Earticle

An enzyme with polySia degrading activity was purified from a culture filtrate of Pseudomonas fluorescens JK-0412 to apparent homogeneity using DEAE-Sepharose CL-6B column chomatography and fast performance liquid chomatography separation on a Mono-Q column. The molecular mass of the purified enzyme (tentatively named Endo-PS) was approximately 20 kDa on SDS-PAGE and 120 kDa on native-PAGE gels, suggesting that the active form is a hexamer. Although 12 residues of the Endo-PS N-terminal amino acid sequence showed 75% homology to the 21 kDa chitin binding protein (CBP21) of Serratia marcescens 2170, no significant similarity to other known proteins was observed. Apparent Km and Vmax values of Endo-PS toward the artificial substrate 4-methylumbelliferyl-sialic acid (4-MU-Neu5Ac) were 0.08 mM and 16 nmol/mg/min, respectively. The enzyme was maximally active at 37oC and pH 8.0. Interestingly, the enzyme was shown to hydrolyze the natural substrate, α2,8-linked polySia (colominic acid), in an endo-acting manner. However, no activity toward α2,3- or α2,6-sialyllactose was observed. Under optimal conditions, oligoSia ranging from 2 to 30 residues long were liberated by the cleavage of polySia, as identified by HPAEC-PED. Therefore, the purified enzyme Endo-PS was found to be a polySia-specific sialidase. This is the first report to describe the properties of a bacterial polySia-specific sialidase. Therefore, this enzyme may be a useful tool for both industrial oligoSia production and research on the structure and biological functions of polySia in nature.

Plant produce complex N-glycans with ß1,2-xylose and core α1,3-fucose residues linked to the conserved core oligosaccharide. Production of heterologous glycoprotein in plants for therapeutic purposes is limited by the presence of plant specific oligosaccharide residues that are considered to arouse immunogenic response in the recipient body. Therefore, efforts for developing humanized N-glycosylation system in plants are required for the large scale production of safe therapeutic glycoproteins. In this regard, we isolated a rice mutant lacking complex N-glycans. However, the mutant showed severe developmental defects such as retarded shoot and root development, failure in tiller formation, and finally resulted in early developmental lethality. It is also shown that the mutant contains low amount of cellulose and high content of lignin. Interestingly, callus induced from the mutant seeds was maintainable and proliferate similarly compared with that of WT despite the size were relatively smaller.

Environmental or physiological influences that induce accumulation of unfolded proteins in the lumen of endoplasmic reticulum (ER) cause ER stress and activate signaling pathway called unfolded protein response (UPR). An ER-located transmembrane receptor protein kinase/ribonuclease called Ire1 plays an essential role in the UPR in yeasts and mammals. However, it has been unclear whether a similar mechanism is applicable to Arabidopsis. To elucidate the role of Arabidopsis IRE1, we performed functional analyses by isolating loss-of-function mutants of IRE1A and IRE1B. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. ire1a/ire1b result in a delayed induction of BiP3 which is well known ER chaperone by DTT treatment, whereas induction of BiP1 in ire1a/ire1b was similar with that of WT. Our result indicate that IRE1A and IRE1B are implicated in unfolded protein response signaling in plants.

N-glycosylation is one of the major post-translational protein modification, which alters physicochemical properties of the protein, affecting the folding, distribution, stability and thus biological function and efficiency of protein. Although the earlier steps in the N-glycosylation pathway leading to the formation of oligomannosidic structures are conserved in plants and mammals, the later steps in the formation of complex N-glycans are quite different from each other. In particular, plant type complex N-glycans are distinctive from those found in mammalian because they contain ß1,2-xylose and core α1,3-fucose residues attached to the pentasaccharide (Man3GlcNAc2) core structure but no sialic acid residues. The presence of ß1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans has long been an irritating limitation in the use of plant-made pharmaceuticals (PMPs) in human therapy, as these N-glycan epitopes are potentially immunogenic in mammals. In this study, to remove the plant specific sugar residues and humanize the N-glycosylation in plant, we isolated mutants of the corresponding plant specific glycosyltransferase genes and used for multiple-mutants construction. The resulting mutants will be transformed by human glycosyltransferases genes to accomplish humanized N-glycosylation in plant.

In plants, glycoproteins have been implicated in a wide variety of cellular processes including production of cell walls, pollination, pathogen defence and cell-to-cell communication. In addition, they have attracted considerable interest from the medical field as the main cause of food and pollen allergies. N-glycosylation is a complex process that encompasses the biosynthesis and modification of sugar moieties in the endoplasmic reticulum (ER) and Golgi. The core oligosaccharide Glc3Man9GlcNAc2 is assembled by a series of membrane-bound glycosyltransferases as the lipid carrier dolichylpyrophosphate-linked glycan in the endoplasmic reticulum (ER). The first step of this assembly pathway on the ER luminal side is mediated by ALG3 (asparagine-linked glycosylation 3), which is a highly conserved reaction among eukaryotic cells. In Arabidopsis ALG3 mutant (alg3), an immature lipid-linked oligosaccharide structure, M5ER, was synthesized and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response and alg3 mutant showed a stress sensitive phenotype. These results indicate that ALG3 is a critical factor for correct N-glycosylation of proteins and is involved in the ER stress response.

High-mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, giving rise to various processing intermediates with exposure of specific glycotopes that are recognized by a series of lectins involved in glycoprotein-fate determination in cells. Atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of energetics of the carbohydrate-lectin interactions. Although carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, it is often hampered by poor spectral resolution and the lack of recombinant technique to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for preparation of a homogeneous high-mannose-type oligosaccharide with 13C labeling using genetically engineered yeast strain. We herein successfully extended this method to overexpression of 13C-lebeled Man9GlcNAc2 (M9) using a newly engineered yeast strain with deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeld M9 in comparison with its processing product M8B, which lacks the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches can interact with the core in both glycoforms and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be associated with the lectins and the glycan-trimming enzymes.

Glycosylation is one of the most abundant protein modifications and plays an important role in many specific biological functions. Various investigations concerning quantitative glycan analysis have been performed because of the importance of the oligosaccharide moiety on glycoproteins in biological systems. There are several technologies in quantifying the N-linked glycans in an effective manner such as 13CH3I and QUIBL within vitro isotopic and isobaric labeling. Endo-β-N-acetylglucosaminidase (Endo-M) hydrolyzes the diacetylchitobiose core of the N-linked glycans bound to the asparagines of various glycoproteins and transfers the intact complex oligosaccharides to suitable acceptors. The transglycosylation activity of Endo-M is dependent on the several experimental parameters such as pH and temperature with the acceptors. As an alternative quantification method of N-linked glycans, we report a chemoenzymatic isotope labeling of fully sialylated N-linked glycans by a transglycosylation reaction using Endo-M and glucose as acceptors. The results provide the use of a quantitative technology for fully sialylated N-linked glycans in biological systems such as the processes of diseases and development of cells.

Lung cancer is the leading cause of cancer death worldwide. Currently, lung cancer is classified into two major types, small-cell lung cancer carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), based on the histological appearance. The histological classification has important implications in the clinical practice guideline and the prediction of the patient prognosis. However, conventional serum markers used in clinical tests are insufficient for clinical demands due to the low sensitivity and the low specificity to distinguish them. We have identified a number of glyco-biomarker candidate molecules from lung cancer cell lines using our developed glycoproteomics technologies such as lectin microarray and LC/MS-based protein analysis. On the validation studies, we found out that the selected molecules showed characteristic lectin biding profiles depending on either SCLC or NSCLC. Therefore, combination of these glyco-biomarkers could be expected to improve the diagnostic accuracy for histological classification in lung cancer compared to protein expression alone.

Cell glycome is defined by the glyco synthesis machinery regulated by harmonized expression of more than 100 glycogenes. The machinery-dependent glycome drastically shifts during cell progression and differentiation in association with tumorigenesis and malformation, and thus it motivates us to discover the disease-related alteration in glycosylation. Glycan-targeted histochemical approaches using lectin and anti-glycogene antibodies have provided some key information to characterize specific histological types of cells in pathology. However, this approach is not suitable for the comprehensive analysis targeting the cell glycome, and thus may fail to provide insight into glycome shift during the disease progression. Several years ago, we developed the methodology for rapid and systematic glycome shift analysis targeting formalin-fixed tissue specimens by means of lectin microarray. The resultant method enabled simultaneous observation of over 40 lectins interacted with glycoproteins in 1 mm2 of the tissue specimens. Recently, we sophisticated this methodology to be suitable for comparative analysis of a series of cells in specific groups isolated from a single tissue specimen by laser microdissection, and now our research has gained interest in the variability and distribution of cell glycome in the tissue, i.e., “tissue glycome mapping”. In this meeting, we will summarize the advantage of this new methodology and its application for glyco-biomarker discovery, as well as the construction of “tissue glycome atlas”.

Glycosylation is essential for proper cell signaling and embryonic development. Changes in glycosylation are often a hallmark of disease states. Cardiovascular disease has become one of the top causes of death. Many studies using rat models have related heart diseases and regulations of heart physiological functions to altered glycosylations. However, the dynamic regulation and molecular mechanism of glycosylation in heart are not clear. Rat is an important model organism for human cardiovascular disease studies. Isolated intact rat hearts are often used in physiological and pathological studies. In situ imaging biomolecules in intact heart avoid possible damage of myocyte and biased loss of cardiac myocytes during enzymatic isolation. Here, we applied the metabolic glycan labeling technique for imaging sialylation and O-linked glycosylation in living cardiomyocytes and intact rat hearts. The effects of unnatural sugars on the function of cardiomyocytes and hearts were evaluated. We found unnatural sugars did not perturb the function of cardiomyocytes and hearts. Glycan imaging revealed an interesting distribution pattern: sialic acid or O- linked glycan enriched at intercalated discs of adult rat cardiomyocytes. We also observed that the glycosylations of cardiomyocytes were altered in isoproterenol-induced cardiac hypertrophy rat models. This work extends the application of metabolic glycan labeling technique to probe glycosylations in rats and provides the first example of using this technique for in situ cellular glycans imaging in mammalian model organisms. The biological significance of altered glycosylations in cardiac hypertrophy heart is under investigation in our lab.

N-linked glycosylation of viral proteins have been implicated in shielding antigen epitopes and blocking neutralizing antibodies production and contribute to the evasion of HCV from the host immune response. In this study, the effects of N-linked glycosylation of Hepatitis C virus (HCV) envelope E1 and E2 proteins, the naturally poor immunogens, on the induction of specific immune response were examined. We constructed plasmids containing the genes encoding both wild type E1E2 proteins and mutated E1E2 proteins in which N-linked glycosylation sites are mutated individually or in combination by site-directed mutagenesis or coupled with CpG, a TLR9 ligand. The immunoreactivity of wild type E1E2 and mutated E1E2 proteins or coupled with immune activator CpG was analyzed in BALB/C mice using a DNA-based vaccination approach. We found that E1E2 specific N-glycosylated mutant induced much higher IgG titer than wild type (WT) E1E2. E1E2 mutant vaccinated mice developed a mixture of IgG1 and IgG2a, but CpG-E1E2 mutant and E1-E2 wild type dominantly developed IgG2a isotype. And CpG-E1E2 mutant significantly stimulated Th1-type response and specific CD8+T cells cytotoxic T lymphocytes (CTL) activities. More important we found that CpG-E1E2 mutant significantly induced much higher neutalizing antibody than E1-E2-WT and had the highest level of neutralizing antibodies among all groups. The monoclonal clonal antibody (McAb) were prepared from CpG-E1E2 mutant immunized mice and the main neutralizing McAb 1C2 had significantly neutralizing Ab activity with blocking HCVcc infection in Huh7.5.1 cells in vitro. Epitope mapping of binding of a neutralizing McAb 1C2 was also analyzed. Our data indicate that the presence of glycans at the surface of HCV envelope proteins could help explain how HCV evades the humoral immune response and why most HCV infections lead to chronicity. CpG have the ability to augment both neutralizing antibody and cellular responses of E1E2 mutant. The CpG-E1E2 mutant holds potential applicant for the development of therapeutic vaccine with enhanced cellular and neutralizing immune responses.

Alpha-L-fucosidase catalyzes the removal of L-Fucose residues from the non-reducing end of glycoconjugates. There are two fucosidases in human, including  α-L-fucosidase-1 (FUCA1) and α-L-fucosidase-2 (FUCA2). Abnormal fucosidase activity has been associated with many diseases, such as fucosidosis (one of the lysosomal storage disorders), hepatocellular cancer and breast cancer. We previously demonstrated that gastric epithelial cells secrete FUCA2 extracellularly upon the infection of Helicobacter pylori, a strategy tomodify the cell surface glycoconjugates to prevent the bacterial adhesion. However the role and physiological function of FUCA2 still remains ambiguous, not mentioning the difficulty that the enzyme is highly unstable and the activity is often too small for detection. We developed an activity-based probe for sensitive detection of the enzyme activity in vitro and in vivo. First of all, the probe was utilized for dot-blot assay to measure the H. pylori-induced fucosidase activity in a quantitative manner. Secondly, the probe provided a real-time detection of the increased fucosidase activity by using confocal microscopy. The result also supported the idea that the bacterial infection not only leads to an increase in the extracellular activity of FUCA2, but also an intracellular increase of fucosidase activity. Western blotting analysis by FUCA1- and FUCA2-specific antibodies further corroborated the aforementioned increase levels of FUCA1 and FUCA2 activities, respectively. Our current emphasis is placed to search for the factors of H. pylori in association with the secretion of fucosidase during infection.

Tuberculosis (TB) has been a major world-wide cause of death for centuries. One-third of the world’s population is infected with Mycobacterium tuberculosis (M.tb), the etiologic agent of TB. The development of potent new anti-TB drugs is urgently needed. The major M.tb surface lipoglycans, mannose-capped Lipoarabinomannan (ManLAM) had immunosuppressive effects during M.tb infection. In this study, aptamer ZXL1 which specifically bound to ManLAM from the virulent strain M.tb H37Rv was screened out by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The binding affinity of ZXL1 to ManLAM was measured as 8.907X10-8 M of quilibrium dissociation constant (Kd) value by surface plasmon resonance (SPR) analysis. We found that aptamer ZXL1 prevented the ManLAM-induced immunosuppressive effects on DCs and inhibited M. tb entry into macrophages. More importantly, we found that single injection of aptamer ZXL1 significantly prolonged the survival rate of infected mice, and prevent the infected rhesus monkeys from weight loss. The Bacterial numbers were significantly lower in the lungs and spleens in ZXL1-treated groups. These results suggest that aptamer ZXL1 can be used as antimycobacterial agent or as TB vaccine adjuvent.

The HIV-1 envelope is a basic lipid bilayer that contains gp120 and gp41 glycoproteins. Several animal models studies have demonstrated that antibodies can provide considerable benefit against HIV or SIV (Simian Immunodeficiency Virus) challenge and prompted an interest in inducing broadly neutralizing antibodies, an ultimate goal in HIV vaccine research. However, the HIV vaccine development has been deterred by the low antigenicity and immunogenicity of the HIV envelope glycoprotein gp120 and the efficient hiding of highly immunogenic epitopes by their surface glycans. Recently, a new class of bNABs (PG9, PG16, PGT121-137, and PGT141-145) isolated from HIV positive donors was shown to be potent in neutralizing primary HIV-1 strains across clades. Here we describe our efforts towards chemical and enzymatic synthesis of various gp-120 related high-mannose, hybrid, and complex type N-Glycans. We studied specificities of various bNABs towards synthetic N-glycans printed on glass slides using carbohydrate microarray. Based on recognition patterns of bNABs to quaternary structure at variable domain (V1/V2 and V3 regions) of gp-120, we created synthetic oligomannose constructs that more effectively mimic the glycan display found on gp120 and further optimized strategies to conjugate these constructs to carrier protein molecule.

Nanoparticles have been extensively investigated for targeted delivery of anticancer drug. Especially, chitosan based nanoparticles is believed as a promising carriers for cancer chemotherapy and imaging. Due to superior biocompatibility, non-cytotoxic, and biologically active properties, chitosan is frequently used as a biomedical materials and drug carriers. Furthermore, chitosan is regarded as an ideal vehicle for delivery of anionic drug or DNA drug due to its positive ionic proeprties. Chitosan is used to make polyion complexes with anionic drug such as all-trans retinoic acid (RA) and these ionic complexes can form nanoparticles in aqueous media We prepared RA-incorporated glycol chitosan (GC) nanoparticles by simple mixing RA into the GC aqueous solution through ion complex formation between RA and GC. Particle sizes of RA-incorporated GC nanoparticles were approximately 300~500 nm. Lyophilized nanoparticles were simply reconstituted into aqueous solution in spite of absence of cryoprotectants. RA-incorporated GC nanoparticles showed similar cytotoxicity against cholangiocarcinoma cells. Furthermore, RA-incorporated GC nanoparticles showed enhanced anti-invasive capacity and anti-migration capacity against HuCC-T1 cholangiocarcinoma cells. We suggest that RA-incorporated GC nanoparticles are promising vehicles for antitumor drug delivery.

Cryptococcus neoformans is an encapsulated basidiomycete causing cryptococcosis in immunocompromised individuals such as AIDS patients. Previous studies show that cell surface mannoproteins of C. neoformans stimulated host T-cell responses indicating that these protein are involved in pathogenicity. However, the structure of N-linked oligosaccharides assembled oncryptococcal cell wall mannoproteins has not yet been elucidated. In order to obtain comprehensive information on the structure of N-linked glycans in various serotypes of C. neoformans, the profiles of N-linked oligosaccharides obtained from cell wall mannoproteins were analysed by MALDI-TOP and HPLC. Exoglycosidase treatment revealed that C. neoformansN-glycans were mostly composed of α1,2- and α1,6-linked mannose residues. Notably, the neutral N-glycans of serotypes A and D were shown to have high-mannose type N-glycans with a β1,2-xylose residue, which is attached to the trimannosyl-core of N-glycans, whereas those of serotype B were shown to be much shorter and devoid of a xylose residue. Further analysis of the acidic N-glycans of C. neoformans indicated the presence of xylose-phosphate residues in the core and outer regions of N-glycans regardless of serotypes, but with quite differential extent. The N-glycan profiles lacking both xylose and xylose-phosphate residues in the C. neoformansuxs1Δ mutant strongly indicated that UDP-xylose is used as a donor sugar for the addition of a xylose or a xylose-phosphate residue to the N-glycans. Our data present the first report on the unique structure of N-glycans in C. neoformans, which is quite different from those of other yeast and fungal species.

Photodynamic therapy (PDT) is a palliative therapy for treatment of unresectable tumor and has been used to cure various kind of tumors. Recently, 5-aminolevulinic acid (ALA) has been used as a pro- photosensitizer, which can be transferred to intercellular protoporphyrin IX (PpIX), which is a strong photosensitizer, via the heme pathway. The main limitation of using ALA in PDT is the hydrophilic properties of ALA, which results in low cellular uptake. Chitosan is known to enhance mucosal delivery drugs and to enhance uptake of anticancer agent into tumor cells In this study, poly(ethylene glycol)-conjugated chitosan was synthesized and ALA-incorporated nanoparticle (CNP-ALA) was prepared for delivery of photosensitizer into human cholangiocarcinoma cells. CT26 murine colon carcinoma cells were treated with 0.1mM CNP-ALA or ALA for 24h. The effect of CNP-ALA was evaluated by monitoring the intra-converted PpIX amount and cell survival after irradiation under different light intensity. Results indicated that CNP-ALA as a nano-photosensitizer enhances intracellular PpIX generation and then cellular phototoxicity. We suggest that CNP-ALA is a promising carriers for photosensitizer to tumor cells.

Nanoparticles or polymeric micelles based on stimuli-responsive drug release have been extensively investigated in recent decades [1,2]. Especially, pH-sensitive drug targeting using pH-sensitive polymers against solid tumor has attracted remarkably since solid tumor has acidic environment [3]. Tumor targeting based on pH-responsive polymeric nanoparticles or micelles is known to have superior antitumor activity against various solid tumors. In this study, we prepared block copolymers composed of poly(L-histidine) (PHS) and dextran (DexPHS) to make pH-responsive polymeric micelles for pH-based tumor targeting. Doxorubicin (DOX) incorporated pH-resposive polymeric micelles of DexPHS block copolymer was prepared by dialysis method. DOX- incorporated polymeric micelles showed that particle size was changed by changes of solution pH, i.e. size of polymeric micelles were increased at acidic pH while they showed reduced particle size at basic pH. Furthermore, drug release was faster at acidic pH than basic pH. These results indicated that DexPHS polymeric micelles have pH-sensitive properties and then drug release was also controlled by pH. Antitumor activity of DOX-incorporated DexPHS polymeric micelles were studied using HCT116 human colon cancer cells. Results indicated that cell viability with polymeric micelle treatment was decreased at acidic pH while DOX treatment did not show significant changes against pH variation. In colculsion, our results indicated that DexPHS polymeric micelles are promising candidates for antitumor drug targeting.

In a recent day, the environmental pollution caused by industrialization, usage of food additives increased and resulting the several types of atopic symptoms. We report the successful application of seaweed and ascidians tunic used as mediator for preventing skin inflammation of human. The current study was investigated to treat the skin inflammation; raw seaweed and ascidians tunic have been used and extracted. A natural ascidians tunic was extracted to get the glycosaminoglycans (GAGs). GAGs were mixed with the extraction of natural seaweed and it tested on the subject. As a result, it is found that the extraction of seaweed and ascidians tunic together refined material was not involved the cytokine inhibitory, although an extraction of seaweed was showed the anti-inflammation effect by the experiment. Ascidians tunic is represents an effect of cell generation and the mechanism of cytokines inhibition are rather than the inflammatory inhibition, at the same time the skin regeneration is considered greater effect. Finally, we found that the atopic skin inflammation can be improved and/or treated by using natural mixture extracts of the seaweed and ascidians tunic.

The development of analytical tools is a vital factor for understanding infection mechanism by pathogenic bacteria or viruses. In the present work, functional carbohydrate microarray combined with fluorescence immunoassay was developed to analyze interactions of Vibrio cholerae toxin (ctx) and carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and its analogues and then their bindings were detected using rabbit anti-ctx antibody and fluorescence dye-conjugated anti-rabbit antibody. The analysis of ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx is GM1 >> GM2 > asialo GM1 ≥ GM3, indicating that two-finger grip formation as well as terminal three monosaccharides are a vital factor in ctx-GM1 interaction, and whole cholera toxin (ctxAB5) has stricter substrate specificity and stronger binding affinity than cholera toxin B subunit (ctxB). From the quantitative analysis, the carbohydrate microarray showed the ctxAB5-GM1 interaction with detection of limit (LOD) of 2 ng/mL (23 pM) using 10 mM GM1 pentasaccharide as recognition element, which is comparable to other reported high sensitive assay tools. The sensitivity of interaction on the carbohydrate microarray was influenced by immobilized GM1 pentasaccharide concentration and optimal GM1 pentasaccharide concentration was about 250-500 μM. This implies that the LOD of interaction on carbohydrate microarray will be enhanced by using optimal GM1 pentasaccharide concentration. In addition, based on the ctx-GM1 interaction with high sensitivity, the carbohydrate microarray can be used to detect actual ctx, which is produced from Vibrio cholerae without cross-reactivity. These results demonstrate that the carbohydrate microarray is suitable to analyze ctx-carbohydrate interactions and detect ctx in the environment.

It has been reported that one downstreameffector produced from glucose is uridine diphosphate-N-acetly glucosamine(UDP-GlcNAc) via the hexoamine biosynthetic pathway (HBP). The dynamic cycle of addition and removal of O-linked-N-acetlyglucosamine (O-GlcNAc) to Ser/Thr residues is involved in regulating nuclear and cytoplasmic proteins. Nucleocytoplasmic O-GlcNAc transferase (ncOGT) adds a single GlcNAc onto hydroxyl groups of serine and threonine residues. Interestingly, O-GlcNAc glycosylation occurs in ncOGT as well and several putative sites have been reported mainly within TPR domain. However, the mechanism by which nuclear translocation of O-GlcNAc transferase is not clear. Here, we identified specific nuclear localization signals (NLS) in O-GlcNAc transferase that is required for nuclear transport. A 3 amino acid domain inserts a non-diffusible protein to the nucleus autonomously. Also, we show that ncOGT binds importin α proteins and the association between importin α proteins and ncOGT is interfered by O-GlcNAcylation on TPR domain. This ongoing effort would give us clear understanding of the key enzyme of O-GlcNAc metabolism.

Surface plasmon resonance (SPR) can provide a kinetic information for an interaction and rapidly monitor any dynamic process, such as adsorption or degradation, without the need for any sample preparation. Herein, we used SPR system to analyze carbohydrate-protein interaction, especially, Vibrio cholera toxin-GM1 pentasaccharide. The understanding of carbohydrate contribution in carbohydrate-protein interaction may provide meaningful information about carbohydrate-related biological system. Interaction between ctxA and ctxB is firstly investigated in this study since the binding is a prerequisite for virulence mechanism. Interaction kinetics between ctxB and GM1 pentasaccharide using the our proposed saccharide immobilization method were evaluated, and the results showed similar degree of kinetics to the previous reports, thereforeour direct immobilization could make SPR analysis simply for carbohydrate-related researches. And the introduction of ctxA during the interaction showed higher affinity than that of ctxB-GM1 although KD constant was 10-times lower than ctxB-GM1 binding. Comparative analyses for GM1 analogues-ctxAB were conducted in order to evaluate kinetic values from structural differences.Although these results did not showsignificant difference, determination of the association constants for ctxAB revealed the following order: LST-b > GM3 > LST-a >asialo GM1 and dissociation constants for ctxAB showed following order: LST-b >asialo GM1 > LST-a > GM3. These results indicate that sialic acid thumb is sufficient for recognition, and the terminal glalactose and GalNAc finger are required to stabilize the ctxAB-ganglioside GM1 interaction. Collectively, direct immobilization of carbohydrate at SPR-based analytical system can evaluate structural contribution of carbohydrate moieties in carbohydrate-protein interactionas well as provide valuable information for understanding the interactions.

Free Oligosaccharides in human milk (HMO) have multiple biological functions, for example, the growth of beneficial bacteria in the gut and the development of brain. It has been known that the concentration of free oligosaccharides in bovine milk and infant formula is relatively low compared with HMO. In thisstudy, we have profiled and quantified free oligosaccharides extracted from various milks and dairy products. HMO and free oligosaccharides extracted from domestic milk (bovine, porcine), commercial milk (milk, low-fat milk), four infant formulas made from goat and bovine milk were used for this study. For quantitative analysis, initially same volume 5ml of milk was taken. Extraction of free oligosaccharides was performed by following steps including the removal of lipids by Folch method and protein precipitation by ethanol. Extracted free oligosaccharides were further purified by solid phase extraction using a porous graphitized carbon cartridge. Purified and enriched free oligosaccharides were redissolved in water prior to mass spectrometry analysis. Overall glycan profiling was obtained by MALDI-TOF/TOF MS. We found that human milk has more free oligosaccharides than the others. In addition, the ion at m/z 730.237 corresponding to [Hex]3[HexNAc]1 is the base peak while the ion at m/z 568.179 corresponding to [Hex]2[HexNAc]1 is the base peak in bovine milk. Interestingly, we found NeucGc in bovine milk as a major glycan, which is not produced in human anymore. Quantitative analysis of targeted glycans was performed by UPLC-Triple quadrupole. This study can be applied as an analytical platform to extract, profile, and quantify free oligosaccharides in milk and dairy products.

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

Recombinant erythropoietin (EPO) is an important biotherapeutic used to treat anemia. However, the quality, safety, and potency of EPO are determined largely by its glycosylation, which is in turn strongly affected by the conditions of the cell culture in which it is produced. Qualitative and quantitative glycomic analyses of glycosylated biosimilars are important for assessing their quality as well as determining batch-to-batch variation during production. We studied overall profiling and comparative quantitation of native N-glycans from three batches of darbepoetin-alfa using MALDI-TOF MS and nano-LC/Q-TOF MS. After detergent removal, denaturation, and enzymatic N-glycan release, N-glycans were purified by graphitized carbon solid phase extraction (SPE). Darbepoetin-alfa was found to contain mostly tetraantennary, tetrasialylated glycans. Sialic acid modifications such as O-acetylation and dehydration were extensively observed, as were low abundances of polylactosamine-containing N-glycans. The correlation coefficients amongst the three batches were above 0.94. We observed similar results from both MALDI-TOF MS and nano-LC/Q-TOF MS. This platform was successfully applied to characterize the glycosylation of a complex biosimilar.

Fucose-containing glycoconjugates play important roles in numerous physiological and pathological processes. Given the biological importance of posttranslational glycosylation, a specific and robust strategy for the identification of fucosylated glycoproteins is highly desirable. In this study, we demonstrate an alternative way of labeling of fucosylated structures by metabolic engineering, using a chemoenzymatic approach. In this approach, the activities of Bacteroides fragilis 9343 L-fucokinase/GDP-fucose pyrophosphorylase and human α1,3-fucosyltransferase 9 are combined in a Namalwa cellular model. Interestingly, this system could be applied to labeling of alkyne-modified fucosylated glycoproteins. N-glycan site mapping and identification was done using an in vitro selective chemical ligation reaction and isotope-coded glycosylation site-specific tagging, subsequent to liquid chromatography-tandem mass spectrometry analysis. This work illustrates the use of a click chemistry-based strategy combined with a glycoproteomic technique to get further insight into the pattern of fucose-mediated biological processes and functions.