Earticle

Polymeric nanoparticles based on natural polymer have been investigated due to low cytotoxicity, biocompatibility, and non-immunogenicity. In particular, chitosan has many advantages in this regard, but has to overcome the obstacles such as low bioavailability and gene expression level. For this object, ligand molecules binding with targeting receptors which are overexpressed in pathogen or tumor cells provides immense opportunities for the concentrative delivery of bioactive cargos into targeting cells and the minimum toxicity in normal cells. Therefore the conjugation of a pathogenic cell-specific ligand molecules to polymeric nanoparticles is an important strategy in the design of drug delivery carrier. The present studies describe a notable use of antifungal peptide as targeting ligand toward fungal pathogen and the applications of new ligands in tumor gene therapy.

Water is a common medium and looks simple, yet its interaction mechanisms with molecules and materials are very complicated. Therefore, intermolecular interactions of load-bearing polysaccharides with water determine the structure and properties of polysaccharides in aqueous systems. In this presentation, the recent advances in understanding metal-ligand interactions, cation-pi interactions, hydrogen bonding of carbohydrates will be presented. We directly have measured these interactions using a surface forces apparatus (SFA) in aqueous solutions and some insights has been discovered. Some case studies which apply the insights from the measured interactions to translate to the design of advanced materials, especially nano-cellulose/chitin based materials, will be also introduced.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder. The main cause of HD is assumed to be the mutation in the gene coding for huntingtin (Htt), and the mutated Htt contains an abnormally expanded polyglutamine stretch (polyQ>35), which tends to aggregate into insoluble amyloid-like fibrils. Many attempts were made to inhibit the polyglutamine-induced protein aggregation, and the non-reducing disaccharide trehalose was reported to alleviate the disease symptoms of HD transgenic mice. However, trehalose is known to be rapidly hydrolysed to glucose by trehalase enzyme present in the small intestine, and its uptake into tissues is regarded to be very low. In addition, there is no evidence that trehalose can cross the blood-brain barrier (BBB). Here we show that BBB-permeable trehalose derivatives could be prepared by applying the delivery methods which are previously developed before, and the derivatives were found to efficiently prevent the aggregation of polyQ in the transfected HEK293 cells. Furthermore, the derivative (TD-G6), when given ad libitum to a transgenic mouse model of HD (Tg R6/2), was found to significantly prolong lifespan, improve motor functions and reduce the inclusion bodies in the mouse brain compared with the trehalose control.

Understanding of the molecular relationships in carbohydrate-protein interactions provides useful information on biological processes in living organisms and is also helpful for development of potent diagonostic systems. Synthesizing the specific complex glycans is important for analysis of interactions between glycans and their binding proteins. Particularly, on-chip enzymatic synthesis of complex glycans can be an efficient and useful tool because this can directly provide diverse glycans to use for analyzing carbohydrate-protein interactions on the chip. We prepared novel on-chip glycan synthesis platform in which glycan-conjugated single-stranded DNA was synthesized using DNA synthesizer and the synthesized carbohydrate-oligonucleotide conjugates (COCs) were immobilized onto the complementary single-stranded DNA-modified glass slide. Due to the property of DNA to be possible for reversible hybridization and denaturation under certain conditions, we effectively recovered the synthesized glycans from the chip, and optimized the synthesis conditions for target complex glycans. We anticipate that this novel on-chip complex glycan synthesis can be successfully used in a number of glycan-related studies including analysis of diverse carbohydrate-protein interactions and high throughput diagnosis.

Aberrant glycosylation in serum proteins are associated with many human diseases, including various types of cancer. In this study, we developed a comprehensive glycomic approach involving immunoaffinity purification, lectin blotting, and advanced MS techniques to discover potential biomarkers for gastrointestinal cancer, especially focus on colon and gastric cancer. Haptoglobin (Hp), a target glycoprotein that displays aberrant glycosylation during cancer development, was initially screened by blotting with various lectins. To discover novel aberrant glycans in colon and gastric cancer, we have analyzed glycosylation status of haptoglobin by lectin blotting. In particular, aleuria aurantia lectin (AAL, which is specific to fucosyl residues) reactivity with cancer Hp was higher than control groups. Moreover, by LC/MS analysis following PNGaseF treatment for purified Hp, several aberrant glycans were detected in Hp from sera of colon and gastric cancer patients. Increased levels of antennary branching and reduced levels of high-mannose type structures were observed in cancer patients. In addition, enhanced fucosylation at Asn 241 of the four potential N-glycosylation sites of Hp in sera of cancer patients was especially identified by site-specific analysis. Our findings indicate that glycome profiling of Hp will be a useful platform for gastrointestinal cancer biomarker discovery, rapidly identifying a number of potential diagnostic markers for colon and gastric cancer.

As a representative new medicine of Korea which has advanced to the global market, `Supect`, the no tolerance and no side-effect medicine for chronic myelogenous leukemia developed by Ilyang Pharmaceutical Co., Ltd. may be mentioned above all without hesitation. 'Supect' which was introduced as a representative role model for R&D and commercialization of innovative new medicine at the 2015 BIO International Convention may be called a success model for development of innovative medicine and new market which wisely overcame domestic and foreign entry barrier of licensing. Showing will to develop new medicine, Government has kept supporting and, in the process, basic research infrastructure has matured much in the industry's research institutes and schools. As technology is built and research ability of companies grows, the outcome of new medicine development will accelerate more and more. Survey of progression stages of new medicine development made by each research institution shows that externally division of roles between institutions involved in new medicine development is necessary for each R&D stage over the whole period and internally technology transfer or cooperative research is imperative between universities and companies or research institutes and companies. Any company cannot secure the overall infrastructure and experiences of alliance and cooperation required for development of globally competitive new medicine in a short term. National support system which can induce more efficient R&D and formation of incubative environment for developing innovative new medicine are necessary. Development of new medicine is progressed as a multidisciplinary R&D over the whole period. Such open innovation strategies that are freely able to operate space-time transcending real-time win-win cooperation with outside institutions possessing technologies are being established as core strategies for developing new medicine. To prevent bottleneck phenomenon occurring when investment on new medicine research is connected to new medicine development, the open innovation system for new medicine development and commercialization in which industry, academics, research community and government can coexist and pursue co-prosperity should be well operated.

Starch, one of the most important energy sources for human, has been used as main or minor ingredients of the varied food systems to the purified (starch itself) and unpurified (cereals, starchy vegetables, and their flours) forms. It also plays important roles in the structure formation, quality maintenance, and cost reduction of the processed foods. Nevertheless, native starch has been first modified in the chemical and physical fashions to resolve its intrinsic demerits: incomplete solubility, uncontrolled starch paste consistency, rapid tendency of its pastes toward gelling and retrogradation, high syneresis, its higher paste opacity, etc. Regarding the nutritional aspects of native starch, the consumers have recently recognized the higher intake of simple sugars (from sucrose and starch) as causing obesity, followed by metabolic disorder. The chemically cross-linked resistant starches, belonging to the category of dietary fibers, are known to be most effective to retard and/or restrict the intake of simple sugars from starchy foods into the body. However, an increased resistance of consumers to chemically-synthesized food additives results in the attempts to exclude and/or reduce the use of chemically-modified starch products for the processed foods in food industries. Thus, many studies have tried to find the optimal compositions of native starches and non-starch polysaccharides as ways of replacing their physical and nutritional functionalities. In this presentation, accordingly, the functions of non-starch polysaccharides will be introduced and discussed on the rheological and physical properties of starch pastes, the digestibility of starches, and the structure restoration and oil uptake reduction of the bakery foods, based on our recent researches.

For decades, quality of starch-based foods has been associated with the in vivo measured glycemic index or the in vitro digestion rate-based categories of rapidly digestible, slowly digestible, and resistant starch (RS). Glycemic index has been related to health-based endpoints mostly through correlative or observational studies with mechanisms proposed, but not well established. Here, we bring forth the concept of locational delivery of glucose from dietary starches to the distal small intestine to elicit an “ileal brake” effect. Both effects slow gastric emptying and, in turn, extend nutrient (i.e., energy) delivery to the body and may decrease appetite and promote weight management. Slowly digestible starches are currently a popular topic of research, though where they are digested and the released-glucose is delivered in the small intestine is not known. A proposal is to further study and establish this mechanism of appetite and food intake regulation so that starch-based ingredients and foods can be developed that promote the ileal brake mechanisms.

Endo-dextranases (EC 3.2.1.11), which hydrolyze α-1,6-glucosidic linkage of dextran at random, are classified into glycoside hydrolase (GH) family 49 and 66 based on the amino-acid sequence similarity. Cyclodextran glucanotransferase (CITase) is also classified into GH family 66. CITase is an interesting enzyme that catalyzes the conversion of dextran to cycloisomaltodextrins (CIs) by intramolecular transglycosylation. CI is of importance by preventing the dental caries since it strongly inhibits insoluble glucan formation by mutansucrase. Although endo-dextranase and CITase belong to GH family 66, their products from dextran are largely different in structure, such as cyclic- or linear-isomaltooligosaccharides, respectively. In this study, it was found that Paenibacillus sp. dextranase, a member of GH family 66, showed both of endo-dextranase activity and CITase activity, allowing us to classify the GH family 66 enzymes to three groups for the first time. These results may contribute to understand of the relationship between the structure and function of GH family 66 enzymes. Recently, several other Bacillus and Paenibacillus CI-producing bacterial strains were found and the larger CI molecules up to CI-17 were isolated from their culture supernatants. In order to produce CIs for commercial scale, a high dextran producing strain Leuconostoc sp. S-51 was obtained and the B. circulans T-3040 strain was mutated to produce CITase with about 110 times the activity of the wild type strain. All CIs well inhibit glucosyltransferase of mutans streptococci and especially CI-10 and larger molecules of CIs have strong inclusion-forming activity such as preservation of Victoria blue color or solubilization of isoflavones. There have been no problems arising from safety tests of CIs inratsand humans. The fact that CIs are present in brown sugar is indicative if their safety as natural oligosaccharides.

Lactulose (4-o-β-D-galactopyranosyl-D-fructose) is a non digestible disaccharide that is used medicinally as a treatment for portal systemic encephalopathy, chronic constipation, and hyperammonemia. In the food industry, the bifidus factor lactulose can be used as an ingredient that has greater sweetness and solubility than lactose. The biological synthesis of lactulose has been studied with β-galactosidases using lactose and fructose as substrate and co-substrate, which exhibits a low conversion yield. In this study, the enzymatic production of lactulose was attempted without the addition of fructose using a thermostable recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus as a new type of lactulose-producing enzyme. The conditions for maximum lactulose production from lactose, as a single substrate, by cellobiose-2-epimerase from C. saccharolyticus were determined to be pH 7.5, 80 C, 700 g/L lactose, and 150 U/mL of enzyme. Under the conditions, the enzyme produced the two bifidus factors lactulose at 408 g/L and epilactose at 107 g/L after 2 h. The yields of lactulose and epilactose from lactose and the productivities of lactulose and epilactose were 58%, 15%, 204 g/L/h, and 54 g/L/h, respectively. This is the first trial of enzymatic synthesis of lactulose using the single substrate lactose. These results will contribute to the industrial production of lactulose from lactose via an enzymatic process.

While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase (GalNAc-T14), is highly correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here we showed that GalNAc-T14 expression was closely associated with the invasive phenotype. Microarray and biochemical analyses revealed that HOXB9 , the expression of which was regulated in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the b-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of b-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and suppressed invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with recurrence-free survival and hazard risk, suggesting that targeting b-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC.

Galectin-3, a member of carbohydrate recognition protein, is often over-expressed in cancerous regions, compared to normal counterparts. Moreover, this altered galectin-3 expression correlates with the aggressiveness of the tumor and the acquisition of the metastatic phenotype, indicating that galectin-3 might modulate tumor progression and influence disease outcome. It is important to note that a change in the subcellular localization of galectin-3 occurs during the transition from normal cells to cancer cells. In this study, it deals with the underlying mechanism that galectin-3 enhances gastric cancer cell survival and motility. Therefore, we suggest that galectin-3 can serve as potential target molecule in the prevention and/or therapy of gastric cancer.

The encapsulated basidiomycetous Cryptococcus neoformans species complex is an opportunistic fungal pathogen causing fatal cryptococcal meningoencephalitis in immunocompromised populations, such as AIDS patients. In this presentation, we report the unique structure and biosynthesis pathway of N-/O-glycans and their physiological roles in C. neoformans. By analyzing oligosaccharide profiles combined with exoglycosidase treatment, C. neoformans was shown to have serotype-specific high-mannose type N-glycans with or without xylose residue. Moreover, comparative analysis of acidic N-glycan profiles from wild-type and och1Δ, mnn2Δ, and uxs1Δ mutant strains strongly indicated the presence of xylose-phosphates attached to mannose residues in the core form and outer chain of N-glycans (1). We further showed that the major C. neoformans O-glycans were short manno-oligosaccharides that were connected mostly by α1,2-linkages but connected by an α 1,6-linkage at the third mannose residue. C. neoformans KTR3 was shown to be responsible for the addition of the second α1,2 mannose residue to the major O-glycans lacking xylose. The ktr3Δ mutant strain displayed attenuated virulence in a mouse model of cryptococcosis, suggesting that the extended structure of O-glycans is required for full pathogenicity of C. neoformans. Intriguingly, C. neoformans HOC1 and HOC3, homologs of the Saccharomyces cerevisiae OCH1 family genes, were shown to transfer the third mannose residue, via an α1,6 linkage, to minor O-glycans containing xylose and to major O-glycans without xylose, respectively, indicating two independent O-glycan biosynthesis pathways involving different sets of processing enzymes in C. neoformans (2). The glycosylation-defective mutant strains developed in our study are currently used for systematic investigation on how structural alterations of N-/O-glycans affect the intensity of virulence and the extent of host immunological interactions in C. neoformans.

O-linked N-acetylglucosamine (O-GlcNAc) modification is unique glycosylations and it can occur exclusively in nucleus and cytoplasm. O-GlcNAc is attached onto a serine or threonine residue of a protein by O-GlcNAc transferase (OGT) and detached by O-GlcNAcase (OGA) under dynamic regulations by a variety of conditions. O-GlcNAcylation is associated with several clinical situations including cancers and inflammatory diseases. The effect of O-GlcNAcylation of subunits of nuclear factor-ĸB on its transcriptional activity has been reported to date. Interestingly, O-GlcNAc modification of NF-ĸB exhibited reciprocal results on its activity depending on different cell types: increased O-GlcNAcylation elevated the activity of NF-ĸB in pancreatic cancer cells, while aortic smooth muscle cells showed negative correlation between O-GlcNAcylation and NF-ĸB activity In Rheumatoid Arthirtis (RA), NF-ĸB is the novel regulator of pro-inflammatory cytokines mediating synovitis, long-term cartilage degradation and bone erosion. We hypothesize that the alteration in the activity of NF-ĸB resulting from its O-GlcNAcylation might affect the severity of RA. There was no report on the effect of O-GlcNAcylation of NF-ĸB on its activity in RA. We used in vitro synoviocytes and in vivo in mice with collagen induced arthritis (CIA) and investigated the inflammatory potential of O-GlcNAcylation of P65, one of subunits of NF-ĸB, in the pathogenesis of RA.

Human α-amylase (EC 3.2.1.1) genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B). Various studies have been conducted on the structure, mutagenesis and kinetic analysis for chemically synthesized substrate of AMY2A. However, characterization of AMY2B has received less attention. In this study, human pancreatic α-amylase (AMY2B) gene was cloned and expressed in Pichia pastoris using the methanol-induced alcohol oxidase (AOX1) promoter. After 48 hours for incubation, the activity of recombinant AMY2B in the culture supernatant reached the peak and purified by Ni-NTA affinity chromatography. The optimum temperature and pH of purified enzymes were similar to those of AMY2A. Furthermore that is consistent with the characteristics of AMY2A. Kinetic analysis for various native starches revealed that chloride ion was required for maximum activity of recombinant AMY2B and increase in kcat values in the presence of NaCl was mostly contributed to the maximum activity.

Lytic polysaccharide monooxygenases (LPMOs) are copper ion containing enzymes to cleave crystalline cellulose or chitin through an oxidative mechanism. Despite the importance of LPMOs for biomass decomposition, basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Generally, LPMO activity has been determined by HPLC analysis of the product by simultaneous action of a LPMO and one of a cellulase and a chitinase depending on LPMO’s substrate specificity. Recently a spectrophotometric method has been developed using the side reaction of LPMOs to produce H2O2 in the absence of polysaccharide substrates. However, both assay methods do not give the information about polysaccharide lysis by only LPMOs. In this study, we developed an assay method for LPMOs based on the consumption of ascorbic acid that was used as an external electron donor for LPMOs. The assay method allowed determining the concentration-dependent activity of a chitin active LPMO from Bacillus atrophaeus 1942. This assay method would be a useful tool for detail characterization of LPMOs.

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

The predominant N-glycan in plants is paucimannosidic N-glycan with core β1,2-xylose and α 1,3-fucose residues (PNGXF). Here, We hypothesized that additions of the core β1,2-xylose, α 1,3-fucose, and 6-arm β1,2-GlcNAc residues to the common acceptor (GlcNAcMan3GlcNAc2) are relatively determined by the different activity and/or substrate occupancy of the enzymes of the corresponding genes in plants. As a result, we describe a mechanism in Arabidopsis thaliana that effectively form the largest N-glycan in plants. Genetic and biochemical evidence suggest that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis thaliana. Moreover, analysis of gnt2 mutant and 35S: GnTII transgenic plants indicates that the additions of the core β1,2-xylose and α1,3-fucose residues to the common acceptor (GlcNAcMan3GlcNAc2) are partially inhibited by the addition of the 6-arm β1,2-GlcNAc residue. Our findings show that plants control the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the general N-glycans with Man3XylFucGlcNAc2 and GlcNAc2Man3XylFucGlcNAc2 structures by hexosaminidase activity and the regulated sharing of the common acceptor GlcNAcMan3GlcNAc2.

Gaucher’s disease is a lysosomal storage disorder by mutations in the gene encoding glucocerebrosidase (GC). Gaucher’s disease is currently treated by enzyme replacement therapy using recombinant GC (CerezymeⓇ) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells have not terminate in mannose residues, which are essential for the biological absorption of GC via macrophage mannose receptors in human patient with Gaucher’s disease, glycan modification in vitro is required in order to expose the mannose residues on the glycans of CerezymeⓇ. Notably, the recombinant human GC (rhGC) expressed in the plant cells unaffectedly contains terminal mannose residues on its complex glycan. Hence, the plant-produced rhGC does not require exposure of mannose residues in vitro, which is a requirement for the production of CerezymeⓇ. However, The presence of β1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans are potentially allergic in mammals. In this study, to remove the plant specific sugar residues and customize the N-glycan structure in plant, we isolated mutants of the corresponding plant specific glycosyltransferase genes and used for multiple-mutants construction. The resulting mutants was transformed by a human α 1,6-fucosyltransferase gene to accomplish customized N-glycosylation in plant. In consequence, the production of a rhGC in a humanized N-glycosylation plant is described.

Multiple earlier studies have shown that interactions between glycans and carbohydrate binding proteins (lectins) displayed on cells surfaces are involved in many important physiological and pathological processes. For example, cell surface lectins in the immune system recognize glycans expressed on the exterior of pathogens and these interactions lead to stimulation of immune responses to pathogens. Mouse SIGN-R1 (SIGN-related 1) is a homolog of human DC-SIGN that is expressed largely on macrophages. This lectin recognizes mannose-rich or fucosylated glycans in a Ca2+- dependent manner. When SIGN-R1 interacts with glycans on host cells, bacteria or viruses, glycan antigens are internalized into cells via lectin-mediated endocytosis to induce immune activation. As a consequence, the lectin hasbecome fascinating markers for recognition and targeting of specific cells to regulate cellular functions and for carbohydrate-based drug discovery. To investigate glycan binding properties of cell surface lectins, we have utilized carbohydrate microarrays which allow high-throughput screening of cell-glycan interactions. Glycan microarrays used in this study were constructed by immobilizing a variety of unmodified carbohydrates onto hydrazide-coated glass slides. SIGN-R1 expressing cells pretreated with Hoechst 33342 were loaded on the glycan microarrays to evaluate the binding properties of the lectin. In addition, purified SIGN-R1 was also applied tothe microarrays for the purpose of comparison. The present study demonstrates the potential of glycan microarrays for profiling of binding specificities of cell surface lectins.

Through binding to free glycans or glycoconjugates, animal cell surface lectins are involved in a wide range of biological processes. It has been known that most animal lectins, including selectins, siglecs (sialic acid-binding immunoglobulin-type lectins), mannose receptors, mannose-6-phosphate receptors, asialoglycoprotein receptors and C-type lectins, act as signal transducers after binding to glycans although intracellular signaling events induced by lectins depend on the nature of glycans, cell type and/or species. In particular, cell-surface lectins in the immune system bind to glycans displayed on the exterior of pathogens and this recognition event stimulates the immune response. For example, mouse SIGN-R1 (SIGN-related 1), a homolog of human DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin), is highly expressed on macrophages in the splenic marginal zone and the medullar lymph nodes. This lectin binds predominantly to mannose-rich or fucosylated glycans in a Ca2+-dependent manner. Once glycans on bacterial cells or viruses interact with SIGN-R1, glycan antigens elicit SIGN-R1 mediated immune activation. To investigate immune activation by lectin mediated endocytosis, we conducted a traditional cell assay to probe six glycan-bovine serum albumin (BSA) conjugates (BSA-Man, BSA-Fuc, BSA-GlcNAc, BSA-Gal, BSA-Mana1,2Man and BSA-Lex) and S. cerevisiae mannan for their abilities to stimulate ROS generation. The results show that incubation with mannan and all of the glycoconjugates except BSA-Gal leads to increases in the intensities of fluorescence signals from cells compared to the untreated control.

Recognition of glycans, which are present in the form of glycoconjugates in cells, by proteins is crucial for a variety of physiological and pathological processes. Accordingly, understanding the role of glycan-mediated binding events and blocking glycan-protein interactions associated with diseases are of great importance for both basic biological research and biomedical applications. To investigate binding of glycans by lectins on the mammalian cell surface,we prepared Lewis antigen conjugated fluorescent magnetic nanoparticles and applied them to detect human DC-SIGN and mouse SIGN-R1 on mammalian cell-surfaces. To our knowledge, glycan-modified fluorescent magnetic nanoparticles have not been used to study glycan-mediated recognition events. When DCEK, DCEK-DC-SIGN, and DCEK-SIGN-R1 cells were incubated with glyconanoparticles, Lea and Leb but not H1 conjugated nanoparticles bind to DC-SIGN and SIGN-R1 expressing cells. Subsequently, glyconanoparticles were internalized by the DC-SIGN and SIGN-R1-expressing cells. However, these events were greatly suppressed by pre-treatment of cells with mannan which is a tight ligand for these lectins, indicating that glyconanoparticles mostly enter cells via lectin-mediated endocytosis. Finally, we found that glyconanoparticles lead to induction of immune responses by examining the production of reactive oxygen species. This study will provide opportunities for developing finely tuned multifunctional nanoparticle-based drug and diagnostic nanoplatforms.

Helicobacter pylori is a Gram-negative, microaerophilic bacterium found in the stomach and causes chronic gastritis and gastric ulcers. It is also associated with the development of duodenal ulcers and stomach cancer. Therefore, sensitive detection of H. pylori is crucial for diagnosis and treatment of H. pylori associated diseases. It has been known that H. pylori expresses a sialic acid-binding adhesin (SabA) and a Leb-binding adhesin (BabA) that adhere, through glycan-adhesin interactions, to the human gastric mucosa for infection. To detect H. pyroli expressing BabA, we prepared fluorescent magnetic glyconanoparticles by conjugating aminoethylated Lea, Leband H1 oligosaccharides to carboxy- containing fluorescent magnetic nanoparticles. The successful preparation of glyconanoparticles was examined by zeta potential and transmission electron microscopy (TEM). When H. pyroli J99 strain which expresses BabA was incubated with three kinds of glyconanoparticles, fluorescence microscopy analysis showed that Leb and H1 conjugated but not Lea conjugated nanoparticles bound to this strain. However, these glyconanoparticles did not recognize H. pyroli strains lacking BabA. Finally, they were employed to enrich BabA expressing H. pyroli by using a magnet. It is expected that fluorescent magnetic glyconanoparticleswill be powerful tools to sensitively detect pathogens including H. pylori.

Rat intestinal mucosal α-glucosidases could be a mammalian enzyme source to be used in in vitro carbohydrate digestion kinetic studies, and to further discern effects of substrate and inhibitor effects on the individual four enzymes – maltase, glucoamylase, sucrase, isomaltase. The commercialized rat intestinal powder enzyme comes with α-amylase contamination, and therefore needs to be purified for in vitro starch digestion. The objective of this study was to separate α-glucosidase from the crude rat intestinal powder by size-exclusion chromatography using a media with fractionation in the range of Mw 1 × 103 to Mw 1 × 105. Elution times of the α-glucosidases and α-amylase was found from 26.5 to 43.0 min ( expected molecular weight, 48-90 kDa, excepting a minor peak at 125 kDa) and from 43.0 to 63.0 min (expected molecular weight, 51–54 kDa), respectively. Good separation was achieved with specific enzyme activities of α-glucosidase (fraction range from 26.5 min to 43.0 min) of 44.7 (unit/mg protein) and negligible α-amylase activity (1.0 unit/mg protein). The results demonstrated that purified α-glucosidases from rat intestinal power with high α -glucosidase activity can be efficiently achieved for in vitro carbohydrate hydrolysis test.

Uridine diphosphate-N-acetyl glucosamine, generated from glucose via the hexosamine biosynthetic pathway, is a substrate for O -linked-N-acetylglucosamine (O -GlcNAc). Addition and removal of O -linked-N-acetylglucosamine to Ser/Thr residues is a dynamic cycle and is involved in the regulation of nuclear and cytoplasmic proteins. Nucleocytoplasmic O -GlcNActransferase (OGT) attaches a single GlcNAc onto hydroxyl groups of serine and threonine residues. Although the cellular localization of OGT is important to regulate a variety of cellular processes, the molecular mechanism regulating the nuclear localization of OGT is unclear. Here, we characterized the NLS motif in OGT and this motif is required for the nuclear import of non-diffusible β -galactosidase. OGT bound the importin α5 protein, and this association was abolished when the NLS motif of ncOGT was mutated or deleted. We also revealed that O -GlcNAcylation on tetratricopeptide repeats (TPR) plays an important role in nuclear localization of OGT. Our finding suggests the mechanism behind how OGT can be localized in the nucleus and cytosol simultaneously.

Multiple earlier studies have shown that interactions between glycans and carbohydrate binding proteins (lectins) displayed on cells surfaces are involved in many important physiological and pathological processes. For example, cell surface lectins in the immune system recognize glycans expressed on the exterior of pathogens and these interactions lead to stimulation of immune responses to pathogens. Mouse SIGN-R1 (SIGN-related 1) is a homolog of human DC-SIGN that is expressed largely on macrophages. This lectin recognizes mannose-rich or fucosylated glycans in a Ca2+- dependent manner. When SIGN-R1 interacts with glycans on host cells, bacteria or viruses, glycan antigens are internalized into cells via lectin-mediated endocytosis to induce immune activation. As a consequence, the lectin hasbecome fascinating markers for recognition and targeting of specific cells to regulate cellular functions and for carbohydrate-based drug discovery. To investigate glycan binding properties of cellsurface lectins, we have utilized carbohydrate microarrays which allow high-throughput screening of cell-glycan interactions. Glycan microarrays used in this study were constructed by immobilizing a variety of unmodified carbohydrates onto hydrazide-coated glass slides. SIGN-R1 expressing cells pretreated with Hoechst 33342 were loaded on the glycan microarrays to evaluate the binding properties of the lectin. In addition, purified SIGN-R1 was also applied to the microarrays for the purpose of comparison. The present study demonstrates the potential of glycan microarrays for profiling of binding specificities of cell surface lectins.

Cell death which is very important in mammals, is orchestrated by two types of death signaling: the Apoptosis (Programmed cell death) and Necroptosis (Programmed necrosis). Apoptosis and Necroptosis share common proteins such as FADD, Caspase-8 and RIP1 kinase in their molecular mechanism. However, only Necroptosis includes RIP3 Kinase dependent signaling. Therefore, RIP3 Kinase is a key determinator of necroptotic cell death. RIP3 kinase which is most important protein in necroptosis is strongly down-regulated their expression in most cancer cell. It's known that DNMT1 (DNA methyltransferase 1) plays an important roles in this process. RIP3 promoter is methylated by DNMT1 in most cancer cell. On the other hand, O-GlcNAc modification is a single sugar modification occurs in cytosol and nucleus. This modification has many roles in cellular responses including nutrient sensing. Here, we identified that cellular global O-GlcNAcylation change affects to RIP3 kinase expression by real time RT-PCR. In cancer HeLa and HepG2, mRNA level of RIP3 kinase is largely increase when OGA inhibitor Thiamet-G was treated. We also confirm that OGA inhibitor Thiamet-G induced the increase of endogenouse RIP3 kinase protein level in HEK293 and HeLa cell by Western blotting. And we will confirm the O-GlcNAcylation of RIP3 kinase using immunoprecipitation and succinylated wheat germ agglutinin (sWGA) precipitation. Ultimately we aim to find the physiological functions of cellular O-GlcNAcylation change in RIP3-mediated necroptosis.

Maackia amurensis leukoagglutinin (MAL) is a glycoprotein and sialic acid-binding lectin that is used widely in the detection and characterization of sialoglycoconjugates and human cancer cells. However, its N-linked glycan structure and role have yet to be determined. The N-linked glycans were analyzed using high-performance liquid chromatography with matrix assisted laser desorption/ionization time-of-flight mass spectrometry, and the secondary structure was investigated using circular dichroismanalysis. A hemagglutination assay was performed. Furthermore, surface plasmon resonance analysis, and fluorescence microscopy and fluorescence-activated cell-sorting analysiswere conducted to assess the sialoglycoprotein-binding ability and its usefulness in the detection of human breast cancer MCF-7 cells, respectively. Analysis of the N-linked glycan structure ofMAL confirmed the presence of eight glycans, comprising two α1,3-fucosylated paucimannosidic-type and six high-mannose-type glycans. Glycan analysis of MAL that had been treated with peptide N-glycosidase F (de-M-MAL) revealed that while the two α1,3-fucosylated paucimannosidic glycans remained attached following the treatment, the six high-mannose-type glycans had been completely cleaved from the original MAL. There were almost no secondary structural changes between MAL and de-M-MAL; however, the lectin activities exhibited by MAL, such as hemagglutination and binding to a sialoglycoprotein, were completely absent in de-M-MAL, and the ability to detect human breast cancer MCF-7 cells was 77% lower in de-M-MAL than in MAL. The high-mannose-type glycans in intact MAL are closely associated with its lectin activities. This is the first report of the N-linked glycan structure of MAL and the effect of high mannose-type glycans on lectin activities.

Ovalbumin (OA) is one of the most abundant of the glycoprotein allergens, and induces a T-helper type 2 immune response that results in an IgE-mediated hypersensitivity. In this study, the terminal carbohydrates of N-glycans from intact OA were cleaved with the exoglycosidases galactosidase, mannosidase, and N-acetylglucosaminidase to generate degalactosylated-OA, demannosylated-OA, and de-N-acetylglucosaminylated-OA, respectively, in order to evaluate their role in allergenicity. The exoglycosidase digestion procedure did not result in either degradation or contamination of the three deglycosylated sample, and the digestion efficiency was confirmed by comparing the results of glycan analysis of the three exoglycosidase-treated OAs with that of glycans of intact OA. Mice were immunized with either intact or exoglycosidase-treated OAs, and their respective allergic reactions were compared. IgE production in the de-N-acetylglucosaminylated-OA group was reduced to 58.8% of that in the intact OA group. In addition, the production levels of the cytokines interleukin-4 and interleukin-5 were significantly reduced in the de-N-acetylglucosaminylated-OA group to 53.4% and 45.8% of the levels in the intact OA group, respectively. However, there were almost no changes (or only slight reductions) in the degalactosylated-OA and demannosylated-OA groups, respectively. These results indicate that cleavage of the terminal carbohydrate, and particularly N-acetylglucosamine, reduces the allergenicity of OA. This is the first report of the effect of cleavage of the terminal carbohydrate on glycoprotein allergenicity.