Earticle

Quercetin is a natural plant flavonoid that has been reported to possess a wide range of beneficial health effects, including anti-cancer and anti-inflammatory activities. Glycosylation of natural flavonoids with various sugar moieties can affect their physical, chemical, and biological properties. In this study, quercetin 3-O-xyloside (Quer-xyl) was enzymatically synthesized, and the immunomodulatory activities of quercetin and Quer-xyl were evaluated and compared. The results showed that Quer-xyl more effectively induced the secretion of TNF-α and IL-6 than quercetin by 2.5 and 1.5-fold, respectively. Quer-xyl dose-dependently induced the inducible nitric oxide synthase (iNOS) expression and increased the production of nitric oxide (NO) 1.3-fold more than quercetin. Quer-xyl also increased the phosphorylation of ASK1 and MAPKs (JNK and p38). Treatment with NQDI-1 (an inhibitor of ASK1) significantly attenuated the Quer-xyl-induced up-regulation of TNF-α secretion. The activation and subsequent nuclear translocation of NF-κB were substantially enhanced upon treatment with Quer-xyl (2.5–20 μM), while NQDI-1 treatment blocked the nuclear translocation of NF-κB. These results demonstrated that Quer-xyl can enhance the early innate immunity more effectively than quercetin by activating macrophages to secrete TNF-α and IL-6 through up-regulation of the redox-dependent ASK1/MAPK/NF-κB signaling pathway, suggesting for the first time that Quer-xyl may represent a new immunostimulator.

Gastric cancer (GC) is one of the most commonly occurring cancers and has the highest overall mortality rate worldwide. Unfortunately, the vast majority of GC patients present with advanced stage disease and the overall prognosis remains very poor. In GC diagnosis, the non-invasive tests are limited due to their lack of sensitivity and specificity. Glycosylation changes have been reported in a wide variety of human diseases, including immune disorders and cancers, and even is associated with malignant transformation. Recently, a targeted glycoproteomic approach has gained considerable attention as a novel method for biomarker discovery to improve the specificity and sensitivity for clinical use. Here, we comprehensively investigated the indirect and direct glycomic profile of a target glycoprotein, serum haptoglobin (Hp) by chip-based nano LC-QTOF MS and MS/MS analysis following antibody-assisted purification. From the results of significant N-glycan variations between GC patients and healthy controls, we conclusively suggest that aberrant glycans of serum Hp are associated with patients with gastric cancer and might be a promising marker for GC screening.

Recently, investigators have successfully developed powerful systems using alternative technologies (ZFNs, TALENs and CRISPR/Cas9 system) to genetically engineer mice much faster and more economically compared to traditional homologous recombination method. Herein, we applied the CRISPR/Cas9 system to generate mice carrying mutations in multiple glyco-genes which are frequently species-specific in human and mouse. An example of species-specific glyco-gene is an inactivating mutation in humans of the gene Cmah, an enzyme responsible for Neu5Gc biosynthesis from the Neu5Ac form of sialic acid. We planned to identify several mouse-specific glyco-gene and edit them to generate mice with human-like glycan. We expect these glycan-humanized mice can hold great promise as a novel model for non-clinical study that is to provide safety information to support the clinical development of the new pharmaceuticals, thereby playing a pivotal role in bridging the translational gap to a successful clinical outcome. In addition, glycan-humanized mouse models can be anticipated to contribute invaluable information to our knowledge of glycobiology and medicine.

The glycosyl modification on protein therapeutics is considered to be a very important attribute due to its strong effect on quality, safety, and potency. Thus, systematic approach is absolutely required for measurement and control of glycosylation from the drug development to post-approval batch release. We have developed stepwise strategies for glycome quality assessment during drug design and manufacture in the level of intact protein, glycopeptide, and glycan. Intact protein analysis was employed for detailed characterization of highly complex micro—heterogeneity, including various glycofrom profiles by high-resolution native mass spectrometry. Site-specific glycosylation with micro & macro glycan heterogeneity can be achieved by glycopeptide analysis which combines molecular size fractionation, multiple proteolysis, and PGC nanoLC MS/MS. This approach could be widely applied for high-quality analytical characterization of glycosylation, starting from early drug development to final lot releas.

Characterizing and monitoring the glycan population of a biotherapeutic protein has continued to present technical and logistical challenges to the biopharmaceutical industry. Improvements to analytical technologies and informatics have made the process quicker and more automated, but overall laboratory workflows for released glycan analysis have continued to be laborious and require high skill to generate accurate and reproducible profiles. Electron transfer dissociation(ETD) is a powerful fragmentation technique known to be particularly useful for determining sites of labile post-translational modifications(PTMs), which are often more difficult to characterize using collision induced dissociation (CID). This seminar offers details of glycopeptide characterization using ETD fragmentation.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder associated with redness, warmth, swelling and acute pain within joint linings. Current means of diagnosing RA involve blood test, x-rays and MRI. Any combinations of these tests, however, fail to live up to consistent accuracy when diagnosing RA, potentially resulting in inadequate or improper therapies administrated by health care professionals. In order to find better means of diagnosing RA, research showed that the release of glycosaminoglycans (GAGs), heterogeneous and linear polysaccharides consisting of disaccharide repeat units, occurred during inflammation and infection. In order to better understand the role GAGs have in RA, we used an LC-MS/MS platform to analyze GAGs derived from healthy and RA infected human sera. We established mass spectrometry methods to quantify GAGs and obtained specific information on their sulfation and/or acetylation patterning at a hexuronic acid and hexosamine. Our investigation discovered statistically significant difference in the sulfation and acetylation patterns between RA and healthy sera. Furthermore, since isomers of the disaccharide exist, we established a MS method that generated diagnostic ions capable of distinguishing the eight additional disaccharide isomers. This mass spectrometry method may further increase the overall accuracy of RA diagnoses.

While it is the DNA sequence which holds the information deciding the amino acid sequence of the resulting proteins, in order to fully comprehend the entirety of the structures and functions of proteins in an organism, further information than just the amino acid sequence is a necessity. Of the numerous modifications that occur on proteins, glycosylation is a field in the limelight in post-genome research. In February of 2003 the Massachusetts Institute of Technology published an article on “10 Emerging Technologies that will change the world” – glycobiology was selected in the field of biotechnology, therefore its potential and merit acknowledged, and since then research in the field has been burgeoning in the USA and Japan. Before Dr. Gerald Hart of Johns Hopkins University School of Medicine had discovered the existence of nuclear and cytosolic O-GlcNAc modification in 1984, complex carbohydrates were known to exist only in the endoplasmic reticulum, the Golgi apparatus and outside the cell, thus until now researches conducted on complex carbohydrates were limited to the endomembrane system. However, when O-GlcNAc modification was revealed to be closely involved with diabetes, Alzheimer’s disease and tumorigenesis, as well as that it competes against phosphatase for the modification sites on serine and threonine residues, the biological functions of O-GlcNAc modification, more specifically its role in cell signal transduction became the focal point of researches on molecular and individual levels. 3% of all glucose that enter the cell pass through the hexosamine biosynthesis pathway and are converted into UDP-GlcNAc. Utilizing UDP-GlcNAc as its substrate, O-GlcNAc modification is a modification unlike its predecessors in that its level is dynamically regulated by O-GlcNAc trasferase (OGT) which attaches O-GlcNAc onto target proteins, and reversibly removed by O-GlcNAcase (OGA). Proteomics and glycomics research technologies have determined that over 1,800 proteins are modified by O-GlcNAc, but due to a slowed development of technological infrastructure with which to recognize and study the function of O-GlcNAc modification, locating O-GlcNAc modification sites of a protein has only recently become an active field of research. There are only around 100 proteins of which the exact modified amino acid and its resulting functions are known – a vastly small number compared to the number of proteins known to possess O-GlcNAc modification as aforementioned.

Glycosylation is one of the most important post-translational modifications in mammalian cells. This catalytic reaction is processed by adding the various functional sugar moieties to protein through ER and Golgi apparatus. Most therapeutic proteins for human diseases are glycosylated and the glycan structures have been shown to affect therapeutic efficacy. In vivo half-life of therapeutic glycoproteins, in particular, is determined mainly by sialic acid capping at the end sites of N-linked glycans. The non-sialylated glycoproteins have significantly shorter in vivo half-life, because the exposed galactose residues are recognized, captured by asialoglycoprotein receptors (ASGPR) in hepatocytes and immediately degraded. Thus, it is greatly important to increase the terminal sialic acid contents of therapeutic glycoproteins for longer acting of therapeutics. In this presentation, the enhancement of sialylation of recombinant human erythropoietin as a model protein in CHO cells through sialylation pathway engineering approach will be discussed with the experimental data.

The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose binding domains has been characterized from Atrina’s foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+–DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.

In the present study, a targeted gene carrier for αvβ3 integrin-overexpressed tumor cells was designed with widely applied materials containing water soluble chitosan(WSC), RGD peptide, and polyethyleneimine(PEI). A study of the endocytic mechanism resulted in the microtubule-dependent macropinocytosis and clathrin-mediated endocytosis. In addition, the PEI/WSC copolymer with dendrimer RGD peptide (four-branched RGD moiety) as a targeting moiety suppressed the growth of a solid tumor mass in an in vivo mouse xenograft model with PC3 cells, prostate tumor cells, by silencing BCL-2 mRNA. This result provides a good candidate with a simple and biocompatible gene carrier. On the other hand, the development of drug delivery system (DDS) for antibiotic therapy may be essential to reduce cytotoxicity and side effects of drug, and to overcome drug-resistant fungal strains. Until now, many researches have been reported to improve the therapeutic index of AmB in PEG-based, lipid-based, and polymer-based AmB formulations. However, their high cost and side effects is mainly limiting factor in public use. The aim of this study was to prove another function of the antifungal peptide as a novel ligand which targets fungal pathogens specifically in infected animal model and to investigate a synergistic action between antifungal peptide and agent. In addition, we proposed a minimum dose of antifungal agent in clinical application via pH-responsive and redox-sensitive drug carrier.

The most plentiful component of exoskeletons in invertebrates such as crustaceans and instects is chitin, which is the second abundant naturally occurring biopolymer by living organisms after cellulose. And Chitosan, one of the most important derivative of chitin, is an appealing biopolymer for the replacement of synthetic plastic compounds and applicable to various biomedical fields. Here, we measured molecular interactions of high molecular weight chitosan (MW ~110k, HMW chitosan) and low molecular weight chitosan (MW ~5k, LMW chitosan) in aqueous solutions by exploring the effects of pH, contact time and degree of acetylation which determines chitin and chitosna using a surface forces apparatus (SFA). SFA is a powerful instrument used to characterize the fundamental intermolecular forces such as electrostatic forces, van der Waals forces, capillary forces, hydrophobic interactions, bio-specific interactions, metal coordination forces as well as friction force in aqueous conditions. The nanomechanical studies of chitosan and chitin using SFA will provide new insight into the development of chitosan/chitin based load-bearing materials and biomedical applications.

Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed GlycoProteome Analyzer (GPA) for automated identification and quantitation of site-specific N-glycosylation including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database constructed, we created novel scoring algorithms with decoy glycopeptides with three steps: 1) selection of N-glycopeptide from 15 glycan-specific oxonium ions using HCD-MS/MS spectra; (M-score); 2) selection of candidates by matching the isotope pattern to intact N-glycopeptides in the GPA-DB (S-score); and 3) identification of N-glycopeptide from CID and HCD-MS/MS fragment ions (Y-score) with (FDR) < 1%. For example, 95 N-glycopeptides from standard α 1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. IQ-GPA allows direct analysis of site-specific N-glycopeptides with estimated FDR ≤ 1% using a decoy database of target glycopeptide candidates from complex glycoprotein mixtures. We automatically identified and simultaneously quantified about 600 site-specific N-glycopeptides from 123 glycoproteins, which mark the largest number reported to date, spanning five orders of magnitude in concentration from immunoglobulin G to AFP in human plasma. Thus, IQ-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes.

Two fungal α-glucosidases from Podospora anserina (PAG) and Schwanniomyces occidentalis (SOG) have different regioselectivity thought their high similarity in amino acid sequence (35%). Native PAG was purified from Podospora anserina and recombinant PAG was expressed using Pichia pastoris. Both of native and recombinant PAG revealed high specificity for α-1,3- and α-1,4-glucosidic linkages in hydrolysis as well as transglycosylation, while SOG preferred α-1,4-glucosidic linkage in hydrolysis and generated α-1,4- and α-1,6-glucosidic linkages in transglycosylation. In order to elucidate the structural elements responsible for different regioselectivity of two enzymes, mutations were introduced on active site of SOG by site-directed mutagenesis. Several loop structures constructing active site, that is, β→α loop 1 and β→α loop 8 from catalytic (β/α)8 barrel domain and N-loop from N-terminal domain, were identified to be important structural determinants of regioselectivity. A quadruple mutant SOG 231F232/W324Y/Y700E/L701Y showed increase of α-1,3-specificity and decrease of α-1,6-specificity in hydrolysis as well as transglycosylation. Consequently, the present study clarified structure-function relationship of fungal α-glucosidases, especially focused on α-1,6- and α-1,3-glucosidic linkages.

Starch structure and reactivity is not fully understood, despite the widespread use of starch and its derivatives in food and non-food industries. This study is focused on the relationship between starch granular and molecular structure, and their impact on the relative extent of reaction (i.e., reactivity). Starch was derivatized with a fluorescent probe (5-[4,6-dichlorotriazinyl]aminofluorescein) as a derivatizing reagent (model derivatization system). The granular locale of reaction, and relative reactivities of debranched starch amylose (AM) and amylopectin (AP) branch chains [long (LC), medium (MC), and short (SC)] were monitored for 24 h via confocal laser scanning microscopy (CLSM) and size-exclusion chromatography (SEC) equipped with refractive index (RI) and fluorescence (FL) detection, respectively. The granular locale of reaction was initially focused at external granule surfaces (≤ 0.5 h), and gradually progressed into granule matrix during latter reaction stages (12-24 h). Starch chain reactivities decreased in the order: AP-LC >> AM, AM-MC > AP-SC, irrespective of the length of reaction. Later, in the modified SEC set up, ‘intermediate material’ was identified (IM, subpopulation of AM/AP-LC, 100 ≤ degree of polymerization <372), which exhibited 5.0-9.4 fold higher reactivity than the overall reactivity calculated across all starch chains. IM was shown to be a primary molecule in the outer layers of starch granules where the reaction was most intensive. Overall findings suggest that granule architecture impacts the relative reactivities and reaction patterns of AM and AP branch chains. The gathered information is expected to improve technical practice and design specific functionality of starch derivative.

Breast milk, the sole source of nourishment for newborns, has been under intense selective pressure over millions of years of evolution to meet the infant’s needs to grow and survive. Interestingly, the third most abundant component class in human milk, oligosaccharides, is both non-nutritive and non-digestible. The oligosaccharides play a key role in creating and maintaining a healthy infant gut microbiota through two established mechanisms: prebiotic effects and competition for specific pathogen. The functions of milk glycans (oligosaccharides, glycoproteins and glycolipids) are related to their specific structures. However, identifying and quantifying those structures has been a major challenge in their analysis due to the complexity of the structures. Therefore, functional studies to date have involved either a limited number of structures or little or no structural information. Novel mass spectrometry-based glycomic tools allowed to differentiate and quantitate not only glycans of different mass but also those with the same masses but different sugar composition and linkages. The advances in glycomic analysis have allowed us to observe the correlation between the consumption of individual glycan structures and gut bacteria population. Animal studies which require sufficient amounts of the material will further reveal how the complex fluid nourishes infants and protects them from disease in human body. This has necessitated research directed towards development of methods for large-scale separation of the biomolecules. Bovine milk is a potentially excellent source of commercially viable analogs to human milk glycansn. We recently isolated and discovered that whey permeate, a secondary product from cheese production, contains oligosaccharides structurally far more similar to human milk oligosaccharides than currently available prebiotics. The combination of advanced analytics with the new engineering capabilities allowed for high molecular resolution and separation techniques that can be scaled- up to semi-industrial and industrial scale for translation of lab-based discoveries.

We have identified an ALG3 homolog (CnALG3), coding for a dolichyl-phosphate-mannose dependent α-1,3-mannosyltransferase, in the human pathogenic yeast Cryptococcus neoformans. The CnALG3 gene encodes a protein of 447 amino acids, showing approximately 33% sequence identity to the homologs of other yeast. For functional analysis of CnALG3, we constructed a null mutant strain and analyzed the N-glycan structures by HPLC and exoglycosidase treatment. CnALG3 was shown to be responsible for the conversion of Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP, the earliest step to attach a mannose to the lipid-linked oligosaccharide in the ER. The Cnalg3Δ mutant strain displayed a moderate defect in capsule biosynthesis, and exhibited more increased sensitivity to oxidative and cell wall stresses compared to the wild-type. The C. neoformans phospholipase PLB1, which is a glycoprotein aiding fungal traversal across the blood-brain barrier, was shown to have truncated N-glycans in the alg3Δ, which showed more apparent decrease than in the och1Δ and mnn2Δ. Moreover, the Cnalg3Δ showed fully attenuated virulence in a mouse model of cryptococcosis, suggesting that the alteration of N-glycan assembly affect considerably pathogenicity of C. neoformans. However, the non-opsonic phagocytosis of Cnalg3Δ was shown to be comparable to that of Cncap59Δ during cryptococcal pathogenesis, indicating that the truncated N-linked glycans may not affect the early steps in interaction with macrophages.

Most of the therapeutic enzymes, which is used for treatment of lysosomal storage diseases, require the glycans containing mannose-6-phosphate (M-6-P). It has been shown that the increased content of M-6-P glycan enhances lysosomal targeting and, therefore, the efficacy of therapeutic enzymes [1]. We have constructed the glyco-engineered yeast harboring very high content of mannosylphosphorylated N-glycans, which can be converted to M-6-P glycan through an uncapping process [2]. In this study, cell wall mannoproteins of the glyco-engineered yeast were extracted by using hot citrate buffer and then digested with pronase, which generated mannosylphosphorylated glycans as attached to asparagine amino acid (Asn). Through the mild acid hydrolysis for uncapping and α(1,2)-mannosidase treatment for trimming, these Asn-linked glycans were converted to Asn-linked M-6-P glycans, which were specifically purified by titanium dioxide resin. The purified Asn-linked M-6-P glycans could be easily conjugated with the crosslinkers containing an amine-reactive group such as a N-hydroxysuccinimide esters which attacks amine group of Asn. Using a chemical group (maleimide, azide or dibenzo-bicyclo-octyne) at the opposite site of the attached cross-linkers, M-6-P glycans were successfully conjugated to target proteins. Proper conjugation of M-6-P glycan to target protein was confirmed by increase of molecular weight in SDS-PAGE and lectin blot using domain 9 of cation-independent M-6-P receptor. Further, M-6-P glycan-conjugated protein was shown to be internalized and targeted to lysosomes of cells.

Genetic engineering approaches to improve therapeutic potentials of mesenchymal stem cells (MSCs) have been made by viral or non-viral gene delivery methods. Viral methods have severe limitations in clinical application due to potential oncogenic, pathogenic, and immunogenic risks while non-viral ones have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery. Conventional cationic liposome method was not effective for MSC transfection to result in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles up to 66% in MSCs without significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression, compared to plasmid microporation. When the MSCs transfected to express firefly luciferase by minicircle microporation were subcutaneously injected to mice, their bioluminescence was monitored for more than a week whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By using minicircle microporation as a non-viral gene delivery, the engineered MSCs to overexpress CXCR4 were shown to have greatly increased homing ability toward the injury site through in vivo bioluminescence imaging in mice, which showed a promise to enhance therapeutic potentials of MSCs in clinical applications.

Cryptococcus neoformans is the opportunistic human-pathogenic fungus causing life-threatening meningoencephalitis in immunocompromised patients, such as patients who acquired AIDS. Several C. neoformans mannoproteins (MPs), such as MP98 and MP88, are reported as key antigens stimulating host CD4(+) T-cell response. In this study, we investigated the effect of altered structure of N-glycans assembled on MPs in interaction with host cells. The expression patterns of MP98(H) and MP88(H) proteins, the his-tagged forms without GPI-anchor, in the wild-type (WT) and various N-/O-glycosylation mutant strains indicated that MP98 is modified mainly by N-glycosylation whereas MP88 is subject to both N-glycosylation and O-mannosylation. The MP98(H) and MP88(H) proteins were purified from WT and a N-glycosylation defective mutant strain, Cnalg3Δ, in the cap59Δ mutant background to facilitate purification of secretory proteins by inhibiting capsule biosynthesis. The in vitro adhesion assay showed that both of the WT-type MPs, bearing high-mannose type N-glycans, adhered efficiently to epithelial lung cells but not to macrophage cells. The MP88(H) secreted from the alg3Δ/cap59Δ strain showed slightly decreased adhesion to lung cells compared to the WT-secreted MP88(H). However, unexpectedly, the MP98(H) protein from the alg3Δ/cap59Δ strain showed rather increased adherence to the lung cells than the WT-secreted MP98(H) protein, indicating that truncated N-glycans rather enhanced adhesion of MP98(H) to epithelial lung cells. These results indicate that altered N-glycan structures of MPs may affect the adhesion efficiency of C. neoformans in a protein-specific way.

Radiotherapy for lymphoma, gynecologic cancers, and sarcomas of the upper abdomen may result in radiation-induced kidney injury. Moreover, it is possible that radiation nephropathy could occur after a nuclear accident. Radiation nephropathy usually progresses very slowly, over a period of several years. The precise pathogenesis and/or mediators involved in radiation nephropathy remain under active investigation. It has been indicated that ionizing radiation induces senescence, which is one of the hallmarks of aging. As the senescent cell fraction following irradiation is known to increase, we investigated whether ionizing radiation causes the renal injury by inducing cellular senescence. In the present study, we evaluated the effects of ionizing radiation on canine renal epithelial cells (MDCK), which predominantly used in in vitro renal cell models. We found that the exposure of ionizing radiation on MDCK cells induced cellular senescence. Notably, cleaved form of Klotho, a renal neuraminidase that is essential for the function of fibroblast growth factor 23, was significantly increased following irradiation in culture media of MDCK cells. Lectin affinity analysis determined that these secreted klotho reduced the sialylated form of epithelial calcium channel (TRPV5), in MDCK cells. Furthermore, Klotho expression was decreased in renal tissues of radiation-exposed mice. Collectively, our data suggest that increase of secreted klotho and their neuraminidase activity to TRPV5 following irradiation may contribute to the development of renal dysfunction through the acceleration of cellular senescence.

It is well recognized that certain plant lectins induce apoptosis of cancer cells by interacting with cell surface glycans. Thus, it is of great importance to develop artificial glycan receptors that functionally resemble natural lectins. In this effort, apoptosis-inducing activity of two synthetic aminopyrroles that act as receptors for mannosides was evaluated. When several cancer cells were incubated with the artificial receptors, it was found that the extent of receptor-induced cell death is greater in cells expressing a high level of high-mannose oligosaccharides than in cells producing lower levels of high-mannose glycans. The ability of synthetic receptors to promote cell death was reduced in the presence of external mannosides. The results suggest that the observed cell death reflects an ability of the receptors to bind mannose expressed on the cell surface. Signaling pathway studies indicate that the synthetic receptors promote JNK activation, induce Bax translocation to the mitochondria, and cause cytochrome c release from the mitochondria into the cytosol, thus promoting caspase- dependent apoptosis. The similar phenomena were also seen in cells incubated with mannose-binding lectin ConA. Our findings serve to highlight what may be an attractive new approach to triggering apoptosis via modes of action that differ from those normally used to promote apoptosis.

Cell surface lectins in the immune system bind to glycans expressed on the surface of pathogens, the event that leads to stimulation of immune responses to pathogens. Mouse SIGN-R1 (SIGN-related 1) is known to recognize mannose-rich and fucose containing glycans in a Ca2+-dependent manner. When SIGN-R1 binds to glycans on host cells, bacteria or viruses, glycan antigens enter cells via lectin-mediated endocytosis to elicit immune activation. We applied carbohydrate microarrays containing various glycans to rapidly identify functional glycans that promote cell surface lectin-associated cellular responses. Because binding of glycan ligands to SIGN-R1 on the cell surface promotes production of reactive oxygen species (ROS), the functional glycans on the microarrays are readily identified by using a ROS fluorescent probe PF1. Glycan microarrays used in this study were fabricated by immobilizing xx (몇개) unmodified glycans on hydrazide-modified glass slides. SIGN-R1 expressing cells pre-treated with PF1 were applied to the glycan microarrays and the fluorescence intensity of the cells were then measured by using a confocal fluorescence microscopy or a microarray scanner. The results of microarray experiments revealed that several Man and Fuc bearing glycans stimulate ROS production, a phenomenon that was abrogated in the presence of a ROS scavenger or an NADPH oxidase inhibitor. The present study demonstrated at the first time that glycan microarrays serve as a useful tool to identify functional glycans that elicit cell surface lectin-associated cellular responses.

Cell surface lectins recognize diverse glycans and these interactions are implicated in a wide range of physiological and pathological processes. Accordingly, understanding lectin-mediated recognition events and blocking glycan-protein interactions associated with diseases are of great importance for both basic biological research and biomedical applications. To investigate recognition of glycans by lectins expressed on mammalian and pathogen cell surfaces, we prepared three Lewis antigen conjugated, fluorescent magnetic nanoparticles (FMNPs). Although glycan- conjugated NPs have been widely used to detect cell surface lectins, dual modal (fluorescent and magnetic) glycoNPs have not been employed to study lectin-associated recognition events. In the initial study, when DCEK, DCEK-DC-SIGN and DCEK-SIGN-R1 cells were incubated with glycosylated FMNPs, Lea and Leb but not H1 conjugated NPs were recognized by DC-SIGN and SIGN-R1 expressing mammalian cells, and, subsequently, entered the cells. However, mannan, which is a tight ligand for DC-SIGN and SIGN-R1, suppressed the binding of glycoNPs to the cells. Next, when H. pyroli J99 strain expressing BabA was incubated with glycoNPs, Leb and H1 conjugated but not Lea conjugated NPs bound to this strain. However, these glycoNPs did not interact with H. pyroli strains lacking BabA. We also used glycoNPs to enrich BabA expressing H. pyroli by using a magnet. The present study show that fluorescent magnetic glyconanoparticles can serve as useful tools to sensitively detect lectins expressed on pathogen and mammalian cell surfaces. This study also provide opportunities for developing finely tuned multifunctional nanoparticle-based drug and diagnostic nanoplatforms.

Glycogen branching enzymes (GBEs) from Escherichia coli (EcBE), Deinococcus geothermalis (DgBE) and Vibrio vulnificus (VvBE) have different characteristics for branching activities, especially in chain-length transferred. In this study, the three GBEs were used to produce cluster-starches from amylopectin, and physicochemical properties of the cluster-starches were intensively analyzed in relation to their molecular structures. Side chain distribution of these cluster-starches obviously shifted to short and medium chains that made a significant decrease in their molecular weight and size. They thereby increased water-solubility without exhibiting viscosity. There was no retrogradation of DgBE- and VvBE-clusters during storage at 4℃ for 17 days. Catalytic efficiency of porcine pancreatic α-amylase (PPA) was lowest for VvBE-cluster. In conditions mimicking the human intestine, EcBE, DgBE and VvBE cluster-starches were digested 100%, 67.1% and 73.0%, respectively, in comparison with that of amylopectin. Consequently, the modified clusters from amylopectin produced by DgBE and especially VvBE may be new functional materials for food and pharmaceutical industries.

Gene therapy is the introduction of genes into cells or tissues to up- or down-regulate genes that can remove the pathological symptoms or activate the immune response via the delivery of DNA or RNA. This treatment has great promise for preventing or curing a wide range of diseases. In the present study, a RGD peptide and PEI grafted water soluble chitosan (RPgWSC) copolymer was synthesized and its efficiency as gene carrier investigated. The physiological characters, in vitro targeted gene transfection, cytotoxicity, blood-compatibility, and cellular distributions were investigated. In particular, a study of the endocytic mechanism resulted in the microtubule-dependent macropinocytosis and clathrin-mediated endocytosis.

In particular, cancer is one of major diseases in studies for gene delivery systems worldwide. Among them, breast cancer is a frequent malignancy worldwide and an important public health problem. In the past years, the molecular understanding of this disease has shed light into its heterogeneity. It is categorized into five groups: human epidermal growth factor receptor 2 [HER2] type, luminal A, luminal B, triple negative, and normal like, based on the cell surface and other molecular markers. The aim of this study is to improve transfection efficiency of gene carrier and to achieve targeted gene therapy for HER2-positive cancer cells, based on water-soluble chitosan (WSC) as a conjugating backbone biopolymer, synthetic peptides with Hexa-histidine-linear octa-arginine (HLR) or haxa-histidine-dendrimer octa-arginine (HDR) which is able to enhance nucleic acid-binding, proton sponge effect, and endosomal escape, LTVSPWY dendrimer peptide (Her2(d2)) which can target to HER2-positive cancer cells, and HnMc antimicrobial peptide which may be able to induce mitochondrial apoptosis.

Glycosylation reactions are widespread in nature, and involved almost all vital processes. Glycosylated compounds directly exert a wide range of functions, including energy storage, maintenance of the cellular integrity, information storage and transfer, molecular recognition, cell-cell interaction, cellular regulation, virulence and chemical defense. Glycosylated compounds are the most structurally diverse biomolecules, and their biosynthesis needs quite complex biological processes orchestrated by many enzyme systems. In Plants, various natural products are produced including diverse flavonoid derivatives. Mostly these metabolites are generally glycosides and are accumulated in the vacuole. Among C-glycosylflavones, comprising the various pharmacological activities, are biosynthesized from flavone via C-glycosylation of 2-hydroxyflavone or flavone. This is mediated by uridine diphosphate (UDP)-sugar dependent glycosyltransferase. The C-glycosyltransferase catalyzes the transfer of the glucose moiety to the aromatic carbon of the acceptor substrate. C-Glycosylflavones are involved in UV protection, defense against pathogens and inhibition of caterpillar growth. In this study, we tried to biosynthesize C-glycosylflavone in vivo and the product was confirmed by LC-MS.

Drug development research requires a large amount of target proteins. Screening of drug target often requires 13C- and 15N- labeled protein, and higher protein expression has great advantages for obtaining isotope-labeled proteins. Among many of the proteins, glycosyltransferases(GTs) are attractive biocatalysts in producing a series of important bioactive natural products. In case of kanamycin, kanF gene encodes the first glycosyltransferase which acts both as glucosyltransferase and N-acetyl-glucosaminyl-transferase in kanamycin biosynthetic pathway of Streptomyces kanamyceticus. The recombinant expression of kanF gene in E. coli BL21 (DE3), BL21pLysS and BL21-CodonPlus® (DE3)-RP under T7 promoter-based system showed the expression in insoluble rather than soluble form. Further analysis of the codons revealed that this gene includes number of rare codons which are difficult to be translated in E. coli. Due to variations in codon usage between E. coli and Streptomyces, with high free energy of secondary structure of mRNA, KanF was not expressed under various condition tested. Thus, using codon optimized gene based on codon usage of E. coli has helped to overcome this problem and led to soluble expression of protein. Thus obtained KanF was used for making in vitro reaction for glycosylation of 2-DOS and the product was confirmed to be 2′ -Deamino-2′-hydroxyparomamine by high resolution Q-TOF mass analysis.

Sialylation regulates the in vivo half-life of recombinant therapeutic glycoproteins, affecting their therapeutic efficacy. Levels of the precursor molecule cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) are considered a limiting factor in the sialylation of glycoproteins. Here, we show that by reducing the amount of intracellular CMP-Neu5Ac consumed for glycosphingolipid (GSL) biosynthesis, we can increase the sialylation of recombinant human erythropoietin (rhEPO) produced in CHO cells. Initially, we found that treating CHO cells with a potent inhibitor of GSL biosynthesis increases the sialylation of the rhEPO they produce. Then, we established a stable CHO cell line that produces rhEPO in the context of repression of the key GSL biosynthetic enzyme UDP-glucose ceramide glucosyltransferase (UGCG). These UGCG-depleted cells show reduced levels of gangliosides and significantly elevated levels of rhEPO sialylation. Upon further analysis of the resulting N-glycosylation pattern, we discovered that the enhanced rhEPO sialylation could be attributed to a decrease in neutral and mono-sialylated N-glycans and an increase in di-sialylated N-glycans. Our results suggest that the therapeutic efficacy of rhEPO produced in CHO cells can be improved by shunting intracellular CMP-Neu5Ac away from GSL biosynthesis and toward glycoprotein sialylation.