Earticle

Human LOX-1/OLR 1 plays a key role in atherogenesis and endothelial dysfunction. The N-glycosylation of LOX-1 has been shown to affect its biological functions in vivo and modulate the pathogenesis of atherosclerosis. However, the N-glycosylation pattern of LOX-1 has not been described yet. The present study was aimed at elucidating the N-glycosylation of recombinanthuman LOX-1 with regard to N-glycan profile and N-glycosylation sites. Here, an approach using nonspecific protease (Pronase E) digestion followed by MALDI-QIT-TOF MS and multistage MS (MS3) analysis is explored to obtain site-specificN- glycosylation information of recombinanthuman LOX-1, in combination with glycan structure confirmation through characterizing released glycans using tandem MS. The results reveal that N-glycans structures as well as their corresponding attached site of LOX-1 can be identified simultaneously by direct MS analysis of glycopeptides from non-specific protease digestion. With this approach, one potential glycosylation site of recombinanthuman LOX-1 on Asn139 is readily identified and found to carry heterogeneous complex type N-glycans. In addition, manual annotation of multistage MS data utilizing diagnostic ions, which were found to be particularly useful in defining the structure of glycopeptides and glycans was addressed for proper spectra interpretation. The findings described herein will shed new light on further research of the structure-function relationships of LOX-1 N-glycan.

Nesseria meningitidis is a type of bacteria which causes meningococcal meningitis and meningococcal septicemia. It often infects children under two years old and causes infant dead. Meningococcal diseases are prevalent in developing countries, like China and Africa. Based on their surface capsular polysaccharides, they are classified into 13 serogroups, while group A, B, C, Y, W135 are the major epidemic strains in the world. Though, some efforts have devoted to develop polysaccharide vaccines against N. meningitidis, but these vaccines often elicit weak immune responses and short immune memories. Therefore, polysaccharide-protein conjugate vaccines were developed to tackle the problem and the immunogenicity improved greatly. However, the compositions of commercial available vaccines are very heterogeneous and the quality is hard to control. In this study, we synthesized the N. meningitidis W135 capsular oligosaccharide in different lengths and acquired the homogeneous form to detect the immunogenicity. Because the N. meningitidis W135 capsular oligosaccharide is silalic acid-galactose repeating structure, we used sialyl phosphate as a donor to conjugate thiol galactoside to form the basic repeat unit. By linking the repeat units, we have obtained various length of oligosaccharide from disaccharide to decasaccharide. We conjugated these different length of oligosaccharides to carrier protein CRM197 (a mutated diphtheria toxin) and injected these conjugates to mice to search for the best vaccine composition.

Sialyltransferase is a glycosyltransferase transferring sialic acid from CMP-Neu5Ac to the non-reducing terminal position of glycoconjugates. Twenty different sialyltransferases containing so-called sialylmotifs are highly conserved from invertebrates to vertebrates. These well conserved sialylmotifs suggested that all sialyltransferases had been evolved from a common ancestor. Many sialyltransferases have been known in vertebrate linage, however, only a few have been characterized in non-vertebrates. In this study, we cloned a gene encoding sialyltransferase (ST) from marine invertebrate organism, Ciona savignyi (Csav), and analyzed its phylogenetic relationship with other animal sialyltransferases. Csav-ST was clustered with ST3Gal subfamily. Csav-ST has two putative transcript isoforms identified in of the sea squirt C. savignyi genome database. To indentify a sialyltransferase activity, Csav-ST3Gal was functionally characterized by using recombinant enzyme expressed in Saccharomyces cerevisiae. Csav-ST3Gal is localized to Golgi membrane when expressed in yeast. Enzymatic assays indicate that Csav-ST3Gal is capable of transferring sialic acids to the glycans attached to asialofetuin. Substrate specificities and kinetic properties indicated that Csav-ST3Gal could prefer O-glycans rather than N-glycan of asialoglycoproteins as substrates. Further, it has been displayed that Csav-ST3Gal catalyzes the formation of α(2,3)-linkage by a lectin blot analysis with Maackia amurensis lectin and by the linkage-specific sialidase treatments. In addition, Csav-ST3Gal has three putative N-glycosylation sites in its stem region, Interestingly, only one site for N-glycosylation was occupied with N-glycan when expressed in yeast. By N-glycan trimming with glycosidases treatments, we determined that this glycan of Csav-ST could not be required for in vitro enzyme activity, because of no difference in non-glycosylated and glycosylated sialyltransferase activities. These results indicate that Csav-ST3Gal could be an common ancestral glycosyltransferase belonged to ST3Gal I/II subfamily, transferring sialic acid to Gal-β1,3-GalNAc moiety.

To diversify the therapeutic uses of quercetin, Escherichia coli was exploited as a production factory, by assembly of various bacterial and plant UDP-xylose sugar biosynthetic genes and glycosyltransferase. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were over-expressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21 (DE3)/Δpgi, E. coli BL21 (DE3)/Δzwf, E. coli BL21 (DE3)/ΔpgiΔzwf, and E. coli BL21 (DE3)/ΔpgiΔzwfΔushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase, and galU integrated E. coli BL21 (DE3)/Δpgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. The highest production of 3-O-xylosyl quercetin was achieved with E. coli BL21 (DE3)/ ΔpgiΔzwfΔushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM) representing 54.75 % bioconversion, which was ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5 ml culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98% when the reaction was scaled up in a 3 L fermentor at 36 h.

Glycolipids offer recognition sites of a variety of proteins such as antibodies and microbial toxins, thereby playing physiological and pathological roles on cell membrane surfaces.Therefore, these glycolipid-protein interaction systems could be potential therapeutic targets for various diseases, including bacterial infections and neurodegenerative disorders [1]. To elucidate the underlying mechanisms of molecular recognition between glycolipids and proteins, we investigatedthe interaction between sarcotoxin IA and lipid Aas a model system to characterize conformational transitions and intermolecular interactions of the membrane-binding peptides promoted on glycolipids. Lipid A is a major component of the outer membrane of Gram-negative bacteria and can be a recognition target in the innate immune system. This molecule can also serve as targets of sarcotoxin IA, which is a 39-residue cecropin-type antibacterial peptide from Sarcophaga peregrina. In order to obtain structural information at atomic level, we expressed sarcotoxin IA peptides in Escherichia coli to achieve 13C- and15N-labeling for detailed NMR analyses. We observed NMR spectral changes of sarcotoxin IA upon interacting with lipid A, which was embeded in micelles composed of dodecylphosphocholine. Our spectroscopic data revealed that the N-terminal segment of sarcotoxin IA was converted from random- coil to an amphiphilic α-helix upon specific binging to lipid A. By inspecting chemical shift perturbation data, we successfully identified key lysine residues involved in interaction with lipid A and consequent antibacterial activity.

Overweight and obesity, which are major risk factors for a number of chronic diseases, including diabetes, cardiovascular diseases and cancer, have become a major health concern nowadays. Thus, the discovery of appetite suppressant property of Hoodia plants growing in the desert of South Africa attracts a great attention and has led to many commercial preparations. A series of pregnane glycosides have been isolated from Hoodia species, among them only P57 (or P57AS3) is reported to have the activity. Animal studies show that P57 increases the content of ATP in hypothalamic neurons and this might be relevant to the appetitive responses. However, the detailed structure-activity relationship and mechanism of action of P57 has yet to be known. Synthesis of P57 has only been mentioned in a patent, in which the aglycone was synthesized from progesterone employing micro-organism transformation to introduce the 12,15-OH for further elaboration and the trisaccharide assembled onto the aglycone via sequential glycosylation with fluoride donors in low yields (<15% for each step). Recently, Miesch et al. reported a synthesis of the aglycone employing a Norrish type I–Prins reaction as the key step for introduction of the 14-OH, albeit in low yield. Herein, we describe an expeditious synthesis of P57 in a linear 20 steps and 2.4% overall yield from digoxin. The present synthesis provides a consulting strategy to other Hoodia saponins and analogues; the availability of these pregnane glycosides (including stereoisomers) shall facilitate the studies on the SAR and mechanisms of the appetite suppressant activity of P57. For a practical synthesis of P57, however, the stereoselectivity of glycoyslation in the present synthesis remains as a challenge to address.

Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD). The clinical severity of EV71central nerve system (CNS) infection is mostly in children less than 5 years-old. Recently, the twoglycosylated membrane proteins SCARB2 and PSGL-1 have been identifiedas the cellular and functional receptors of EV71.But the role of glycosylation in EV71 infection remainsunclear. In this study, we demonstrated thatthe attachment of EV71 to two susceptible cell lines werediminished after the removal of cell surface sialic acids. Pre-incubatecells with sialic acid specific lectins, MAA and SNA,reducedthe binding of EV71. Additionally, we found that SCARB2 was a sialylated glycoprotein,andthe interaction of SCARB2and EV71was decreased after desialylation. Based on these results, we concluded that the attachment of EV71 to cell surface was assisted bysialic acids.Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71.

Anti-inflammatory effects of low-molecular weight chitosan oligosaccharides (LM-COS) prepared from high-molecular weight chitosan by enzymatic digestion were investigated against allergic reaction and allergic asthma in vivo and in vitro. Allergic asthma is an inflammatory disease of the airways associated with enhanced degranulation and cytokine generation. The LM-COS (<1 kDa), consisting of glucosamine (GlcN)n, n = 3-5, were capable of inhibiting both antigen-stimulated degranulation and cytokine generation in rat basophilic leukemia RBL-2H3 cells. The protective effect of LM-COS against ovalbumin (OVA)-induced lung inflammation in asthma model mice was also examined. Oral administration of LM-COS (16 mg/kg body weight/day) resulted in a significant reduction in both mRNA and protein levels of interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor (TNF)-αin the lung tissue and bronchoalveolar lavage fluid (BALF). The protein levels of IL-4, IL-13 and TNF-αin BALF were decreased by 5.8-fold, 3.0-fold and 9.9-fold, respectively, compared to those in the OVA-sensitized/challenged asthma control group. These results suggest that oral administration of LM-COS is effective in alleviating the allergic inflammation in vivo and thus can be a good source material for the development of a potent therapeutic agent against mast cell-mediated allergic inflammatory responses and airway inflammation in allergic inflammatory diseases, including asthma.

For better understanding the molecular basis of the mechanism underlying oligosaccharides functions, it is quite desirable to gain detail information on their conformational dynamics in solution. However, the conformational analysis of oligosaccharides is still a remaining challenge, because of their branched covalent structures and dynamic conformational multiplicities, which hinder conventional analytical methods. For conformational characterization of flexible oligosaccharides, their structures should not be dealt with as a single well-defined global free energy minimum but as an ensemble of low energy conformers. Hence, we have been developing a methodology for evaluating a dynamic ensemble of oligosaccharide conformations by employing paramagnetic-assisted NMR methods based on paramagnetic effects such as pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) in conjunction with molecular dynamics (MD) simulations [1,2]. We applied this approach to the conformational dynamics analyses of branched GM2 tetrasaccharide, βGalNAc-(1-4)-[αNeu5Ac-(2-3)]-βGal-(1-4)-βGlc, which share the common core structure of gangliosides forming an integral part of cellular membranes. We synthesized the GM2 tetrasaccharide covalently attached to a lanthanide chelating-tag (Fig. 1). Upon complexation with paramagnetic lanthanide ions, the tagged sugar exhibited NMR spectral changes due to PCSs. The observed PCS values were in excellent agreement with those back-calculated from the 3D ensemble models derived from the MD calculations. In addition, we have developed spin-labeling method of gangliosides for their NMR analyses in amphipathic environments. These methodologies open a new prospect for conformational analyses of dynamic structures of gangliosides toward decoding glycocodes from the 3D structural aspects.

β-O-linked N-acetylglucosamine(O-GlcNAc) is dynamic post-translational modification in nucleus and cytosol and it is regulated by O-GlcNAc transferase(OGT) and O-GlcNAcase. Many proteins, such as transcription factors, enzymes, cytoskeletal proteins, ribosomal proteins and chaperones have been identified to be modified with O-GlcNAc. O-GlcNAc modification modifies at their Ser/Thr residues and affects their functions. The hexosamine biosynthesis pathway takes about 5% of glucose in total glucose flux, and it’s final product is UDP-GlcNAc, which is source of O-GlcNAc modification. O-GlcNAc modification is reported to involve in skeletal muscle metabolism and development process. We studied whether O-GlcNAc modification is involved in differentiation in myoblast C2C12 cell. We observed that total O-GlcNAc modification level was dynamically changed during myogenesis. After treatment NButGT, we observed that myogenin expression level decreased. Using RT PCR, we found that mRNA level of myogenin decrease after treatment NButGT. And we found that the promoter activity of myogenin decreases after NButGT using reporter gene assay. For finding the promoter region affected O -GlcNAc , we did reporter gene assay using shorter length promoter and found at least 169 base pair upstream region of myogenin is affected by O-GlcNAc. And using this region, we found that Mef2c protein can be important transcription factor regulated by O-GlcNAc using avidin-biotin complex DNA binding assay. We confirm that Mef2c is O-GlcNAcylated using immunoprecipitation and we found several O-GlcNAcylated sites on Mef2c using mass spectrometry. Based on the result, we made the Mef2c point mutants converted from Serine, Threonine of O-GlcNAcylated sites to Alanine. Additionally, we found O-GlcNAc modification could regulate DNA binding affinity and transcriptional complex formation.

Lipid rafts have been assumed to work as a platform where protein receptors, cholesterols and gangliosides (representatively GM3 and GM1) assemble and interact for efficient signal transduction. According to the observation of these raft molecules by single molecule tracking technique, raft components were frequently but very transiently recruited to the cluster of GPI-anchored proteins formed upon binding of extracellular signaling molecules [1]. However, behaviors and functions of gangliosides in living cell membranes have never been extensively investigated because of the lack of their fluorescent probes that behave as equivalents of native gangliosides. To address this issue, we intend to develope novel fluorescent GM3 and GM1 probes for single molecule tracking, in which glycan parts are site-specifically labeled with various fluorescent dyes and evaluate the functionality of the fluorescent gangliosides. We designed the replacement of the C9 hydroxyl groups of sialic acid of GM3 and GM1 with amino groups to introduce fluorescent dyes. The synthesis of glycan parts of GM3 and GM1 were successfully achieved by using a Neu-Gal unit having a trifluoroacetamide at the C9 position. Next, the glycan parts were glycosidated with the Glc-Cer acceptor developed by our research group [2], yielding the skeletons of GM3 and GM1, respectively, which was followed by the conversion of trifluoroacetamide groups into amino groups. Finally the amino GM3 and GM1 were conjugated with fluorescent dyes through amide linkages, producing the targeted fluorescent gangliosides GM3 and GM1. The synthesized gangliosides were subjected to biophysical evaluations; DRM analysis and single molecule observation of colocalization with raft molecules (GPI-anchored protein, CD59 and epidermal growth factor receptor). Results obtained in these evaluations demonstrated great influence of the loaded position and polarity of fluorescent dye on the raftphilicity of gangliosides. Furthermore, the Kd value of the fluorescent GM1 probe for cholera toxin B subunit (CTXB) was comparable to that of native GM1, indicating that dye did not interfere the binding to CTXB. These results strongly suggest that the fluorescent gangliosides can be used to identify behaviors and functions of gangliosides in raft domains.

Chitosan is one of the most widely used structural polymers for biomedical applications because it has many favorable properties. However, one of the most critical drawbacks regarding the use of chitosan as a biomedical material is its poor mechanical properties in wet conditions. Here, we designed a method to improve the mechanical properties of chitosan in wet conditions and minimized the swelling behavior of chitosan film due to water adsorption by mimicking the sclerotization of insect cuticles and squid beaks, i.e., catechol-meditated cross-linking. The biomimetic chitosan composite film was prepared by mixing chitosan with L-3,4-dihydroxyphenylalanine (DOPA) as a catecholic cross-linker and sodium periodate as an oxidant. The catechol-meditated cross-linking provided a 7-fold enhancement in the stiffness in wet conditions compared to pure chitosan films and reduced the swelling behavior of the chitosan film. This strategy expands the possible applications for the use of chitosan composites as load-bearing biomaterials.

Glycosylation in the nervous system is thought to play a particularly important and dynamic role in both vertebrates and invertebrates. Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc:polypeptideα-N-acetylgalactosaminyltransferases (ppGalNAc-Ts, EC 2.4.1.41) which contains 20 members. ppGalNAc-T13 is one of the essential isoform, and has been found highly and restrictively expressed in the brain and neurons [1]. In this study, we found that the mRNA and protein expression of ppGalNAc-T13 was dramatically increasing during the all-trans retinoic acid (ATRA) induced neural differentiation of embryonic carcinoma P19 cells, mouse brain development and primary neuronal cultures. ppGalNAc-T13 was localized in axons of the neurons derived from P19 cells and the O-glycosylation level was simultaneously increased during the ATRA induced neural differentiation. Furthermore, up-regulation of ppGalNAc-T13 induced the formation of lamellipodia and a higher O-glycosylation level. These results imply that the high expression of ppGalNAc-T13 in neurons may play a regulatory role in the neural differentiation. Study to explore the roles of ppGalNAc-T13 in regulating neural differentiation is under way.

Fukutin is a putative glycosyltransferase protein that is involved in the glycosylation of α-dystroglycan, and highly expressed in skeletal muscle, heart and brain. Therefore, the protein could play an important role in muscle and brain development. Mutations in this gene have been reported as a cause of Fukuyama congenital muscular dystrophy (FCMD), Walker-Warburg syndrome (WWS), limb-girdle muscular dystrophy type 2M (LGMD2M), and dilated cardiomyopathy type 1X (CMD1X). However, the biological functions of this protein and molecular mechanisms of these genetic diseases remain elusive. To serve disease models for functional study, we disrupted the fukutin gene using a transcription activator like effector nuclease (TALEN) that is an artificial restriction enzyme composed of DNA binding domain and Fok I nuclease. TALENs induce site-specific DNA double-strand breaks (DSBs) in the genome, whose repair via error-pron non-homologous end-joining gives rise to targeted mutations. We used a surrogate reporter to enrich cells that contain TALEN-induced mutations in HeLa cells and isolated gene knockout clones. We propose that engineered TALENs are powerful tools for functional studies of glycosylation in cell lines.

Glycan play key roles in protein folding, cell-cell recognition, cancer metastasis, and the immune system. In cell, glycosylation comprise that many kinds of glycan structures associated with a specific glycoprotein have different functions. So, the biological meaning of glycosylation has made them a prime target for discovering biomarker in diseases. Identifying the numerous glycan, we have used new method to assess the diversity of the N-linked oligosaccharides without derivatization has been developed using on-line nano-liquid chromatography (nanoLC) and high resolution time-of-flight mass spectrometry. In three times MS/MS results, chips offered good sensitivity and reproducibility in separating a intact protein. In tandem mass spectra, glycan fragment ions were identified as N-glycan sequences for mining glycoproteome. As a result, we have identified several N-glycan structures from monoclonal antibody.

Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control (QC) system, which discriminates and excludes misfolded malfunctional glycoproteins from correctly folded one. In the QC system, UDP-glucose:glycoprotein glucosyltransferase (UGGT) recognizes misfolded glycoprotein bearing high-mannose type N-glycan and glucosylate it so that the misfolded proteins can interact with molecular chaperones calnexin/calreticulin to attain correctly folded structure. As chemical tools to study glycoprotein quality control system at molecular level, we systematically synthesized misfolded homogeneous glycoproteins bearing high-mannose type oligosaccharide. Interleukin 8 (IL-8) is a nonglycosylated protein consisted of 72 amino acid residues and two disulfide bonds between Cys7–Cys34 and Cys9–Cys50. As a model, we incorporated high-mannose type (Man9GlcNAc2) oligosaccharide at the Asn36. The full-length glycosylpolypeptide chain was successfully synthesized by native chemical ligation between N-terminal 33-amino acid peptide-thioester and C-terminal 39-amino acid glycopeptide, which was prepared with Fmoc-Asn derivative having high-mannose oligosaccharide on the side chain.Extensive folding experiments of chemically synthesized homogeneous IL-8 glycopeptide yielded correctly folded glycoprotein with native disulfide bond patterns as well as misfolded glycoproteins with non-native disulfide bond patterns and a disulfide bond-linked misfolded homodimer. Other misfolded glycoprotein models with one and no disulfide bond and glycopeptides consisted of C-terminal 39-amino acid residues were also prepared. Transfer of glucose residue to these homogeneous glycoprotein analogues by UGGT was analyzed by using LC-MS. This assay proved that the critical endoplasmic reticulum folding sensor enzyme, UGGT recognizes misfolded glycosyl-IL-8s with different preferences. The most favored substrate was a homodimer which exhibits molten globule-like hydrophobic nature, and the least favored substrate was a correctly folded glycosyl-IL-8. Glycoproteins and glycopeptides having non-native disulfide bonds were also favored substrates.

The incidence of cholangiocarcinoma (CCA), a bile duct cancer, is high in the northeastern part of Thailand. At present, there is no known marker that can be used as an early marker for CCA. The surveillance of people at risk of CCA is the effective strategy to detect patients with early CCA, the stage at which surgery for completely cure can be offered. There is accumulated evidence indicated that people with chronic inflammation of bile duct epithelia and peribiliary fibrosis have high risk of CCA. Recently, a high resolution ultrasonography of peri-portal fibrosis was shown to be the acceptable approach to identify people at high risk or early CCA. However, the ultrasonography has to be conducted and determined by an experience radiologist and the operation cannot be offered for people in rural area. In this study, we aimed to find a suitable serum marker that can distinguish people who has benign bilirary diseases (BBD) or CCA from healthy subjects, liver fluke infected persons and patients with other gastro-intestinal cancers. Serum was electrophoresesed in a 12% SDS-polyacrylamide gel electrophoresis and ConA-binding glycoproteins were determined using lectin-biotin/avidin-peroxidase system. ConA blotting of serum from 29 CCA and 23 benign biliary diseases were compared with those from healthy persons (n = 20), active liver fluke infected people (n =10), patients with hepatoma (n =15) and other gastro-intestinal cancers (n =20). A high molecular weight of ConA binding glycoprotein was frequently detected in 85% of serum from CCA and BBD comparing to 24.6% of all serum from the control group. This marker can identify person who had CCA or BBD with a 81.6% sensitivity, 75.4% specificity, 73.3% and 85.9% positive and negative predictive values, respectively. The value of this ConA-specific marker in differentiating people who are at risk of CCA should be re-investigated in a larger cohort including people from the villages that are in the endemic area of liver fluke infection and patients with various pathological conditions.

Cholangiocarcinoma (CC) is a lethal malignancy which exhibits asymptomatic growth infiltrating the surrounding structures, and thus,CC is usually detected at an advanced stage. The mainstay of treatment for CC is complete resection with negative surgical margins. Therefore, its diagnosis at a relatively early stage is demanded for performing the surgical resection. Since the definitive CC diagnosis relies on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. In the previous meeting, we reported that Wisteria floribunda agglutinin (WFA) is the best probe lectin which reliably distinguishes between CC and normal bile duct epithelia in tissue sections. Moreover, L1 cell adhesion molecule (L1CAM), CA125, and maspin were assigned as the reliable CC marker candidates by WFA-assisted glycoproteomics and immunohistochemistry. In this meeting, we will introducethe verification and validation process in one of the above candidates, L1CAM. Since the serum concentration of L1CAM was low as described in other reports (< 5 ng/mL), we firstly constructed a highly-sensitive detection system to confirm the existence of L1CAM in both bile and serum of CC patients with immunoprecipitation and western blotting. We then performed highly-sensitive glycan profiling with antibody-assisted lectin microarray (limit of detection: 25 pg) and confirmed WFA-positivity of biliary L1CAM from the CC patients. The subsequent validation study using bile samples from CC patients (n = 29) and patients with benign bile duct diseases (n = 29) showed that WFA-positive L1CAM distinguished CC from the benigndiseases with good specificity (sensitivity = 0.66, specificity = 0.93, overall accuracy = 0.79, area under the receiver operating curve [AUC] = 0.82). The combined use of the L1CAM assay with the highly-sensitive assay detecting WFA-positive sialylated mucin 1 (WFA-sialyl MUC1), a reliable CC marker (Matsuda A., et al., Hepatology, 2010), sufficiently improved the diagnostic accuracy of CC (overall accuracy = 0.84, AUC = 0.93). This combination assay using WFA–L1CAM and WFA–sialyl MUC1 will possibly be a useful serological test with enhanced reliability.

Fucosyltransferases (FucTs) usually catalyze the final step of glycosylation and are critical to many biological processes. High levels of specific FucT activities are often associated with various cancers. Here we report the development of a chemoenzymatic method for synthesizing a library of GDP-L-fucose derivatives, followed by in situ screening for inhibitory activity against bacterial and human -1,3-FucTs (4). Several compounds incorporating appropriate hydrophobic moieties were identified from the initial screening. These were individually synthesized, purified and characterized in detail for their inhibition kinetics. Compound 5 had a Ki of 29 nM for human FucT-VI, and is 269 and 11 times more selective than for Helicobacter pylori FucT (Ki = 7.8 □ M) and for human FucT-V (Ki = 0.31 □ M).

Glycosphingolipids including gangliosides play important regulatory roles in cell prolifera-tion and differentiation. UDP-glucose:ceramide glucosyltransferase(Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs(shRNAs) to evaluate the roles of glycosphingolipids in proliferation and gangliosdies differentiation of PK15. HPTLC/immunofluorescence analyses of shRNA transfected PK15 revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM1 and GD1b. Furthermore, MTT and western blot/immunofluorescence analyses demonstreated that inhibition of the Ugcg expression in PK15 resulted in decrease of cell proliferation. In addition to we showed that inflammatory respon-ses in PK15 and gangliosides knock-down PK15 cells stimulated with Escherichia coli lipopolysaccharide(LPS). The gangliosides GM3, GM2, GD3, GD1a and GD1b were detected in normal PK15 cell. However almost gangliosides undetected in gangliosides knock-down PK15 cell. In addition we found that the expression levels of TNF-α, indu-cible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and we measured the production of NO. The expression of inflammatory proteins, TNF- α, iNOS and COX-2 the elevated concentration in gangliosides knock-down PK15 cell. These results suggest that gangliosides interact with components of the proinflammatory response pathway and might, therefore, be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

Glycosphingolipids including gangliosides play important regulatory roles in cell prolifera-tion and differentiation. UDP-glucose:ceramide glucosyltransferase(Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs(shRNAs) to evaluate the roles of glycosphingolipids in proliferation and gangliosdies differentiation of PK15. HPTLC/immunofluorescence analyses of shRNA transfected PK15 revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM1 and GD1b. Furthermore, MTT and western blot/immunofluorescence analyses demonstreated that inhibition of the Ugcg expression in PK15 resulted in decrease of cell proliferation. In addition to we showed that inflammatory respon-ses in PK15 and gangliosides knock-down PK15 cells stimulated with Escherichia coli lipopolysaccharide(LPS). The gangliosides GM3, GM2, GD3, GD1a and GD1b were detected in normal PK15 cell. However almost gangliosides undetected in gangliosides knock-down PK15 cell. In addition we found that the expression levels of TNF-α, indu-cible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX -2) and we measured the production of NO. The expression of inflammatory proteins, TNF- α, iNOS and COX-2 the elevated concentration in gangliosides knock-down PK15 cell. These results suggest that gangliosides interact with components of the proinflammatory response pathway and might, therefore, be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

Norovirus (NoV) is a major causative agent of nonbacterial acute, epidemic gastroenteritis, and often widespread in winter. Although most NoV infections are cured without serious symptoms, it could cause severe symptoms in elderlies and young children. The initial infection event is adhesion of NoV to glycans expressed in the epidermal cells of the small intestine. We have identified several NoV strains that show affinity to Le

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) remains a major global health problem. Human ficolin2 (L-ficolin/P35) is a recently identified lectin complement pathway activator present in normal plasma associated with infectious diseases, however, little is known about the roles and mechanisms of ficolin2 during M.tb infection.We describe in this study for the first time L-ficolin expression levels in tuberculosis (TB) patients. We found that serum levels of ficolin2 of 107 pulmonary TB patients were much lower compared with 107 healthy controls, and recurrence TB patients have even lower levels than onset TB patients. In vitro analysis showed that Ficolin2 bound to virulent M.tb H37Rv strain much stronger than to non-virulent M. bovis BCG and M. smegmatis. Ficolin2/ficolin A bound to the H37Rv surface glycolipid portion and blocked H37Rv infection to human lung A549 cells. We further determined that the extrogenous ficolin2 had remarkable protecting effects against virulent H37Rv strain infections in C57BL/6J mice. FicolinA (ficolin2 like molecule) knockout mice had greatly increased susceptibility to the H37Rv infection. Ficolin2 activated macrophages via phosphorylation of JNK and stimulated IFN-□, IL-17, TNF-□ and nitric oxide (NO) secretions. Ficolin2 also stimulated IFN-□ production of CD8+T cells, but not CD4+T cells. The opsono-phagocytosis was promoted by ficolin2 as well. Our data demonstrate that ficolin2 can defend against virulent M.tb H37Rv infection both in vitro and in vivo mainly via activating macrophage proinflammatory cytokines IFN-□ and NO production and opsonophagocytosis, and its insufficiency is associated with more susceptible infections in human.

Chitooligosaccharide (COS) is derived from chitosan and has low molecular weight. Since chitosan is only soluble in acidic water, COS has advantages such as excellent solubility in deionized water, ease of chemical modification, altered functionality and enhanced bioactivity. Especially, COS is regarded to have enhanced anti-carcinogenecity, anti-microbial activity, and, especially, enhanced mucosal delivery of bioactive agents. In this study, we prepared photosensitizer-incorporated COS nanoparticles through ion complex formation between COS and photosensitizer, Ce6. Since COS has cationic properties, ion complexes can be formed with anionic drug, DNA and photosensitizer. Photosentizer-incorporated COS (ChitoCe6) has small particle sizes less than 100 nm at TEM observation and particle size measurement. They enhances Ce6 delivery to HCT116 human colon carcinoma cells rather than Ce6 itself. Furthermore, Ce6-incorporated COS nanoparticles generated ROS higher than Ce6 itself in spite of anti-oxidant properties of COS. Ce6- incorporated COS nanoparticles enhances phototoxicity against tumor cells and induced apoptosis/necrosis of tumor cells. We suggest that COS nanoparticles is simple and promising vehicles for photosensitizer delivery.

Cell-surface glycans play key roles in mediating various molecular recognition events; aberrant glycosylation is implicated in disease progression. Therefore, probing the dynamic changes of glycan biosynthesis and structures is of great importance for augmenting our understanding of glycobiology and improving disease diagnosis and therapeutics. The metabolic glycan labeling was an appealing approach to incorporate specially designed carbohydrate analogs into the glycans, which enables the detection and imaging of the glycans in cells and living animals. However, one major bottleneck of this method is cell- type selectivity. Herein, we present the cell-specific metabolic glycan labeling using ligand-targeted liposomes to deliver unnatural sugars to target cells in a cell-surface receptor dependent manner. In this strategy, sugar analogs are encapsulated in ligand-targeted liposomes. The ligands bind to specific cell-surface receptors that are only expressed or up-regulated in target cells, which mediate the intracellular delivery of azidosugars via endocytosis. The delivered azidosugars are metabolically incorporated into cell-surface glycans and detected or imaged using a bioorthogonal reaction. The application of this strategy includes the facile introduction of myriad alternative ligands, as well as the cell-specific or tissue-specific imaging and detection of glycosylation in vivo.

Dendritic cells (DCs) are professional antigen-presenting cells that serve as messengers between innate and adaptive immunity. Due to their unique roles, DCs are under active investigations as the target for antigen delivery in vaccination against human immunodeficiency virus (HIV), cancer and autoimmune diseases. However, there’s yet no perfect strategy for a cost-effective, high-selective and time-saving detection and isolation of DCs from a complex cell population. Adhesion molecules such as DC-specific intracellular adhesion molecule-3 grabbing non-integrin (DC- SIGN) on the surface of DCs play a critical role in establishing contact between the activated DCs and the naïve T cells through interactions with intercellular adhesion molecule-3 on T cells. DC-SIGN is a member of the C-type lectin receptors and can recognize antigens bearing mannose-rich or fucosylated glycans, as well as Lewis X (LeX). Here, we present the fabrication of magnetic nanoparticles coated with multivalent LeX glycans using the Cu (I)-catalyzed azide-alkyne cycloaddition. The resulting nanoparticles are selective and biocompatible, serving as a highly efficient tool for DC capture detection and enrichment.

The capsular polysaccharide (CPS) of pyogenic liver abscessKlebsiella pneumoniaeconsists of repeating units of the trisaccharide (→3)-□-D-Glc-(1→4)-[2,3-(S)-pyruvate]-□-D-GlcA-(1→4)-□-L-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C2-OH or C3-OH of fucose. The present study investigates how CPS activates human monocyte-derived dendritic cells (DCs). Our experimental results show that CPS activates DCs by (1) increasing the expression of CD11c, CD40, CD80, CD83, CD86, and MHC-II (2) increasing the production of TNF-□, IL-1, IL-6, and IL-12p70 (3) increasing DC-elicited allogeneic T-cell proliferation, and (4) increasing the DC-driven Th1 response. In addition, CPS activates DCs through TLR4 and the pyruvation and the acetylation of CPS are important for its cytokine induction activity. Further, our results show that CPS activates TNF-□ and IL-6 secretion through JNK1/2, p38, NF-B,- PKC and ROS pathways in DCs.

The oligosaccharides (OS) in milk are known to play essential roles in the development of the infant. However, the carbohydrate composition of milk from different animal sources as well as human milk varies. Formulated infant milkpowder made from cow milk lacks certain bioactive carbohydrate structures and shows limited amounts of N-acetylneuraminic acid and fucose, compared to human milk. Our strategy to discover the difference of bioactive carbohydrate structures in milk and the potential for the development of novel functional dairy products is based on the analysis of glycosylation profiles of free and protein bound OS with a unique chromatography-based methodology. This approach is currently also adopted for a broader range of foodstuff, which shows carbohydrate related biological functions. Additionally, the level of nutritional components (e.g. lipids) has been found to influence many biological events. The effect of the nutritional level on glycan structures is also of great interest and to monitor the structural changes, and to reveal the underlying biochemical mechanisms is another target of our research.

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.