amino acid long gene generated ~45 kDa N-terminal his-tag protein. The protein has been purified and used for the in vitro glycosylation of phloretin. Moreover, the enzyme was also applied for the in vivo glycosylation of the same phloretin molecule. The in vitro and in vivo study found that YjiC can glycosylate at different positions of hydroxyl groups of phloretin molecule very efficiently with conversion rate of ~98%. Four different products has been identified from the in vitro as well as in vivo reaction as phloretin-2’-glucoside, phloretin-4’-glucoside, and two different diglucosides of phloretin. All the products have been confirmed by TLC, HPLC, LC-MS, and former two products has been further confirmed by NMR analysis.

The glycosylation process helps in stabilization, detoxification and solubilization of many compounds. Apigenin, which is a class of flavonoid, known for its high antioxidant activity and contains hydroxyl groups at C-5, C-7 and C-4' positions, has been glycosylated using YjiC, a glycosyltransferase obtained from Bacillus licheniformis DSM 13, which is an industrial organism used for the manufacture of enzymes, antibiotics, and chemicals. YjiC consists of 1,191 bp open reading frame encoding a 44.7 kDa protein. UDP-D-glucose has been used as a sugar donor. Thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectorscopy (ESI-MS-MS) analyses of in-vitro reaction products revealed that YjiC could glycosylate apigenin. The positon of glycosylation in apigenin is yet to be determined.

Sialic acid-binding immunoglobulin-like lectin (Siglec) 7 is one of the sialic acid binding protein that plays an important role in immune system. Siglec 7 is expressed on natural killer cells and displays a unique ligand binding properties comparing to the other members of the Siglec family, specifically binds to  2,8-disialyl residues. The precise structural analysis of Siglec 7 has been already performed, and amino acid residues involved in the ligand binding are reported. In such context, we set out the first total chemical synthesis of Siglec 7 carbohydrate binding domain (CBD) which consists of 127 amino acid residues. Since chemical synthesis can easily introduce unnatural amino acids, we envisioned that we could produce an artificial sialic acid binding lectin based on this chemical synthetic strategy. The synthesis of Siglec 7 CBD was carried out by convergent total chemical synthesis. The whole polypeptide was assembled from 5 peptide segments (Figure), which were synthesized by conventional solid phase peptide synthesis. We applied native chemical ligation (NCL) for the coupling of these segments sequentially from C-terminus, combined with alkylation with haloacetoamide or desulfuration reaction. These reactions can provide pseudo-Gln residue or Ala residue from Cys residues at the ligation sites. Based on these strategies we have successfully obtained the full-length polypeptide of Siglec 7 CBD and currently are investigating the in vitro folding step.

GlycoNAVI is a computer software suite containing databases, desktop applications, web services and other useful tools that has been developed to provide a comprehensive support for carbohydrate research. GlycoNAVI database, one of the components, consists of multiple databases relevant to glyco -organic chemistry such as organic synthesis methods (reactions), abbreviations (synonyms), molecular information and so on. All information in the database is gathered from numerous literatures related to carbohydrates. The functions in the database provide a number of search options for researchers to quickly access the desired information. The web retrieval system, for instance, allows researchers to input ID number or Keyword as a search query. The system also has the function to show the graphical images of chemical structure so that an easy access to the desired molecular information is possible. On the other hand, a molecular structure is often used as a search query in a conventional database of compounds. However; in this system, a researcher is able to use CSEditor, a desktop application with user-friendly graphical interface, to perform a search for chemical structure. CSEditor allows researchers to easily depict a chemical structure and the depicted structure can be used as a query for partial or exact matching search. Furthermore, by selecting the molecule in the search result, the details regarding chemical reactions including the molecule on the query are easily obtained. GlycoNAVI database is an inclusive set of information which opens the door to all carbohydrate researchers including the ones that are unfamiliar with chemical structures because CSEditor, an easy editing tool of carbohydrates, is available and always comes along the suite.

Glycoprotiens are responsible in many biological events. However oligosaccharides covalently attached to these proteins always exhibit heterogeneity. This has been a hindrance to investigate what oligosaccharide structure is essential for the individual biological event. Therefore we have examined the chemical synthesis of several glycoproteins such as cytokines consist of 70 to 166 amino acid residues. [1-3] However, glycoproteins used in the cell-cell communication or immune response exhibit larger molecular weight comparing with those of cytokines we have synthesized. Unfortunately, the synthesis of such large and homogeneous glycoproteins is still far from our current achievements, because we have not established an efficient method for the synthesis of large glycoproteins yet. Native chemical ligation[4] is a powerful method to couple two polypeptide chains through a native amide bond. This ligation takes place between peptide-C-terminal thioester and cysteine at the N-terminal of another peptide. Using this ligation between glycopeptide-thioester synthesized by chemical method and long peptide prepared from E. coli expression, we have examined the synthesis of large glycoprotein. In this presentation, we would discuss the first synthesis of large and homogeneous glycosylpolypeptide chain (25 kDa) consists of over 200 amino acid residues.

Colorectal cancer (CRC) is one of the most prevalent cancers in the world with high mortality and morbidity rates. In this study, we have performed comparative proteomic profiling of sera from CRC patients at stage I (n=17), stage II (n=40), stage III (n=24) and healthy subjects (n=25) to gain a global view of protein expression change during CRC tumorigenesis and provide potential targets for CRC diagnosis and treatment. As a result, a total of 93 proteins were found differentially expressed in CRC patients with a label-free quantitative APXE method. After GO and KEGG pathway analysis, those proteins most frequently involved in ECM-receptor interaction, complement and coagulation cascades. As important as components of ECM, we found several collagens in CRC serum had been changed from tumor stage I to IV. And the validation of collagen I (COL1) at RNA and protein expression level shown extremely comparable to pooled serum proteomic results using independent 26 paired tumor and matched normal colorectal tissues. Those findings indicated that the change of collagen I observed in serum were indeed from pathogenic lesion of colorectal tissue. Moreover, we further investigated serum levels of COL1, PICP (the synthesis indicator) and CTx (the breakdown indicator) in 77 CRC patients and 33 normal controls by ELISA. The results showed PICP and CTx were better for discriminating normal from cancer groups as well as non-metastatic from metastatic tumor than COL1. Finally, we evaluated the expression of MMPs in paired tumor and normal tissues from patients with different stages. Notably, the expression of MMP1, 7 and 14 were remarkably enhanced in carcinoma tissues and the trend were parallel with the progression of tumor stage. The expression of E-cadherin and CDX2, which had been considered as targets of COL1 in cell models, were also verified in tissues and displayed decrease in tumor. Overall, COL1 might be affected by MMP1, 7, 14 and had effects on cell adhesion and differentiation through E-cadherin and CDX2.

Propylene glycol alginate sodium sulfate (PSS) is a kind of sulfated polysaccharide that is derived from seaweed extract sodium alginate through hydrolysis and esterification. PSS is a heparinoid drug, mainly used for the prophylaxis and treatment of ischemic cardiovascular and cerebrovascular diseases in clinical in China. In order to develop and utilize PSS better, a new fluorescent labeling method was established to study the pharmacokinetic parameters of PSS. A rapid and sensitive fluorescent labeling method was developed and validated for microanalysis of marine sulfated polysaccharide PSS in rat plasma. 1, 6-diaminohexane was chosen as the spacer arm to link PSS with FITC. The fluorescent labeled PSS (F-PSS) was identified through its spectroscopic properties. The labeling method showed good linearity, precision, recovery and stability, which suggested that it was sensitive and reliable. The pharmacokinetic results showed that the absolute bioavailability of F-PSS was 8.39% and the other parameters were tested after oral and intravenous administration of F-PSS. The labeling method could be successfully applied to the investigation of the absorption and metabolism of PSS in future. Moreover, the labeling method could be also applied to pharmacokinetic studies of other polysaccharides in biological samples.

Cell surface carbohydrates play significant roles in a number of biologically importantprocesses. Heparan sulfate (HS), for instance, is a ubiquitously distributed polysulfatedpolysaccharide that is involved, among others, in the initial step of herpes simplex virus type1 (HSV-1) infection. The virus interacts with cell surface HS to facilitate host cellattachment and entry. 3-O-Sulfonated HS was discovered to function as an HSV-1 entryreceptor. A complete understanding of these interactions necessitates the chemical synthesisof such oligosaccharides, but remains challenging. Here, we present a convenient approachfor the synthesis of two irregular 3-O-sulfonated HS octasaccharides making use of a keydisaccharide intermediate to acquire different building blocks for the oligosaccharide chainassembly. Despite substantial structural differences, the prepared 3-O-sulfonated sugarsblocked viral infection in a dosage-dependent manner with remarkable similarity.