Earticle

Insect cell lines have been used extensively for the production of numerous functional eukaryotic proteins. However, insect cell systems have limited commercial use due to a lack of complex glycosylation. Glycosylation is very crucial factor for use of pharmaceutical proteins. While, baculovirus is used as a strong gene delivery carrier for transfection of insect cells. Especially, Drosophila S2 cells have been developed as a plasmid-based and non-lytic expression system using recombinant baculovirus, infects into insect cells and does not require any selection markers. A gene encoding human erythropoietin (hEPO) or human 90K (h90K) fused with a hexa- histidine (His6) tag under the control of the Drosophila originated metallothionein promoter (pMT) was stably transfected into Drosophila S2 cells. In the preliminary studies, we found that expressed proteins had simple N-glycan structures instead of complex structures. We thought that this result caused by N-acetylglucosaminidase (GlcNAcase) through enzyme assay experiment. Therefore, this research will propose that suppression of GlcNAcase and several expression of N-glycosyltransferases using recombinant baculovirus- based multiple protein expression platform.

We describe a simple and rapid chemical strategy for preparing carbohydrate microarrays and utilize these arrays for the characterization of carbohydrate-protein interactions. Carbohydrate microarrays were prepared by reductive amination-mediated immobilization of amine-modified carbohydrate onto NHS-activated glass slide. The carbohydrate conjugates were confirmed by MALDI-TOF MS and 1H NMR. Carbohydrate microarray presenting six disaccharide were evaluated by profiling the binding specificities of several lectins. The results showed that the array selectively bound the corresponding lectins with relative binding affinities. We identified lectin and carbohydrate detection limit concentration on this array using WGA(Wheat Germ Agglutinin) as lectin and N,N'-diacetylchitobiose(GlcNAcβ-12Man) as carbohydrate. We also evaluated interactions between cholera toxin B subunit and GM1, GM1 analogues such as asialo GM1, GM3 on this array. As a result, GM1 pentasaccharide showed a strong affinity and specificity in interaction, but asialo GM1 and GM3, which is lacking of terminal Neu5Ac and galactose-N-acetylgalactosamine, respectively, showed a low affinity. We proposed that this carbohydrate microarray system could be applicable for feasible studies, like sensing of pathogenic bacterium or detection of toxin protein.

The glycosylation of therapeutic glycoproteins can affect their efficacy, stability, solubility, and half-life. Especially, the analysis of mono- and oligosaccharide on the glycoproteins are an important part of glycoprotein characterization, because sialylation or fucosylation in oligosaccharides often causes dramatic changes in the function of glycoproteins. Analyzing the composition of monosaccharides, such as that of neutral, amino sugars and sialic acids, is the first step for elucidating the structure of glycan attached to glycoproteins. In the present study, monosaccharides obtained from glycorproteins were analyzed using an enzyme-linked lectinsorbent assay(ELLA) and high-performance anion exchange chromatography with pulsed amperometric detection(HPAEC-PAD). Peroxidase-labeled lectins such as concanavalin A, Ricinus communis agglutinin, and soybean agglutinin were used for ELLA, since they specifically bind to the monosaccharide residue most frequently encountered in a glycan. We also prepared pyridylamino-oligosaccharides from therapeutic glycoproteins, and their structural analysis was performed using 3D mapping method. The present results demonstrate that ELLA, HPAEC-PAD, and 3D mapping are very effective methods for rapidly estimating the types and relative amounts of monosaccharides and structural analysis of oligosaccharide in intact glycoproteins.

Even though more than 90 lectins have been extracted from legumes, few of them bind specifically to sialic acid derivatives. In the present study, a sialic acid-binding protein has been purified from the bark of legume Maackia fauriei using fetuin-affinity chromatography. The eluted fractions from affinity column exhibited hemagglutination activity, thus they are designated as M. fauriei agglutinin (MFA). A typical purification method could produce at least 25.5 mg of pure MFA with a specific activity of 40.2 and a recovery yield of no less than 4.9%. A single N-terminal 20-amino acid sequence of MFA, SDELSFNINNFVPNQADLLF, was determined on an automatic Edman degradation amino acid sequencer, and it exhibits a high homology with lectins from M. amurensis which binds to sialylated oligosaccharide Neu5Aca2-3Galb1-3(Neu5Aca2-6)GalNAc. The hemagglutination activity of MFA with human erythrocytes was specifically inhibited by sialylated trisaccharide Neu5Aca2-3Galb1-4GlcNAc, but it was not inhibited by either Neu5Aca2-6Galb1-4GlcNAc or Neu5Aca2-3Galb1-4Glc/Neu5Aca2-6Galb1-4Glc. This activity of MFA was dependent on divalent cations. Many plant lectins are cytotoxic against cancer cells, with the effect differing with cell type; however, the mechanisms of action remain poorly understood. MFA exerts cytotoxic effects on human breast cancer MCF-7 cells, human melanoma G-361 cells, and human liver cancer SNU-449 cell lines but had no effect on the human colorectal cancer SNU-C1 cell line. It is especially noteworthy that the deleterious effect of MFA on the viability of MCF-7 was greater than that of MAH or wheat germ agglutinin.

Insect cell expression system using baculovirus has several benefits from high capacity, flexibility, safety to humans and glycosylation capability. Thus, the baculovirus insect cell system has been widely used for production of recombinant protein. In this study, the insect cell expression system was designed to produce anti-cancer mAb CO17-1A, which recognizes the antigen GA733 highly expressed on the surface membrane of colorectal carcinoma cells. The heavy (HC) and light chain (LC) genes of the mAb CO17-1A were cloned under the control of two different promoters, P10 and PPH, respectively, on baculovirus expression pFastBacTM Dual vector. The gene expression cassettes carrying HC and LC genes were transferred into a parent bacmid in E.coli (DH10Bac). The bacmid was transfected to Sf9 insect cells to generate the baculovirus expressing mAb CO17-1A. Western blot and immunoflouorescence analyses confirmed expression and secretion of mAb CO17-1A in the virus infected insect cells. The optimum condition for mAb expression was optimized at 24, 48 and 72hr after the virus infection with MOI (optimum virus concentration) ranging (0.2, 1 and 5). Expression of mAb CO17-1A in insect cells significantly increased at both 48 and 72hr after transfection with the MOI 1. HPLC chromatography revealed that the insect-derived mAb CO17-1A had insect specific glycan structures. Cell ELISA showed that the purified mAb from insect cell cultured media had a specific binding activity to SW948 human colorectal cancer cell. These results indicated that the baculovirus insect cell system is able to express, assemble, and secrete functional full size monoclonal antibody with insect specific glycosylation.

N-glycosylation is a 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. Plant type complex N-glycans are distinctive from those found in mammalian because they contain β1,2-xylose and core α 1,3-fucose residues attached to the pentasaccharide (Man3GlcNAc2) core structure but no sialic acid residues. The presence of β1,2-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 (α1,3-fucosyltransferase I, α1,3-fucosyltransferase II, β 1,2-xylosyltransferase, β1,3-galactosyltransferase and α1,4-fucosyltransferase). We made double, triple and quadruple mutants by crossing the mutants, and quintuple mutant is on the construction. The triple (fuct1/fuct2/xylt) and quadruple (fuct1/fuct2/xylt/fuct3) mutants do not show significant developmental defects in a normal growth condition and they do not produce the plant specific sugar residues on the N-glycan. The resulting mutant will be transformed by human α1,6-fucosyltransferase and β1,4-galactosyltransferase genes to accomplish further humanized N-glycosylation in plant. [Supported by BK21 program]

A number of environmental and physiological stimuli, such as perturbation in calcium homeostasis, shift of endoplasmic reticulum (ER) redox potential, altered glycosylation and elevated protein synthesis lead to accumulation of unfolded or misfolded proteins in the ER lumen, and subsequently impose stress to ER. Such condition activates a set of signaling pathway termed the unfolded protein response (UPR). To date, a large number of components for UPR signaling pathway has been isolated and characterized in yeast or mammals. Plants also show a quite similar response compared to the UPR in yeast and mammals when they are exposure to the stimuli. However, the plant UPR and its signaling pathway are rarely studied. In this report, we present our research regarding to the characterization of a UPR signaling component AtXBP1 in Arabidopsis. AtXBP1 is alternatively spliced generating two isoforms; long isoform (AtXBP1L) and short isoform (AtXBP1S). Overexpression of AtXBP1S has shown to up regulate the expression of ER stress responsive genes. Futhermore, expression of the luciferase gene, driven by BiP3 promoter, under the ER stress condition has been significantly decreased in the atxbp1 mutant. Electrophoretic mobility shift assay revealed that only AtXBP1S is able to interact with putative XBP1 binding cis-elements in AtBiP2 and AtBiP3 promoters. These results support our hypothesis that the AtXBP1 is an important regulatory component in the UPR signaling in Arabidopsis. [Supported by BK21 program]

Surface (or biosurface) organic chemistry is an emerging research field in organic chemistry, the ultimate goal of which is to control the interfaces between biological and non-biological systems at the molecular level for the fundamental understanding of biological interactions at interfaces and for the potential applications to (nano)biotechnology and biomedical sciences. The term, “biosurface”, implies that the field focuses on surfaces (or interfaces in general) and the surfaces are functional in the interactions with biological entities, such as biomolecules (DNAs, proteins, and polysaccharides) and cells. The biological interactions would occur at nonbiological (in other words, man-made) surfaces via either organic or inorganic functionalities, and in the field of biosurface organic chemistry, organic functional groups are designed and introduced onto man-made surfaces to control the biological interactions. Biosurface organic chemistry is composed of three mutually interacting research fields: surface organic chemistry, bioconjugation, and micro/nanofabrication. One of the aims of surface organic chemistry is to control the physicochemical properties of man-made surfaces by the functionalization of surfaces, yielding “tailor-made” surfaces. The tailor-made surfaces could be either “static”, playing any designated roles, or “dynamic”, playing switchable roles in response to demands. In this talk, I will present some of research results in the field of biosurface organic chemistry. Because of the wide use of self-assembled monolayers (SAMs) in surface sciences and technologies, a large portion of the review contains research results based on SAMs.

Plant genetic engineering led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current animal expression systems. In this study, the expression level of mAbP SO57 between with and without Lys-Asp-Glu-Leu (KDEL) was compared in transgenic plant. PCR and PT-PCR analyse showed stable gene insertion transcription of heavy and light chain genes of mAbP SO57 with or without KDEL in plant, respectively. We validated expression of mAbP SO57 by western blot. Western blot showed the significantly higher expression level of mAbP SO57 with KDEL compared to without KDEL. Flow cytometry (FACS) analysis showed that the Fc domains of both purified mAbP and mammalian-derived mAb (mAbM) evidenced similar binding activity to the FcγRI receptor (CD64). High performance liquid chromatography (HPLC) analysed, glycosylation patterns of mAbP SO57 with or without KDEL. The mAbP SO57 with KDEL had glycan profile with both oligomannose type (47.6%) and golgi type (52.4%), while the mAbP SO57 without KDEL had only golgi type (100%) glycans. Neutralizing analysis with rabies virus CVS-11 showed the similar neutralizing activity between mAbP SO57 with and without KDEL. These results suggest the potential of mAbP SO57 for rabies immunotherapy, regardless of plant specific glycan structures.

The mAb CO17-1A recognizes the colorectal antigen GA733-2E, which highly is expressed on the surface membrane of human colorectal carcinoma cells. We have successfully developed plant system for production of anti-colorectal cancer monoclonal antibody, mAb CO17-1A. Plant expression system for mAb production has several advantages in terms of the lack of animal pathogenic contaminants, low cost, ease of scale-up for agricultural production, and efficient post-translational glycosylation modifications compared to the conventional fermentation methods. KDEL (Lys-Asp-Glu-Leu), ER retention signal attached to C-terminus of protein might induce high protein expression and the glycoprotein carrying oligomannose glycans. In this study, the expression level of mAb CO17-1A was compared between mAbs fused with and without KDEL in plant. PCR analysis showed stable gene insertion of heavy and light chain genes of mAb CO17-1A with or without KDEL. We validated the expression of mAb CO17-1A by western blot and its subcellular localization under the confocal microscopy. Western blot showed the significantly increased expression level of mAb CO17-1A with KDEL compared to without KDEL. Cell ELISA with human colorectal carcinoma cell line confirmed that expression of the mAb with KDEL was significantly higher than that of mAb without KDEL. The glycosylation analysis revealed that mAb CO17-1A without KDEL had plant specific glycans structure while mAb CO17-1A with KDEL had mainly oligomannose type glycans. Taken together, these studies suggest that KDEL affects expression of mAb and its glycan structures.

The doxorubicin biosynthetic gene cluster in Streptomyces peucetius ATCC 27952 was found to contain a TDP-D-glucose 4,6-dehydratase gene, dnsM, putatively involved in the biosynthesis of 2,3,6-trideoxy-3-aminohexose, daunosamine. But the gene contains a frameshift in the DNA sequence that caused the premature termination of translation. In pursuit of another TDP-D-glucose dehydratase in S. peucetius, we discovered a homologue gene, rmbB, whose deduced product exhibit a high sequence similarity to a number of TDP-D-glucose 4,6-dehydratases. The gene was found to be located within a putative rhamnose biosynthetic gene cluster at another locus of the genome. The rmbB protein was verified to be functional by enzyme assay as it converted TDP-D-glucose into TDP-4-dehydro-6-deoxy-D-glucose. Inactivation of rmbB from S. peucetius genome abolished the production of doxorubicin. Consequently, rmbB was proved to be essential for the biosynthesis of doxorubicin.

Sialic acid containing structures in eukaryotic systems play important roles in a variety of physiological and pathological processes, including cell–cell interactions, inflammation, fertilization, viral infection, differentiation, malignancies, and cell signaling etc. Especially sialic acid derivatives product in living system are spotlighted because sialic acid is an intermediate to inhibit viral disease like SARS, bird flu infection is reported. Although, sialytransferase has been expressed in E.coli, because of its toxicity many problems arise in use to animal or human. In this study, to express sialytransferase gene from Pasteurella multocida in yeast, a non-toxic widely used strain. Cloning was carried out in yeast shuttle vector pYES2 and expressed. Enzymatic activity was analyzed by TLC and Bio-LC.

The deoxysugar biosynthesis gene cluster of calicheamicin contains calS9, which encodes UDP-D-glucose decarboxylase and catalyzes the conversion of UDP-glucuronic acid to UDP-xylose in the presence of NAD+ cofactor. The calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21(DE3). An enzymatic assay was carried out with the purified CalS9. A one-pot assay was also developed using thymidyl kinase, acetyl kinase, CalS7 and CalS8 to convert UMP to UDP-xylose via UDP-D-glucose and UDP-glucuronic acid. The reaction products were extracted and analyzed by HPLC and ESI-MS for UDP-D-glucose, UDP-glucuronic acid and UDP-xylose, respectively. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by homologous recombination and the S. sp. GerSM2 mutant, which could not produce dihydrochalcomycin, was obtained. calS7, calS8 and calS9 genes were cloned in integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in S. sp. GerSM2. Finally, the novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was analyzed by LC-MS.

Aminoglycosides are antibiotics used to treat many types of bacterial infections, particularly those caused by gram negative bacteria. Kanamycin which is isolated from Streptomyces kanamyceticus, is an aminoglycoside antibiotic, used to treat a wide variety of infections. Kanamycin contains kanosamine, a major part plays key factor in biological control of plant disease and highly inhibitory to plant-pathogenic oomycetes is glycosylated from UDP-kanosamine to 2-DOS. Here we tried to one pot enzymatic synthesis of UDP-kanosamine from the cheap substrate UMP as a starting precursor via UDP-glucose and UDP 3-keto glucose. UDP-kanosamine is synthesized in two steps. First, we synthesize UDP-glucose from UMP using ACK, TMK and GalU enzyme and second, from UDP-glucose to UDP-3-keto-glucose an intermediate for UDP-kanosamine. To synthesize UDP-kanosamine, two major enzymes KanC (UDP-glucose dehydrogenase) and KanD (aminotransferase) were cloned in pET32a+ and the soluble protein was expressed and enzymatic assay was carried out and analyzed.

The intricate system of serum complement proteins provides resistance to infection. The spleen and complement C3 provide resistance against blood-borne S. pneumoniae infection. To better understand the mechanisms involved, we studied SIGN-R1, a lectin that captures microbial polysaccharides in spleen marginal zone. We found that SIGN-R1 directly bound the complement C1 subcomponent, C1q, and assembled a C3 convertase, but without the traditional requirement for either antibody or factor B. Therefore the transmembrane lectin SIGN-R1 contributes to innate resistance by an unusual C3 activation pathway, unraveling the novel and 4th complement activation pathway (1) It has been reported that sialylation on Fc doamin of immunogloublin is critical for the effect of intravenous immunoglobulin (IVIG) and seems to be mediated by lectins which are expressed on splenic marginal zone macrophages (2,3). And they demonstrated that SIGN-R1 preferentially binds to 2,6-sialylated Fc compared with similarly sialylated, biantennary glycoproteins, thus suggesting that a specific binding site is created by the sialylation of IgG Fc (4). Moreover, they showed that a human homologue of SIGN-R1, DC-SIGN, displays a similar binding specificity to SIGN-R1 but differs in its cellular distribution, potentially accounting for some of the species differences observed in IVIG protection (5). However, the function of DC-SIGN in IVIG effects is controversial now, given that DC-SIGN and alpha2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells (6). Autoantibody is the principal mediators of autoimmune disease. IVIG is a milestone of the therapy of autoimmune disease. Now we hypothesize that complement systems are essential for the recognition of the sialylated Fc domain of immunoglobulin by SIGN-R1 or DC-SIGN, which lead to various IVIG effects or anti-inflammatory effects. In our further studies, we hope to further characterize the SIGN-R1-mediated IVIG effects and these works could lead to develop a potential therapeutic target against several autoimmune diseases.

Seaweed extracts have recently been found to have biological activities including effects on the immune system, anticoagulant system, cancer, etc. Capsosiphon fulvescens is a green alga that is widely distributed in the Southern coastal area of Korea. Crude polysaccharides were obtained from this seaweed collected at a coastal area of Wando, Korea, mainly by dilute acid extraction, ethanol precipitation and CaCl2 precipitation. It was further purified by DEAE-cellulose column chromatography and its chemical composition, structural analysis, immunostimulating activity, anticoagulation activity, anticancer activity and osteogenesis was determined. From HPLC analysis molecular mass of this polysaccharide was determined to be approximately 385 kDa. Elemental analysis of CPS, The estimation of only four elements, that is carbon(31.9398% w/w), hydrogen(4.9022% w/w), nitrogen(0.6318% w/w) and sulfur(6.2166% w/w), was undertaken. CPS stimulated the production of TNF-α to a level 80 times greater than the normal. Also CPS induced IL-6 secretion from RAW264.7 cells in a dose-dependent manner. Moreover, Cf–PS treatment increased the expressions of cyclooxygenase-2 (COX-2) and the inducible form of nitric oxide (iNOS). The CPS could significantly increase anticoagulant activity, which was especially characterized by activated partial thrombloplastin time (APTT) and thrombloplatin time (TT). Two hundred ug/ml of CPS delayed the APTT up to 61 s. And TT of CPS reached up to 32 s. MTT assay showed that the CPS significantly inhibited the proliferation of cultured human MKN-45 gastric cancer cells in a dose-dependent manner. Also nitrite plays an important role in the formation of carcinogenic nitrosamine. One percentage of CPS eliminated the nitrite up to approximately 50%. CPS plays a role for osteoblastic bone metastasis by promoting both osteoblasts proliferation and apoptosis of osteoclasts. One mg/ml of CPS increased the osteoblast cell growth up to approximately 150% and decreased the osteoclast cell growth up to approximately 60% by induction of apoptosis.

Sialyltransferases add CMP-sialic acid to the terminal portions of the sialylated glycolipids (gangliosides) or to the N- or O-linked sugar chains of glycoproteins. Here, we focused on alpha-2,3 sialyltransferase VI (ST3GAL6) as a transporter that add sialic acid to N-glycan of glycoproteins. Our study showed that amyloid β-peptide 1-42(Aβ1-42)-induced cell death of SK-N-SH human neuroblastoma cells and SB203580, a p38 MAPK inhibitor protect against Aβ1-42 toxicity. During Aβ1-42 induced-apotosis, we found that alpha-2,3 sialyltransferase VI was decreased almost 50% by 20uM Aβ1-42 and alpha-2,3 linked sialic acid on total protein decreased. Also, cell viability was increased by SB203580 in Aβ 1-42-induced cell death for about 30%. Collectively, our findings suggest that p38 MAPK signaling pathway is related with alpha-2,3 sialyltransferase VI expression in beta-amyloid induced apoptosis.

The crushed fruiting body of Alaskan Phellinus pini (Brot.) Murrillwas extracted in boiling water for 4 h with the yield of 20.5% in dry mass. From which the ethanol precipitate (EP) and supernatant fraction (ES) were obtained through 75% ethanol precipitation with the yield of 43.3% and 14.2 % in dry mass, respectively. From the EP preparation, two major carbohydrate-positive fractions, named EP-AV1 and EP-AV2, were purified by mainly Sepharose CL-4B column. Both EP-AV1 and EP-AV2 inhibited the plaque formation by CVB3 on HeLa cells by 32% and 84%, respectively, at 1 mg/mL, with EC50 of over 1 mg/ml(EP-AV1) and 0.576 mg/ml (EP-AV2), and the antiviral activity of these two polysaccharides against HSV-1 in Vero cells, inhibited in a dose-dependent manner at much lower concentrations (1 to 50 μg/ml), with EC50 of 0.20 μg/ml (EP-AV1) and 0.21 μg/ml (EP-AV2), respectively. Also, EP-AV1 and EP-AV2 inhibited neuraminidase activity by 27.4% and 52.4% at 1.7 mg/ml, and 44.1% and 73% at 3.4 mg/ml, respectively. EP-AV1 was shown to be a heteropolysaccharide containing glucose as the main sugar residue with mole percentage of 53.4% and other sugars like galactose (19.2%), xylose (17%), mannose (5.8%), and fucose (4.6%). EP-AV2 was also a heteropolysaccharide with glucose as the main sugar (56.1%) and other minor sugars similar to that of EP-AV1 but relatively lower proportion of galactose (10.3%). The HPAEC-PAD analysis after laminarinase digestion showed that both polysaccharides contain β-1,3-linked glucose residues, suggesting that these polysaccharides are a β-(1,3)-glucan. The results of this study suggest that these water-soluble polysaccharides of Alaskan P. pini fruiting body can be a strong candidate for the development of a potent broad-range antiviral agent.

Fucoidan was purified from the sporophyll of Korean Undaria pinnatifida and low-molecular weight fuco-oligosaccharides (LMFOs), ranging from 305 to 3,749 Da, were obtained by enzymic digestion of the purified fucoidan by Sphingomonas paucimobilis PF-1 (KCTC 11130BP). The enzyme was partially purified through 3 steps, including ammonium sulfate fractionation and affinity column chromatography. The enzyme was purified at 2.9% (activity/activity) yield from the concentrated supernatant of the sonicated PF-1. SDS-PAGE analysis of the partially enzyme showed three main band (34, 80 and 100 kDa) but still not purified to homogeneity. However, Native electrophoresis gel shown in gave the two main band at 30 and 60 kDa. The results presented here suggest that the 60 kDa subunit forms 2 species, namely 80-, 100 kDa subunits. The enzyme activity was assayed at different pH ranging from 3.0 to 8.0. The optimal activity was absorbed at pH 5.6. The enzyme was active in the range of pH 5-6, but the activity decreased at above pH 7 and below pH 4. When assayed at pH 5.6, crude enzyme showed its optimal activity at 30 ℃, and then sharply decreased by 30 ℃. When the incubation temperature was increased to 37, 42 ℃, there was decreased about 40-50 % of original activity the retained under the same conditions. The intact fucoidan and its LMFOs were compared for their anticoagulanting activities. Intact fucoidan prolonged activated partial thromboplastin time (APTT) at concentrations of 5 and 10 ug/ml, which was about 2.3- and 4.2- fold of the control (clotting time 90 and 168.4s each). LMFOs showed at concentrations of 100 and 200 ug/ml, which was about 2.3- and 3.9- fold of the control. Intact fucoidan prolonged thrombin time (TT) at concentrations of 5 and 10 ug/ml, which was about 42 and 68.7 s, respectively. It was about 2.8- and 4.6- fold. LMFOs approximately 1.7- and 2.6-fold at 50 and 100 ug/ml compared to that of control (14.8 s), respectively. LMFOs did not show any affect on prothrombin time (PT) at different concentrations compared with control but intact fucoidan effect increased with increasing concentration (10, 20, 50, 100 μg).

Sialylation of the brain cortex and hippocampus tissues and their total proteins from normal mouse (SamR1) and Alzheimer model mouse (SamP8) were comparatively analyzed by a combination of SDS-PAGE, Emerald Pro-Q dye staining, lectin histochemistry and lectin blotting. Lectin histochemistry using TRITC-labeled SNA-I, specific for α-2,6-linked sialic acid, and FITC-labeled MAA, specific for α-2,3-linked sialic acid, showed that both α2,3-and α2,6-linked sialic acid residues are usually expressed with no significant variations in cortex and hippocampus tissues of normal and Alzheimer model mouse brain. SDS-PAGE and Emerald Pro-Q dye staining of the total proteins from each tissue also showed that glycosylation of the most major proteins of both mouse brain tissues are not significantly changed. However, lectin blotting of total proteins showed significant variation in sialylation of most major glycoproteins; especially, significant decreased expression of α 2,3-linked sialic acid residues in proteins with 110, 90, 68, 49, 43 and 36 kDa of both tissues of Alzheimer mouse brain was observed. The specific roles of these glycoproteins showing significant degree of alterations in sialylation in AD mouse remain to be elucidated

Streptococcus pneumoniae, the major causative agent for pneumonia, otitis media and meningitis, produces surface exoglycosidases such as neuraminidase (NanA), β-galactosidase (BgaA) and N-acetylglucosaminidase (StrH). These enzymes play critical roles for colonization and pathogenesis by sequentially hydrolyzing various glycoconjugates to penetrate host defense molecules, to expose binding sites on the surface of the epithelial cells or to obtain monosaccharides for growth. In this study, we characterized a novel β-galactosidase encoded by the bgaC gene of S. pneumoniae. The recombinant BgaC protein exhibited a highly regio- and sugar-specific hydrolysis activity for Galβ(1,3)GlcNAc moiety of oligosaccharides. Interestingly, the BgaC was shown to be expressed as a surface protein, even though it does not have a typical signal sequence or membrane anchorage motif. The bgaC mutant strains did not show detectable changes in growth or morphology compared to wild type stains when cultivated under normal laboratory conditions. However, the bgaC mutant showed higher colonization levels at 6 and 12 h post-infection in vivo than the wild type. Our data strongly indicate that S. pneumoniae BgaC is a surface-associated β-galactosidase with a specific hydrolysis activity that contributes significantly to the adherence and invasion of pneumococci in vivo and in vitro.

Thermus caldophilus GK24 (TCA) is an extremothermophile growing at 70℃ ,providing industrially important enzymes. From TCA genome sequence, all of gene sresponsible for carbohydrate biosynthetic network were identified .Among them, 120 enzyme genes have been expressed in E. coli and their reactions were characterized. In this lecture, Thermus-derived Carbohydrate Biosynthetic Network’ will be introduced, particularly focused on in vitro operation of a-glucan network’, glycolysis and pentose-phosphate pathway. 1) ‘a-Glucan Network’ showed to be the practical synthetic methods of various sugars such as trehalose, fructose, phosphosugars, sugar-nucleotides, a-glucan and so on. 2) in vitro Glycolysis using glycolytic enzymes have been operated for synthesizing pyruvate from glucose or glucose-1-phosphate. Accordingly, we could have managed to operate the glycolysis in flask. 3) And one pot reaction containing a-glucan phosphorylase, phospho glucomutase, glucose-6-phosphate isomerase, transaldolase and transketolase could synthesize sedoheptulose-7-phosphate from starch. Overall, ‘in vitro Cell Factory’ utilizing thermostable carbohydrate enzymes turned out to be a practical synthetic model to produce C3-C9 sugars, phosphosugars, sugar-nucleotides and biopolymers.

Glycan microarrays have received great attention as high-throughput analytic tools in studies of carbohydrate-mediated biological processes. Most of the methods employed to fabricate glycan microarrays rely on the immobilization of modified glycans on the properly derivatized surfaces. This immobilization strategy requires the availability of modified glycans whose syntheses in many cases are time-consuming and difficult. We have developed a simple and direct immobilization technique that involves a one-step, site-specific attachment of diverse unmodified glycans to the hydrazide-derivatized glass surface. To demonstrate the generality of this direct immobilization method, we examined its use for the construction of carbohydrate microarrays containing a variety of glycans. The results of protein and cell-binding experiments indicate that the glycan microarrays, prepared by using this methodology, are applicable to the rapid evaluation of glycan-mediated biomolecular interactions and the determination of quantitative binding affinities between carbohydrates and proteins.

Properly protected hexasaccharide was synthesized by employing the glycosyl phthalate method from a trisaccharide acceptor and a trisaccharide donor, both of which were obtained by deprotection of a common trisaccharide precursor. Trisaccharide was prepared by assembly of three monosaccharide building blocks through two β-mannosyl linkages. The mannosyl phthalate donor for the disaccharide synthesis and the CB glycoside donor for the trisaccharide synthesis were achieved in the 4, 6-O-benzylidene directed β -mannosylation.

The unique trisaccharide repeating unit, [→3)-α-D-Rhap-(1→2)-α-D-Manp3CMe-(1→3)-α -L-Rhap-(1→], of the O-polysaccharide of the lipopolysaccharide from Danish Helicobacter pylori strains has been synthesized. Key steps include the coupling of three monosaccharide moieties by glycosylations employing the 2'-carboxybenzylglycoside method and stereoselective epoxide ring opening to prepare the novel branched sugar, 3-C-methyl-D-mannose, which has not been found in Nature before. furthermore, the synthesis of a hexasaccharide and a nonasaccharide were successfully achieved by dimerization or trimerization of the repeating units of trisaccharide employing the 2'-carboxybenzyl glycoside method.

The direct β-mannosylation with C-1 hydroxy sugars would provide a complimentary or efficient strategy for β-mannosylation, in that anomeric derivatization, isolation of the donor and activation and coupling should be carried out as separate steps. Herein we describe a novel β-mannosylation method. Reaction of C-1 hydroxy sugars with phthalic achydride in the presence of DBU at RT in CH2Cl2 and sequential addition of Tf2O, DTBMP and ROH without isolation of any intermediates to afford β-mannopyranoside. We have applied the successful one-pot dehydrative glycosylation method to the stereoselective β -mannosylation with C-1 hydroxy sugars to the synthesis of tetrasaccharide.

Glycosaminoglycans (GAGs) are unbranched, polydisperse, acidic polysaccharides, often covalently linked to a protein core to form proteoglycans (PGs). The most common GAGs are heparin, heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS). Heparin, an anticoagulant isolated from animal tissue, is an important and chemically unique polysaccharide of considerable biological significance. In order to understand the complicated structure of GAGs, GAG lyases from microorganisms have been widely used and their reaction products were analyzed using various HPLC methods. We have cloned and expressed GAG lyases of Bacteroides thetaitaomicron in E.coli. In collaboration with Dr Mirek Cygler’s group, the tertiary structures of recombinant chondroitinases ABC and heparinase have been solved. These things will be presented. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure a-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Its structure is related to heparin and heparan sulfate but is distinctly different from all known members of these classes of glycosaminoglycans. Because of its structural similarities to heparin, a diverse array of biological activities and functions have been studied. Because of its high molecular weight and copper binding affinity, specific proteins in the plasma could be purified by affinity chromatography.

Human mesenchymal stem cells (hMSCs) have been considered to be alternative sources of adult stem cells because of their potential to trans-differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in neural differentiation of hMSCs. When hMSCs were cultured in neural differentiation condition, expression of neural cell maker genes such as nestin, MAP-2 and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analyses showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hMSCs. We found a significant increase in GD3 and GD1a expression during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hMSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg). Ugcg knockdown hMSCs were not able to differentiate into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hMSCs.

Ganglioside GQ1b has important functions in neuronal differentiation and neuronal development processs. The regulation of ganglioside levels is relationship to the induction of neuronal cell differentiation. In the previous study, we showed that expression of GT1b and GQ1b gangliosides in undifferentiated mouse embryonic stem cell were exhibit during their neural differentiation. We evaluated the co-localization of ganglioside GQ1b and neuroanl markers in an in vitor model of neuronal differentiation in mouse embryonic stem and found that neuronal differentiation was facilitated by the presence of increased gangliosides, especially GQ1b, due to daunorubicin. Furthermore, we investigated that important effect of GQ1b by overexpression and konckdown GQ1b in the neural differentiate. First, we designed a primer of a2,8-sialyltransferase (ST8Sia V) for producing GQ1b. In this study, we inserted ST8Sia V gene to overexpression (pEGFP-C2), and knock-down (pLKO.1 puro ShRNA) vectors. We will confirm the efficient transfectionof mouse embryonic stem cell by green fluorescent protein (GFP) and expressin of ganglioside GQ1b by immunofluorescence staining analyses.