Earticle

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.

Many natural polysaccharides and derivatives isolated from various organisms such as marine seaweeds, mushrooms, plants, and microorganisms have attracted considerable attention in recent years as a rich and promising source of bioactive natural compounds in the fields of pharmacology and health beneficial foods due to their diverse biological activities, including antioxidant, antitumor, immunomodulatory, anti-inflammation, anticoagulant and antithrombotic, antibacterial, and antiviral activities. Among natural polysaccharides, sulfated polysaccharides (SPS) that contain hemi-ester sulfate groups in their sugar residues are commonly found in three major groups of marine algae, red algae (Rhodophyta), brown algae (Phaeophyta) and green algae (Chlorophyta). The major SPS of red algae are galactans commercially known as agar and carrageenan, and those of brown algae are fucans, including fucoidan, ascophyllan, sargassan, and glucuronoxylofucan. In contrast to those of red and green algae, the major SPS of green algae are more heterogeneous in sugar compositions, three main groups are glucuronoxylorhamnans, glucuronoxylorhamnogalactans and xyloarabinogalactans. Apart from industrial uses, these algal polysaccharides have emerged in recent years as a rich and important source of bioactive natural compounds, due to their diverse pharmacological activities including immune-modulatory, anticoagulant, antithrombotic, anti-mutagenic, antioxidant, anti-inflammatory, antitumour, antiprotozoal, antimicrobial, antiviral activities, etc, which are prerequisites of pharmaceutical and medicinal applications. For example, fucoidans, one of the most extensively studied SPS isolated from brown seaweeds, are known to exhibit a wide range of physiological and biological activities, thus medically useful activities, such as anti-inflammatory, antiviral, anticoagulant, antitumor, antioxidant, and antiangiogenesis activities. For these reasons, production and applications of these acidic polysaccharides as therapeutic agents have been increasingly important topics of intensive researches. Anticoagulating and antithrombotic activities are among the most widely studied properties of SPS for alternative sources of heparinoid-active compounds as therapeutic agents, due to several side effects of currently used heparin. Many SPS isolated from marine seaweeds have been reported for their antiviral activities against animal viruses, such as HSV types 1 and 2, human immunodeficiency virus type-1 (HIV-1), influenza virus, and human cytomegalovirus (HCMV). Although the use of these polysaccharides and oligosaccharides as drugs is in its infancy, currently, some are already in various phases of clinical trials as microbicides. An Undaria pinnatifida extract comprising predorminantly sulfated galactofucan was patented for its in vivo antiviral activity including HSV-1. Therefore, the potential of these SPS derived from marine algae as therapeutic agents in, e.g. antiviral medication, cancer therapy or blood clotting surveillance, becomes of great interest. Generally, these biological activities are related to structure, composition and sulphate substitution of polysaccharide macromolecules. However, it is now commonly accepted that the structures and chemical composition, including sugar composition, degree of sulfation, and molecular mass of these algal polysaccharides vary depending on the algal species, their harvesting time and regions of cultivation as well and thus must give rise to variation in the degree of most biological and pharmacological activities. Therefore, the SPS of Korean seaweeds may have different structures and thus different bioactivities from those of similar seaweeds harvested in other countries. In this context, in the course of our long-term research goal in searching for new and especially carbohydrate-based drug candidates and/or health beneficial foods materials from natural compounds, we have isolated and purified sulphated polysaccharides (SPS) from Korean seaweeds, Undaria pinnatifida and Capsosiphon fulvescens, and characterized their structures and biological activities such as immunomodulating, antiviral, anticoagulant, and antitumor activities. On the other hand, in spite of their potential as biologically active compounds, the high molecular mass and viscous nature of polysaccharides has hampered their application especially as a therapeutic agent. Therefore, a reliable SPS-degrading enzyme preparation would be highly desirable to obtain low-molecular weight oligosaccharides and thereby help overcome these problems. However, available data on, for example, purified fucoidanases remain scarce and no convenient commercial sources with fucoidanolytic activity are yet available for the degradation of these polysaccharides. Recently, we isolated and identified a marine bacterial strain that degrades the fucoidan (MF, Miyeokgui fucoidan) that we purified from the sporophyll of Korean Undaria pinnatifida. Subsequently, we purified this enzyme and produced low-molecular weight galactofuco-oligosaccharides (LMFOs), ranging from 1,389 to 3,749 Da and evaluated and compared their pharmacological activities, including immunomodulating, antiviral, anticoagulant, and antitumor activities, to those of intact high-molecular weight fucoidan, MF. Meanwhile, we have been working on other polysacchardies isolated from hyaluronic acids (HA), mushrooms, and plants like mulberry fruits. Some data on the structures and biological activities including immunomodulating, anti-inflammatory and anti-asthma, and anti-obesity effects of these polysaccharides will be discussed as well. (Recent publications on structures and biological activities of polysaccharides and oligosaccharides)

The International Society of Rare monosaccharide (ISRS) defines rare monosaccharides as monosaccharides and their derivatives that rarely exist in nature. Rare monosaccharides have recently gained increasing attention due to their possible applications as low-calorie sweeteners, bulking agents, antioxidants, inhibitors of microbial growth and glycosidases, nucleoside analogues, and immunosuppressants. Generally, rare monosaccharides have been mainly produced by chemical synthesis. However, the chemical processes have many disadvantages, including low overall yield, many reaction steps, complex purification steps, and by-product formation. Because of them, the bio-conversion of rare monosaccharides by enzymes have been recently attracted much attention as one of alternative production methods for rare monosaccharides to overcome these disadvantages. Among various enzymes, enzymes related sugar metabolism such as sugar phosphate isomerases and sugar isomerases have been paid much attention for the bio-conversion of rare monosaccharides because they are involved interconversion of various types of phosphate sugar or monosaccharides in the sugar metabolism. D-ribose-5-phosphate isomerase and D-lyxose isomerase in the enzymes related sugar metabolism have been reported especially useful enzymes to produce rare monosaccharides with their broad substrate specificities. From the study of the substrate specificities of the sugar isomerases, that has been known the configuration of hydroxyl group on the monosaccharides is one of the most important factor on the enzyme activity. The D-ribose-5-phospahte isomerase has enzyme activity for the aldose substrates with the C2 and C3 hydroxyl groups in the right-hand configuration (Fischer projections). In the case of D-lyxose isomerase, the aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration are the most proper substrates. The substrate specificities of the both enzymes could be applied to produce various kinds of rare keto- and aldo-monosaccharides having various biological functions.

As one of the most important compounds in nature, carbohydrates have attracted much attention in the fields of chemical biology, therapeutics, synthetic chemistry and so on. During the last 15 years, engineered glycosidases, including glycosynthases, thioglycoligases and O-glycoligases, have emerged as very powerful tools to address the synthesis of various functional carbohydrates, such as oligosaccharides, glycolipids and glycoproteins. These mutated glycosidase, whose catalytic residue (nucleophile or general acid/base) is replaced by an inactive residue, loss their hydrolytic activity and show significantly transglycosylation activities. Many elegant cases have been reported so far. Here, the different catalytic mechanisms and advances of engineered glycosidases in the synthesis of carbohydrates were summarized.

Pigmentation is the process of melanin synthesis by melanocytes within the skin and hair follicles. The melanin synthesized plays an important role in protecting the skin from the harmful effects of ultraviolet (UV) irradiation. However, excessive synthesis and uneven distribution of melanin can lead to esthetic problems. We synthesized several materials with and without kojic acids moiety which showed potent tyrosinase inhibitory activities. These molecules show good activity in antioxidant test. In some cases, treatment of melan-a cells with these compounds decrease in protein and m-RNA levels of tyrosinase along with tyrosine-rlated protein. These finding suggest that depigmenting materials is can be used as a potent whitening agent.

We developed a method that uses lectin affinity chromatography to enrich glycosylated proteins where the modification terminates with N-acetyl-D-glucosamine (GlcNAc), and used this method in conjunction with ETD MS/MS to identify Arabidopsis proteins with this type of modification. We found forty-eight proteins with N-linked modification consisting of single GlcNAc attached by an N-linkage to the Asn of the Asn-X-Ser/Thr sequon that is commonly modified with larger N-linked oligosaccharides. An enzymatic and chemical labeling method that changes the mobility of GlcNAc modified protein during PAGE through addition of PEG was employed to investigate the origin N-GlcNAc modifications on the β-thioglucoside glucohydrolases (TGG1 and TGG2), which are both modified with multiple N-linked oligosaccharides. It has been hypothesized that N-GlcNAc modifications are produced when ENGase remove N-linked oligosaccharides. Arabidopsis has two ENGases (AtENGase85A and B). Through the analysis of AtENGase85A, AtENGase85B single and AtENGase85A/B double mutants, we discovered that the N-GlcNAc modification of TGG2 was greatly reduced by AtENGase85A, but TGG1 was not affected in any of the mutants. These results support that ENGase producing N-GlcNAc modifications by hydrolyze N-linked oligosaccharides but also suggest that some Arabidopsis N-GlcNAc modifications may be generated by another mechanism. Since N-GlcNAc modification was detected at only one site on each myrosinase, the production of the N-GlcNAc modification may be regulated.

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 biomedical agents. To analyze their interaction, first, we modified diverse carbohydrate containing thiol group that was directly immobilized on gold cantilever and electrode. Herein, as the method to characterize interactive formation and binding specificity, we used atomic force microscopy (AFM) and electrochemical detection system. As measuring unbinding force from GM1-Vibrio cholera toxin as well as the analogue analyses, we understood how structural and binding positional differences in complex carbohydrate affect the interaction with protein and surmise that the GM1-ctxAB complex makes a “two-finger grip” formation through the conformational change of a flexible carbohydrate. In addition, we found that a potential shift at the maximum current occurred upon interaction with cholera toxin proteins through electrochemical detection system. By comparing results for different sizes of GM1 analogues, we surmise that the potential shift is closely associated with the specificity for the carbohydrate-protein interaction. In conclusion, using AFM force analysis, we successfully quantified and characterized the interactive configuration of carbohydrate-protein molecules and a carbohydrate-based electrochemical system could be used a tool for specific carbohydrate-protein interaction.

A 4-α-glucanotransferase (DgαGT EC 2.4.1.25, 4αGTase) gene was cloned from Deinococcus geothermalis and expressed in Escherichia coli, then recombinant protein was purified using nickel affinity chromatography. 4αGTase possesses intermolecular and intramolecular glucan transfer activity. 4αGTase have been used to produce cycloamylose(CA) from amylose by intramolecular glucan transfer activity. CA production by DgαGT was compared with those by Thermus scotoductus 4αGTase (TSαGT) and Escherichia coli 4αGTase (MalQ). Optimal CA production conditions were 50℃ and pH 6.0. After the 4αGTase treatment on amylose, residual linear maltooligosaccharides were removed by β-amylase. CA was analyzed using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectroscopy. DgαGT produced CA with similar yield 53% to the other enzymes but needed less time to start CA production. Degree of polymerization (DP) of CA produced by DgαGT ranged broadly from DP 5 to DP 43. Further analyses need to be conducted for concluding prominent characteristics of DgαGT among bacterial αGTs.

Obtaining chitosan oligosaccharide having higher molecular weight than the usually obtained oligosaccharides by enzymatic digestion of high molecular weight chitosan was performed in vitro using beta-cyclodextrin (beta-CD) as inhibitor for chitosanase hydrolysis activity. The concentration of beta-CD:chitosan was 5:1 respectively. Beta-CD is cyclic oligosaccharide consists of eight glucopyranose units linked by α-1,4 glycosidic bonds. It was capable of inhibiting chitosanase activity to 59.2% less activity at 37oC after 2 hr incubation and we got chitosan oligosaccharide with higher molecular weight that can be separated by HPLC. The antibacterial activity of this chitosan oligosaccharide was also investigated. By condensing the products of reaction mixture having 2.5% beta-CD+0.5% chitosan+chitosanase 5 times less volume to get final concentration of chitosan 2.5%, this sample demonstrated a remarkable antibacterial activity.

It has been reported that one of the downstream molecules generated from glucose is uridine diphosphate-N-acetly glucosamine(UDP-GlcNAc) via the hexoamine biopsynthetic 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 itself as well. O-GlcNAcylation on N-terminal domain of tetratricopeptide (TPR) repeats plays an important role in nuclear localization of ncOGT. Specific nuclear localization signals (NLS) in ncOGT has not been identified. We characterized the three amino acid motif as NLS because this motif is required for the nuclear import of non-diffusible β-galactosidase. Also, we show that ncOGT binds kayropherin α proteins, and the association between kayopherin α proteins and ncOGT is interfered by O-GlcNAcylation on TPR domain. Our finding suggests the mechanism how ncOGT can be localized in the nucleus and cytosol at the same time. Therefore our data may contribute to better understanding of the key enzyme of O-GlcNAc metabolism.

O-GlcNAc is a carbohydrate post-translational modification occurs on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. O-GlcNAcase (OGA) is the enzyme that catalyzes the removal of O-GlcNAc moiety on its substrates. It has two isoforms, full OGA and variant OGA, and in early reports it is known that full OGA exists both in cytosol and nucleus, primarily to the cytoplasm. However, there are two opposite reports about the subcellular localization of two OGA isoforms by different research groups. The first group figured out that full OGA localizes in cytoplasm, whereas variant OGA exists within nucleus in 2001. However, recently the other group reported that full OGA localized diffusely throughout the nucleus and cytoplasm, whereas variant OGA resides with lipid droplets in cytosol. So these two finding motivates our laboratory to commence this work as to nucleocytoplasmic shuttling mechanism of two OGA isoforms. We also focus on the various post-translational modifications on OGAs. Full OGA should weigh 103 kDa, but it weighs approximately 130kDa in reality. So we speculate that various modifications might be related to OGA’s subcellular distribution. To verify and get the answers to several issues about OGAs, we first overexpressed two FLAG tagged-OGA isoforms in HeLa cells to trace the subcellular localization of them. Then we made three C-terminal domain deletion mutants to know the critical domain for its localization. And we also check modification occurred on OGAs by using immune-precipitation. Consequentially we can get schematic drawing as to OGA’s nucleocytoplasmic shuttling, and we expect that this research makes it easy to understand and approach O-GlcNAc modification.

Hyaluronan (also called hyaluronic acid or hyaluronate) is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. Hyaluronan, a core component of the extracellular matrix, comprises a repeating oligosaccharides of N-acetylglucosamine and D-glucuronic acid. The purpose of this study was to investigate the anti-adipogenic effect of low-molecular weight hyaluronan (LMW-HA, 43,500 Da) on 3T3-L1 preadipocyte cells. LMW-HA did not show cytotoxicity on 3T3-L1 preadipocyte cells up to 200 mg/ml. Treatment of 3T3-L1 cells maintained in adipocyte-induction media with 25, 50, 100, and 200 mg/ml of LMW-HA resulted in significant decrease in intracellular lipid drops by 75.5, 65.4, 58.0, and 53.5%, respectively, suggesting that LMW-HA has anti-adipogenic effect on 3T3-L1 adipocyte differentiation. Triglyceride (TG) levels in the cells treated with varying concentrations of LMW-HA (25, 50, 100, 200 μg/ml) were significantly decreased in a dose-dependent manner, with 53% reduction at 200 μg/ml. However, incubation of cells with LWM-HA (25, 50, 100, and 200 mg/ml) significantly reduced the mRNA level of PPAR-γ by 81, 69, 58, and 56%, respectively, and also aP2 by 81, 67, 56, and 55%, respectively, compared to untreated control group. In addition, the protein levels of PPAR-γ, C/EBP-α and aP2 in LMW-HA treated groups also decreased in a dose-dependent manner. PPAR-γ, C/EBP-α and aP2 are known as the major adipocyte-specific genes. Therefore, these results suggested that LMW-HA has anti-obesity effect by inhibiting adipocyte differentiation through down-regulation of PPAR-γ, C/EBP-α and aP2 expression.

Seaweed extracts have been reported for their diverse biological and pharmacological activities including immunomodulating, anticoagulaning, anticancer activities, etc. Capsosiphon fulvescens (CF) is a green alga that is widely ditributed in the Southern coastal area of Korea. Crude polysaccharides were obtained from the CF collected at a coastal area of Wando, Korea by extraction in acidic condition (0.01 N HCl) and 75% ethanol precipitaion. After removal of alginate by CaCl2 precipitaion, it was futher purified by DEAE-cellulose column chromatography and its chemical components and anticancer activity was determined. Monosaccharide composition analysis by TLC and HPEAC-PAD showed that this polysaccharide consist of Rha, Man and Xyl as the major neutral sugars, with the mole ratios of 4.1:1.0:3.7, respectively, suggesting that it is a mannoxylorhamnan type polysaccharide. This polysaccharide (1 mg/ml) was shown to effectively scavenge nitrite up to 40% at pH 3. The effects of this polysccharide on the cell viability of 4 cancer cell types, human prostate cancer cell (PC-3), human lung cancer cell (A-549), human colon cancer cell (HT-29), and human gastric cancer cell (MK-N-45), were evaluated by MTT assay. It showed most significant cytotoxicity against HT-29 cells among cancer cell lines tested. This polysaccharide significantly induced the cleavage of PARP, caspase 8 and caspase 9, and also enhanced the phosphorylation of p38 in a dose-dependant manner. However, it was shown to reduce the AKT phosphorylation and mitochondrial membrane charge causing mitochondrial dysfunction. Taken collectively, these results demonstated that a watersoluble polysaccharide isolated from the sporophyll of Korean Capsosiphon fulvescens inhibits proliferation of HT-29 cells by activation of apoptotic pathway, suggesting that this polysaccharide can be a good candidate for the development of a potent anti-cancer agent against human colon cancer.

In spite of an increasing interest in fucoidans as biologically active compounds, no convenient commercial sources with fucoidanase activity are yet available. A soil fungal strain that showed confluent growth on a minimal medium containing Miyeokgui fucoidan (MF), prepared from Korean Undaria pinnatifida sporophylls, as the sole carbon source was isolated and identified based on a 28SrDNA sequence analysis as a strain of Coniochaeta velutina, and thus tentatively named Coniochaeta velutina PF-2. A cell-free culture supernatant and intact cells of strain PF-2 were examined for the presence of fucoidanolytic activity. The increase in reducing sugars when adding the intact cell into the reaction mixture containing MF as the enzyme substrate was negligible, suggesting that the intact cell did not contain any fucoidan-degrading activities. The culture supernatant depolymerized MF (2,100 kDa) to one major medium-molecular mass fucose-containing oligosaccharides (MMFO-1), having molecular weight ~ 165.5 kDa. However, supernatant neither released the monomer L-fucose from the fucoidan nor hydrolyzed the chromogenic substrate p-nitrophenyl-α-L-fucoside, indicating that the enzyme may be an endo-acting fucoidanase rather than an α-L-fucosidase. HPAEC-PAD analysis of the TFA-hydrolyzed MMFO-1 demonstrated that the MMFO-1 consists of fucose, galactose, xylose and mannose, 504:413:4:4 respectively (mole ratios). The FT-IR spectra of MF and MMFO-1 showed no significant structural difference except for about 10% reduced level of sulfate esters in MMFO-1. This would appear to be the first report on fucoidanolytic activity by fungal strain Coniochaeta species. Moreover, this enzyme activity may be very useful for structural analyses of fucose-containing polysaccharides and the production of bioactive galactofuco-oligosaccharides.

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13% of chicken egg-white proteins and is known to be a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. This study used a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion to investigate the N-glycan type and branching pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative amount of each glycan. Seventeen N-glycans were identified, including 11 glycans (1core structure and 10 complex-type oligosaccharides) that are common to both OT and OM. Six characteristic glycans (two truncated structures, one complex-type oligosaccharide, and three hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures : mono- (13.2%), bi- (23.9%), tri- (9.0%), tetra- (2.7%), and penta- (2.8%) antennary oligosaccharides; whereas OM contains mostly tri- (33.5%) and penta- (31.2%) antennary oligosaccharides. The N-glycan-containing bisecting N-acetylglucosamine comprised 43.4% and 79.8% of the total glycans in OT and OM, respectively. Moreover, circular dichroism analysis revealed that the secondary structure of the deglycosylated OT differs markedly from that of the intact protein. To our knowledge this is the first study to compare N-glycans between OT and OM.

The carbohydrates in natural plant materials obtained from roots, leaves, stem barks, and seed were investigated. All of the plant materials were dried, and their constituents were isolated by room temperature-water or hot-water extraction. Ethanol precipitation was also used to produce solutions containing the constituents for analysis. Mono- and disaccharides were separated using a high-pH anion-exchange chromatography system equipped with a CarboPac PA10 column and an AminoTrap column working at an isocratic NaOH concentration of 20 mM and at a flow rate of 0.5 ml/min at room temperature for 40 min. Carbohydrates were detected by a pulsed amperometric detector. The concentration of each carbohydrate was quantified by calculation from a calibration curve constructed using a standard mixture containing twofold dilutions from 1,600 pmol of each carbohydrate. Enzymatic deglycosylation of the plant extracts was also performed. Namely, each sample was incubated in the presence of 40 μM trypsin and 40 μM chymotrypsin in 10 mM Tris-HCl buffer (pH 8.0) at 37°C for 18 h. N-glycans were then released from glycopeptides after reaction with 0.1 mU of glycoamidaseA in citrate-phosphate buffer (pH 5.0) at 37°C for 18h. Peptides and salts were removed using graphitized carbon cartridges. The released glycans were lyophilized and stored at –20°C. The N-glycan structures were analyzed by normal-phase high-performance liquid chromatography of 2-aminobenzamide-labeled glycans in combination with exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The functions of the identified and isolated chemical constituents were also investigated.

Plant expression system for mass production of highly valuable recombinant therapeutic proteins has several advantages over other existing expression systems with economical and safety issues. Immunotherapy of multiple monoclonal antibodies, which can recognize multiple targeting including specific proteins and their glycans highly expressed on the surface of cancer cells, can be an efficient treatment compared to a single targeting immunotherapy using a single antibody. In this study, we have established plant production system to express two different targeting monoclonal antibodies in a single transgenic plant through crossing fertilization between two different transgenic plants expressing anti-colorectal cancer mAb CO17-1A (mAb CO) and anti-breast cancer mAbBR55 (mAb BR), respectively. The F1 seedlings were obtained cross fertilization between the two transgenic parental plants. The presence, transcription, and protein expression of heavy chain (HC) and light chain (LC) genes of both mAbs in the seedlings were investigated by PCR and immunoblot analyses, respectively. The seedlings with presence and transcription of HC and LC genes of both mAbs were selected, and the selected seedlings were confirmed to have relatively stronger density of HC and LC protein bands compared to the transgenic plant expressing only each mAb. N-glycan structure analysis will be conducted to confirm the glycan structure of multiple monoclonal antibodies mAbP CO x BR. These results indicate that the F1 seedling plant with carrying both mAb genes was established. Taken together, plant crossing fertilization can be applied to generate an efficient production system expressing multiple monoclonal antibodies for immunotherapy in a single plant.

Genome editing is one of the most demanding tools in mammalian cells to unravel the role of genes and to implement gene therapy of genetic diseases. Zinc finger nuclease is an engineered restriction enzyme which is constructed by combining an array of zinc finger DNA-binding motifs in the N- terminus and the FokⅠ endonuclease in C-terminus. Almost every ZFN recognizes three consecutive nucleotides starting with guanine in a context-dependent manner, thereby limiting the use of ZFN in genome editing. To address this challenge, we replaced ZFNs with a gene-specific nucleotides, which were connected to FokⅠ nuclease by using (his)6-tag and GS linkers. We tested in vitro whether this engineered FokⅠ shows a site-specific nuclease activity. For this, the engineered FokⅠ and nucleotide complex was incubated with template DNA which was produced from 70-mer and 100-mer nucleotides base-paired each other. The results indicate that the complex showed a site-specific nuclease activity, which suggests that the nucleotide-based FokⅠ can be used for the universal genome editing.

The development of cancer biomarker is of a great promise to conquer cancer since it leads to an early detection and provide an opportunity for better cancer treatment. Validation is a time-consuming step for biomarker developments requiring investigation of biomarker candidates using thousands or more of biosamples, which is why a sensitive, multiplexing validation method is necessary. To address this challenge, we are developing as antibody-based validation method, in which antibody with oxidized N-glycans was covalently linked to an identifiable DNA tag for use of the template in the transcription by T7 polymerase. The transcripts will be quantified and used for an indicator of the amounts of the antigen bound to the DNA-tagged antibody. This strategy will be multiplexed for an establishment of a novel validation method.

Anoikis is an anchorage-dependent cell death. Anoikis resistance is known to be a crucial step for cancer metastasis. We have previously identified that N-Acetylglucosaminyl transfer ase-V (GnT-V) is a key protein inducing metastasis in human colon cancer cells. And the secretion of galectin-3 binding protein (LGALS3BP) was lowered in GnT-V overexpressing WiDr cells. In this study, we hypothesized that GnT-V has an inhibitory effect on anoikis of cancer cells, while LGALS3BP stimulates the apoptotic process. To prove this, we established the stable transfectants (WiDr:GnT-V), the control cells (WiDr:mock). As a result, GnT-V was observed to mitigate anoikis and promote cell aggregation in soft- agar plate system. Among human colon cancer cells tested, HCT116 cells formed aggregates poorly and thus showed low cell viability. Of note, the secretion of LGALS3BP was higher than any other colon cancer cellline, which is in line with the result that anoikis resistance was observed in LGALS3BP-suppressed WiDr cells. Taken together, these results may suggest that GnT-V induces anoikis resistance of cancer cells either alone or in cooperation with the target molecules, such as LGALS3BP.

Colorectal cancer is the third most commonly diagnosed cancer in the world, nearly all patients diagnosed with this cancer die from it. Antibodies are glycoprotein molecules, which can efficiently recognize and eliminate specific pathogenic and disease antigens. Antibody researches for the last several decades have demonstrated the potential of therapeutic antibodies to fight cancer. Monoclonal antibody (mAb) CO17-1A recognizes the tumor-associated antigen GA733-2, a cell surface glycoprotein highly expressed in colorectal carcinoma cell, which is applicable for preventing and curing colorectal cancer. We have currently established baculovirus insect cell expression system to produce anti-colorectal cancer mAb CO17-1A. In this study, mAb CO17-1A was expressed in the transgenic insect cell line SWT4, which has humanized glycosylation processing pathway. Immunoblot confirmed that mAb CO17-1A properly expressed in SWT4. mAb CO17-1A was purified using protein G affinity column. In addition, Maldi-TOF verified that the mAb fused to KDEL, ER retention signal had high mannose type of glycan structure whereas the mAb without KDEL had partially humanized glycan structure. These results suggest that the insect cell expression system with the SWT4 possibly can be used as a useful alternative way to produce full-size mAb with humanized glycan structures for cancer immunotherapy.

The antigen GA733 is a cell-surface highly expressed glycoprotein on most human colorectal carcinomas. GA733 can be characterized as a cancer vaccine. In this study, GA733 was fused to the human immunoglobulin IgG Fc fragment to become recombinant gene GA733-Fc. Based on this, 4 recombinant genes were constructed as follows: GA733-Fc with signal peptide sequence and fusion of ER retention sequence (KDEL) (spGA733-FcK), GA733-Fc with signal sequence (spGA733-Fc), GA733-Fc fused to ER retention sequence (GA733-FcK) without signal peptide and GA733-Fc without signal peptide. Baculovirus-insect cell expression system is widely used for the high level production of recombinant proteins especially for glycoproteins. Constructed 4 recombinant genes were cloned to baculovirus express vectors. DH10Bac E.coli.-mediated transformation was used to generate recombinant bacmid DNA. Recombinant DNA was confirmed by PCR. Insect cell was transfected by bacmid to produce the recombinant baculovirus infects insect cell to produce recombinant protein. Western blot and sandwich ELISA showed the expression of recombinant proteins. Each cell lines (sf9 and HighFive) differed in recombinant proteins production level and protein secretion capability. N-Glycosylation analysis showed the function of signal peptide and ER retention sequence (KDEL). Taken together, baculovirus-insect cell system can be used to express recombinant GA733-Fc glycoproteins.

O-antigen of Vibrio cholerae O1 is controlled by ABC transporter-dependent pathway. O-antigen synthesis controlled by this pathway is initiated by a homolog of WecA, which catalyzes the transfer of GlcNAc-1-P from UDP-GlcNAc (donor) to undecaprenyl phosphate (acceptor). However, it speculates that an initial O-antigen synthesis of V. cholerae O1 is regulated by a homolog of WbaP (undecaprenyl phosphate galactose-1-phosphate tranferase). In this work, we characterized new glycosyltransferase WbeW from V. cholerae O1. Sequence and topology analyses of WbeW revealed that the protein might be a member of polyisoprenyl phosphate hexose-1-phosphate tranferase (PHPT) family. Through studies of subcellular location of His6-WbeW protein and its derivatives, which deleted with several amino acids from N-terminus, we found that the His6-WbeW protein was associated to membrane and its membrane association might be affected by diverse factors as well as α-helix transmembrane domain. The activity assay showed that the protein catalyzes the transfer of galactose-1-phosphate from UDP-Gal to undecaprenyl phosphate. This is the first report that an initial glycan of O-antigen formed by ABC-transporter-dependent pathway is galactose but not N-acetylglucosamine. This indicates that there might be a new mechanism of ABC-transporter-dependent pathway-controlled O-antigen synthesis.

Surface plasmon resonance (SPR) can provide kinetic information about an interaction, and it can also be used to rapidly monitor dynamic processes, such as adsorption and degradation, without the need for sample labeling. Here, we employed SPR to analyze carbohydrate-protein interactions, particularly GM1-related carbohydrate-Vibrio cholera toxin interactions. The interaction between cholera toxin subunits A (ctxA) and B (ctxB) was similar to general ligand-receptor interactions. After the direct immobilization of thiol-containing GM1 pentasaccharide on a gold surface, the GM1-ctxB interaction kinetics were evaluated, and they showed a similar degree of kinetics as reported in previous reports. We found that ctxA had a high affinity for the GM1-ctxAB complex, although its equilibrium dissociation constant was 10-times lower than that of GM1-ctxB binding. Comparative analyses for GM1-related carbohydrates-ctxAB interactions were also conducted to determine the kinetic values of several GM1 analogues with different structures, although their kinetic values were one-order of magnitude lower than those of the GM1-ctxAB interaction. The kinetic analysis results for the interactions of GM1 analogues and ctxAB indicated that the sialic acid thumb is important for recognition, and the terminal galactose and N-acetylgalactosamine finger are required to stabilize the GM1-ctxAB interaction. Taken together, our results indicate that the direct immobilization of carbohydrate in an SPR-based analytical system can be used to evaluate the structural contribution of carbohydrate moieties in carbohydrate-protein interactions, as well as provide valuable information that can be used to understand the interactions.

Fucoidans, a group of fucose-enriched sulfated polysaccharides isolated from brown algae and marine invertebrates, have been shown to exert anticancer activity in several types of human cancer, including leukemia, breast cancer, and lung adenocarcinoma cells. In the present study, the anticancer activity of the fucoidan (MF) extracted from the sporophyll (called Miyeokgui in Korea) of brown seaweed Undariapinnatifidawas investigated in human prostate cancer PC-3 cells. PC-3 cells exposed to fucoidan displayed growth inhibition and several typical features of apoptotic cells, such as activation of caspases and mitochondrial dysfunction including depolarization of the mitochondrial membrane potential (MMP, Δψm), and concomitant decreased expression of antiapoptotic Bcl-2 family proteins. Furthermore, treatment of PC-3 cells with fucoidan (10, 50, 100, 200 μg/ml) resulted in a dose dependent induction of MAP kinases including extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 and an inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway. These result revealed that the Miyeokgui fucoidan has a potent anticancer activity in vitro against human prostate cancer by triggering apoptotic cell death through activation of MAPKs (ERK, JNK, p38) and inactivation of PI3K/Akt pathways as well as mithchondrial dysfuction.

Methylation and glycosylation are two different but vital modification processes in plants to generate diversified secondary metabolites. Methylation increases the lipophilicity of compounds whereas glycosylation enhances the solubility. Most of the methylated and glycosylated natural compounds are significantly active against pathogens and cancers. In this study, an O-methyltransferase gene, designated as SpOMT-2884, was identified from Streptomyces peucetius ATCC 27952. To find the exact substrates for SpOMT-2884, we tested several compounds in-vitro. As a result SpOMT-2884 catalyzed O-methylation of flavonoids such a 7,8-dihydroxyflavone (7,8-DHF), quercetin, luteolin, fisetin and rutin. 7,8-DHF was found to be the best substrate. We further proceeded for in vivo biotransformation of 7,8-DHF where we used E. coli BL21 (DE3) expressing SpOMT-2884 cell, as a biocatalyst for the production of methylated derivative of 7,8-DHF. The supplementation of 0.2 mM of 7, 8-DHF in the growing induced culture of E.coli BL21 (DE3) harboring pET28-SpOMT-2884 recombinant resulted in the production of 7-methyl-8-hydroxyflavone which was confirmed by HPLC (Rt: 17 min), high resolution LC-QTOF-ESI/MS (m/z+ 269.08) and NMR spectroscopy. Further, this enzymatically synthesized methylated derivative of 7, 8-DHF was used as a substrate in vitro for glycosylation by Yjic, a glycosyltransferase from Bacillus licheniformis DSM13. This in-vitro reaction mixture analysis revealed the presence of glycosylated product which was confirmed by HPLC and LC-QTOF-ESI/MS (m/z+ 431.13). The glycosylation of the target was further supported by our findings from in silico docking analysis. Inparticular, this study demonstrated the potential for enzymatic biosynthesis of novel methylated cum glycosylated 7,8-DHF.

We successfully produced astragalin (AST) from regiospecific modifications of naringenin (NRN) in Escherichia coli BL21(DE3). The exogenously supplied NRN was converted into dihydrokaempferol (DHK) and then kaempferol (KMF) in the presence of flavanone-3-hydroxylase (f3h) and flavonone synthase (fls1) from Arabidopsis thaliana, respectively. KMF was further modified to produce AST by 3-O-glucosylation utilizing the endogeneous UDP-glucose in presence of UGT78K1 from Glycine max. To increase the intracellular UDP-glucose concentration by channeling the carbon flux toward UDP-glucose at the branch point of glucose-6-phosphate (G6P), the chromosomal glucose phosphate isomerase (pgi) and D-glucose-6-phosphate dehydrogenase (zwf) were knocked-out in E. coli BL21(DE3). The two enzymes directly involved in the synthesis of UDP-glucose from G6P, phosphoglucomutase (nfa44530) from Nocardia farcinia and glucose-1-phosphate uridylyltransferase (galU) from E. coli K12 were overexpressed, which successfully diverted the carbon flow from glycolysis to the synthesis of UDP-glucose. Furthermore, to prevent the dissociation of UDP-glucose into UDP and glucose, the UDP-glucose hydrolase (ushA) was deleted. The E. coliΔpgiΔzwfΔushA mutant harboring the UDP-glucose biosynthetic pathway and the aforementioned genes for the regiospecific glucosylation produced 109.3 mg/L (244μM) of AST representing 48.8% conversion from 500 μM of NRN in 60 h without any supplementation of extracellular UDP-glucose.

Glycodiversification and glycorandomization, invaluable tools for generating biochemical diversity, can be catalyzed by flexible glycosyltransferases (GTs), which attach various activated sugar “donors” onto different type of “acceptor” molecules. Among discovered and successfully developed protein engineering techniques, creation of hybrid protein have been evaluated as very powerful approach to produce enzyme with new activity. Theoretically, hybrid protein was built up by different fragments from various parent molecules. Particularly, engineered GTs have been used as wonderful catalysts for making new glycosylated compound, for example, the generation of new hybrid glycopeptides or glycosylation of scopoletin by GT from Arabidopsisthalianaorproducing novel urdamycin derivatives, etc. Applying protein modeling, we have constructed a hybrid protein HT1 that is composed of DnrS-derived N-terminal and DesVII-originated C-terminal. The enzyme activity was tested using various activated sugar produced Escherichia coli system (such as TDP-rhamnose, UDP-xylose and UDP-6-deoxy-allose) in combination with the transformation of phenolic substrate (flavonoid, stilbene, quinone, etc). The initial result showed that hybrid protein significant change in regio-specific activity in comparison with parent molecule (DnrSandDesVII), particularly, enabling produce flavonol and flavanone-O-rhamnoside such as quercetin, kaempferol, myricetin, andnaringenin-O-rhamnoside. The experiment of glyco-diversification is continuously carrying out to determine the level of enzyme’s promiscuity and which type of the most favorite substrate was recruited. Therefore, the chimeric protein HT1 is apromising tool to divert the natural products. Furthermore it is possible to improve or alter enzyme activity by domain swapping from far related glycosyltransferases.