Earticle

We have previously shown that the glycoengineered methylotrophic yeast Hansenula polymorpha strains, the Δoch1 and Δoch1Δalg3 double deletion strains, with the targeted expression of Aspergillus saitoi α-1,2-mannosidase in the ER, were able to produce human mannose-type N-glycans (Man5GlcNAc2 or core Man3GlcNAc2)1, 2. Here we report the further modification of yeast glycosylation pathway to synthesize the complex-type N-glycans with a terminal N-acetyl glucosamine in both the glycoengineered ΔHpoch1 and ΔHpoch1 ΔHpalg3 strains, respectively. First, several combinatorial synthetic leaders were constructed to localize efficiently active human β -1,2 N-acetyl glucosaminyl transferase I (GnT I) in the Golgi apparatus of yeast. The short, medium, and long N-terminal leader sequences with the various length of stem region of yeast type II membrane proteins (ScMnn9, HpOch1, and HpOcr1), located in the early Golgi compartment, were fused in-frame to the catalytic domain of human GnT I lacking its own N-terminal leader sequence. The GnT I constructs combined with various yeast N-terminal leader sequences were introduced into the H. polymorpha och1 and och1 alg3 strains carrying the ER-targeted α-1,2 mannosidase, and the obtained transformants were screened for proper localization of GnT1 into the Golgi compartment by the size fractional centrifugation and Western blotting. Subsequently, the production of the complex-type glycans with monoantennary N-acetyl glucosamine was analyzed by a capillary electrophoresis of ATPS-labeled cell wall glycans. Our data strongly suggested that the ΔHpoch1 single deletion strain would be a better host for the production of human complex-type N-glycans than the ΔHpoch1 ΔHpalg3 double deletion strain in the respect of the glycosylation site occupancy and the byproduct Hex6GlcNAcs formation.

Cell surface gangliosides, which exist in glycosphingolipids (GSLs)-enriched domains, play important regulatory roles in cell proliferation and differentiation .Recently, gangliosides are known as important role in growth of neuronal progenitor cells. In this study, the relationship between gangliosides expression and neuronal cell development was investigated using an in vitro model of neuronal differentiation from human mesenchymal stem cells (hMSCs) that are able to differentiate into a range of specific cell types in vitro and in vivo. First, to verify the isolated population contained pure hMSCs, we performed positive and negative characterization by fluorescence- activated cell sorting (FACs) analysis, thus we identified pure hMSCs. Next, hMSCs were differentiated into neuronal cell withα-MEM + 20%FBS + 1mM β-mercaptoethanol (BME) for 24h and transferred to serum free α-MEM + 2mM BME for 5 hour. Neuronal induced cells maintenance with α-MEM + 10%FBS + 2% DMSO +200uM β- hydroxyanisole (BHA) + 10ng/mL bFGF + 10ng/mL EGF + 25ng/mL NGF for 2weeks. High-performance thin-layer chromatography (HPTLC) showed that gangliosides GM3, GD3 and GD1a expressed in differentiated into neuronal cells for 2weeks, especially GD3 increased comparison to control cells. Immunofluorescence staining also agreed with the results of HPTLC assay. These differentially expressed gangliosides suggest that gangliosides may have specific functions in stem cells and during neuronal differentiation. Therefore, these result also suggest that regulation of gangliosides expression have used as the marker for differentiated neuronal cell from hMSCs.

Metabolic engineering is a powerful tool for improvement and introduction of new cellular processes in a host strain which is mostly done by genetic engineering. We constructed E. coliΔpgi mutant by disrupting the pgi and inserting neomycin/kanamycin resistant marker. The pgi encoded enzyme glucose phosphate isomerase (Pgi) is responsible for the conversion of glucose-6-phosphate to fructose-6-phosphate and vice-versa. The mutant is a useful host for expression of a gene or gene cluster in which glucose-6-phosphate serves as a precursor. 2-deoxy-scyllo-inosose (DOI) is a first intermediate in biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside such as ribostamycin, neomycin and butirosin. Subsequent amination, dehydrogenation and again amination of DOI results DOS, another stable intermediate to be isolated. N-acetylglucosaminylation to DOS by a glycosyltransferase and deacetylation by a deacetylase gives paromamine, first and stable pseudosaccharide intermediate. In this study five genes required for the conversion of glucose-6-phosphate to paromamine were taken from butirosin gene cluster and cloned in expression vectors. The recombinants were transformed in E. coli BL21 (DE3) and its mutant E. coliΔpgi successively and together so as to get the desired product. After fermentation the products were extracted and analyzed by TLC and HPLC-MS.

Sialylated sugar chains are present at the cell surface of various animal species. Due to their position, they are thought to serve important roles in a large variety of biological functions such as cell–cell and cell–substrate interactions, bacterial and virus adhesion, and protein targeting. We present a bio-conversion process for the conversion of lactose, N-acetyl- glucosamine and CMP into 2,3-sialyllactose with six enzymes. This process consists of two steps; the first step is from N-acetyl-glucosamine and CMP to CMP-neuraminic acid with five enzymes (GlcNAc-2-epimerase, NeuAc aldolase, CMP kinase, acetate kinase and CMP-NeuAc synthetase) including ATP recycle system to yield over 95% by one-pot reaction and the second step is from lactose and CMP-neuraminic acid with sialyltransferase to yield over 90%. The second step can apply to synthesize the various sialyllactose (sialyloligosaccharide) depend on acceptors (lactose and N- acetyl-lactosamine) and sialyltransferase (2,3-sialyltransferase or 2,6- sialyltransferase).

About 2~5% of extra-cellular glucose is converted into UDP-GlcNAc through hexosamine biosynthesis pathway(HBP). UDP-GlcNAc could be used for O-GlcNAc modification of nucleo-cytoplasmic proteins by O-GlcNAc transferase(OGT). At glucose deprivation condition, generally, the levels of UDP-GlcNAc and O-GlcNAc modification of proteins decrease. However, in A549, non-small cell lung carcinoma (NSCLC), O-GlcNAc modification increased in response to glucose deprivation in a time dependant manner. On the other hand, the levels of OGT and O-GlcNAcase(OGA) were not changeable at this condition. Moreover the activity of GFAT, the first and rate-limiting enzyme in the HBP, increased steadily but the activity of OGA decreased in glucose starvation condition. Also we found that O-GlcNAc modification increased in response to glucose starvation through degradation of glycogen in several cell lines including A549 which store glycogen. We used sWGA precipitation method and MALDI -MS to identify the proteins in which O-GlcNAc modification increased at glucose deprivation condition. In view of the results so far achieved, we could identify several cytoskeletons, heat shock proteins, ribosomal proteins etc. we will focus on revealing how the O-GlcNAc modification protects some proteins from degradation in response to glucose deprivation.

β-O-linked N-acetylglucosamine (O-GlcNAc) addition is dynamic post-translational modification in nucleocytoplasmic proteins such as transcription factors, cytoskeletal proteins, and various enzymes. These proteins are modified with O-GlcNAc on their Ser/Thr residues and this event changes their intracellular functions, including transcription, proliferation, apoptosis, cell signaling. This modification takes place at the same sites as does phosphrylation or at adjacent residues. Like phosphorylation, protein O-GlcNAcylation dramatically alters the post translational fate and function of target proteins. Knock-out of the O-GlcNAc transferase (the nucleocytoplasmic enzymes for the addition of O-GlcNAc) result in stem cell and embryonic lethality so O-GlcNAc metabolism in Drosophila melanogaster will likely provide important clues to the cellular functions of O-GlcNAc modification. In order to detect O-linked GlcNAc modifications in Drosophila SL2 cell, immuno- blotting were performed with CTD 110.6 antibody. O-GlcNAcylated proteins in Drosophila SL2 cell were analyzed using two-dimensional gel electrophoresis and MALDI-TOF-MS. As a result, ATP synthase β subunit was identified as a novel O- GlcNAcylated protein in Drosophila SL2 cell. To confirm O-GlcNAcylation, immuno- blotting was performed with ATP synthase β antibody after SWGA lectin precipitation. Also, immuno-blotting was performed with ATP synthase β antibody and CTD 110.6 antibody after immuno-precipitation with CTD 110.6 antibody and ATP synthase β antibody, respectively. ATP synthase β subunit is encoded in the nucleus, synthesized in the cytosol and imported in to the mitochondria. ATP synthase β was identified in the mitochondria when we performed immunoblotting with CTD 110.6 antibody after organelle fractionation. Thus we will focus on revealing how ATP synthase β is modified with O-GlcNAc and what the functional roles of O-GlcNAcylation on ATP synthase β are.

Glycosylation of flavonoid play crucial roles in stabilization of antocyanins and cyanidins; storage of flavonoid and terpenoids; and regulation of hormones. In addition, glycosylation has been recognized as one of the important mechanisms for detoxification of exogenous compounds. Here in this research, we have metabolically engineered the E. coli BL21DE3 (Δ pgi mutant) host to generate four different engineered host to produce glycosylated flavonoid. E. coli BL21DE3 (Δ pgi mutant) was engineered by integration of GalU, expression of CalS8 (dehydrogenase) and CalS9 (decarboxylase) together by cloning in pDuet/ampr vector and expression of four different 3-O-glycosyltransferase and 7-O- glycosyltransferase gene from Arabidopsis thaliana. Engineered hosts are expected to produce glucosyl as well as xylosyl glycosylated flavonoids which are characterized by HPLC as well as LC-MS analysis.

25 bacterial strains that secrete mucous materials were isolated from sediment obtained from King George Island, Antarctica. Seven of these strains proved capable of producing cryoprotective exopolysaccharides. The strain KOPRI 21653 was selected for the further study. KOPRI 21653 was identified as Pseudoalteromonas arctica as the result of 16S rRNA analysis. The exopolysaccharide, P-21653, was purified by Cetylpyridinium chloride (CPC) and protease treatment from the KOPRI 21653 culture. The sugar components of P-21653 determined to be galactose and glucose, at a ratio of 1:1.5, by GC-MS analysis. The cryoprotective effect of P-21653 was determined by a red blood cell (RBC) LDH assay. In the presence of 0.5% (w/v) P-21653, the Damaged cell ratio of RBC was 20.83±2.83%, after three repeated freeze-thaw cycles. The damaged cell ratio of RBC increased to 26.1%, in five repeated cycle conditions in same concentration of P-21653. However, the damaged cell ratios of RBC were increased (20.83±2.83 - 42.11±7.27 %) in the presence of 0.5 - 0.2% (w/v) P-21653. In addition, at lower concentrations of P-21653 (0.2 - 0.5%), the damaged cell ratios of RBC were more less than generally employed as a RBC cryoprotectant (glycerol), which was utilized at the recommended concentrations (40%). The biochemical characteristics of exo-polysaccharide P-21653 reflect that this compound may be developed as a useful cryo-protectant for use in medical applications.

Glycan recognitionleading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Firstly, total N-glycans from membrane glycoproteins derived from specific pathogen-free miniature pig kidney were qualitatively and quantitatively identified by MALDI-TOF, negative ion ESI MS/MS and normal-phase HPLC (NP-HPLC) combined with exoglycosidase digestion. Over 100 N-glycans, including sialylated and neutral types, were identified. Without any derivatization steps, the characterizedpig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.

tAlong with the traditional yeast Saccharomyces cerevisiae, nonconventional yeast species are becoming increasingly attractive hosts for the secretory production of recombinant proteins. Glycosylations of secretory proteins in yeast species occur on asparagine (N) and/or serine/threonine (O) residues through the secretory pathway. The establishment of methods to assess the degree and size of N- or O-glycosylation is particularly useful in the development of expression systems for therapeutic proteins. Whereas the structure of N-linked oligosaccharides of various yeast species have been well characterized1,2, the structural analysis of yeast O-linked oligosaccharides has been hampered due to lack of a universal method to release O-glycans enzymatically. Here, we tried to define O-linked glycan profiling of cell wall mannoproteins and secretory protein chitinase derived from the two nonconventional yeast species, the methylotrophic yeast Hansenula polymorpha and dimorphic yeast Yarrowia lipolytica, based on the ‘ammonia-based β-elimination’ method. The released O-linked oligosaccharides were effectively isolated, labeled with fluorescence tags, and then analyzed using HPLC and CE-based DNA sequencer. The results of α-mannosidase treatment experiment showed that the O-linked glycans from both yeasts, H. polymorpha and Y. lipolytica, are mainly composed of two to five α-mannoses.

Kanamycins, pseudotrisaccharides antibiotics, have three closely related structural form namely kanamycin A, kanamycin B and kanamycin C in which 2-DOS is substituted at the C-4 position by an aminoglucose functionality: 6-amino-6- deoxy-D-glucose, neosamine, and D -glucosamine respectively and at the C-6 position by 3-amino-3-deoxy-D-glucose (kanosamine). Here, we have Engineered Streptomyces lividans TK24 by expressing two sets of rebombinant plasmids pSK-7 and pSK-17 responsible for the production of paromamine and kanamycin C respectively. Bio-active secondary metabolite from pSK-17/SL against Bacillus subtilis and ESI/MS,LC/MS and ESI/MS/MS at 485 [M+H]+ were the evidences for the production of Kanamycin C.

It has been reported that one downstream effector produced from glucose is uridine diphosphate-N-acetly glucosamine (UDP-GlcNAc) via the hexoamine biosynthetic pathway (HBP). The dynamic cycle of addition and removal of O-linked-N-acetlyglucosamine (O-GlcNAc) to Ser/Thr residues is involved in regulating nuclear and cytoplasmic proteins. Nucleocytoplasmic O-linked GlcNAc transferases(ncOGT) which add a single GlcNAc to hydroxyl groups of serine and threonine residues. ncOGT is characterized by an amino terminuns bearing tetratricopeptide repeats (TPR), possible nuclear trafficking motifs, and a catalytic domain within its C-terminus. Interestingly, O-GlcNAc glycosylation occurs in nucelocytoplsmic O-linked GlcNAc transferase (ncOGT) as well and several sites have been identified mainly within TPR domain. We found O-GlcNAcylated site in ncOGT outside of TPR by using Q-TOF MS. Now we focus on the activities and function of ncOGT by mutagenesis studies. This ongoing effort would give us clear understanding of the key enzyme of O-GlcNAc metabolism and how the O-GlcNAc modification may be regulated.

Glycan microarrays have been used recently as an advanced technology for the high-throughput analysis of protein-carbohydrate interactions and for the detection of cells and pathogens. A major driving force behind the expanded use of this microarray technology is the development of new experimental protocols in which they can be applied. As a continuous effort to extend the use of this technology, carbohydrate microarrays were applied for assaying glycosyltransferase activities. The microarray-based approach requires only a small amount of immobilized acceptor substrates (picomoles), and enzymatic catalytic activities are facilely analyzed by measuring the amount of a product formed in a time-dependent manner. The level of time-dependent product conversion is determined by using fluorescence detection of lectin recognition of carbohydrate products. Carbohydrate microarrays immobilized twenty glycan probes were prepared by immobilizing hydrazide-conjugated carbohydrates on epoxide-coated glass slides. Treatment of glycan microarrays with β-1,4-galactosyltransferase in the presence of UDP-Gal and Mn2+ showed that α- and β-GlcNAc were converted to α- and β-LacNAc, respectively. In addition, this experiment indicated that β-GlcNAc was a better substrate than α-GlcNAc for this enzyme, which was also confirmed by HPLC analysis of enzymatic product of each substrate and solution-based assay. Furthermore, quantitative binding affinities (Kd values) between α-LacNAc or β-LacNAc and RCA120 were determined by using carbohydrate microarrays. The Kd values (34 nM for α-LacNAc and 33 nM for β-LacNAc) determined from carbohydrate microarrays were similar to those (26.3 nM for α-LacNAc and 25.3 nM for β-LacNAc) obtained by using a conventional SPR technology. These findings should open new applications of carbohydrate microarrays in the field of glycomics research.

Ganglioside GM3 inhibits growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Previously, we showed that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation and increases the PTEN expression and the accumulation of p53 protein by the PTEN-mediated inhibition of the PI-3K/Akt/Mdm2 signaling pathway in HCT116 colorectal cancer cells, confirming that GM3 functions as the p53 protein stabilizer. Consequently, GM3 induces p53-depentent cyclin-dependent kinase (CDK) inhibitor (CKI) p21WAF1 expression in the wild type of p53-positive HCT116 colorectal cancer cells, but not in the p53-negative HCT116 cells lacking p53 (HCT116 p53-/-). Moreover, the data herein clearly show that GM3 induces the production of the CDK inhibitor p21WAF1 at the transcriptional level, as evidenced by the p21WAF1 promoter-driven luciferase reporter plasmid (full-length p21WAF1 promoter and a construct lacking the p53 binding sites). Furthermore, the down-regulation of the cyclin E/CDK2 complex as well as the up-regulation of the CDK inhibitor p21WAF1 were clearly observed in GM3-treated HCT116 cells, but the down-regulation of cyclin D1/CDK4 was not. These results suggest that GM3 induces PTEN gene expression and p53-dependent p21WAF1 accumulation, thus leading to cell cycle arrest. On the other hand, GM3 induces transcription factor AP-2α-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5’-flanking region of the PTEN gene showed that region between -1175 and -1077 from translational initiation site, which contains AP-2α binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis and chromatin immunoprecipitation assay obviously indicated that the AP-2α is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2α diminished the enhancement of AP-2α and PTEN expression in GM3-induced colon cancer cells. The transient expression of AP-2α also results in the induction of PTEN transcription in AP-2α-negative colon cancer cells. Additionally, GM3 induced AP-2α-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2α transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of PTEN gene [Choi et al., 2008, Glycobiology]. Using phage display method, we have found for the first time that GM3 directly interacts with VEGFR-2, which inhibits VEGF/VEGFR-2-mediated biological function of vascular endothelial cell and angiogenesis both in vitro and in vivo, the growth of primary tumors in mice inoculated with tumor cells and VEGF-stimulated microvessel permeability in mouse skin capillaries. These results suggest that GM3 are angiogenic inhibitor, and might be a therapeutic avenue for anti-angiogenesis and that GM3 represents a physiological modulator of cancer cell proliferation and, therefore, may have potential for use in colorectal cancer therapy.

Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. Ceramide accumulation in cells induces apoptosis, and its glycosylation make it possible to escape from ceramide-induced apoptosis. In some cases, it was reported GCS can lead to multidrug resistance of cancer cells and its depletion cause neuronal defect in brains. Previous report indicates Glycolipids, especially glucosylceramide (GluCer), Lactosylceramide (LacCer) and GM3 ganglioside was accumulated in atherosclerotic lesion. In the atherosclerosis, TNF-α was a prominent inducer of proliferation and migration of vascular smooth muscle cells (VSMC). Reasonably TNF-α does not stimulate apoptotic signals in VSMCs. In our results, we found TNF-α induced the expression of GCS and stimulated ganglioside production in HASMCs. GCS expression was inhibited by a MAPKinase inhibitor, U0126. In addition, PDMP, a GCS inhibitor, suppressed the TNF-α induced proliferation of HASMCs. Taken together, it is supposed GCS is a gatekeeper of TNF-α induced cell proliferation and ganglioside production in atherosclerotic lesion.

The induction of hST3Gal V and reduction of membrane type sialidase are directly linked for expression of differentiation maker protein, CD41b surface antigen. When membrane type sialidase gene is expressed in phorbol 12-myristate 13-acetate-treated cells, Neu3 inhibited the PMA-induced ERK1/2 and p38 MAPK phosphorylation in the K562 cells. Down-regulation of expression of CD41b surface antigen was dependent on expression of Neu3 gene. However, a Neu 3 inhibitor Neu5Ac2en induced morphological changes, showing megakaryocytic differentiation of K562 cells, with expression of CD41b surface antigen, while a specific glucosylceramide synthase inhibitor PDMP inhibited megakaryocytic differentiation of K562 cells. The molecular mechanisms involved in Neu 3 -involved inhibition of CD41b surface antigen expression in K562 cells have been suggested: the Neu 3 degrades membrane sialic acids and the resulting signal pathway of the PKC/ERKs/ p38 MAPK is down-regulated, decreasing the CD41b expression and inhibiting the megakaryocytic differentiation of K562 cells.

Aminoglycosides are a group of antibiotics that are used to treat certain bacterial infections. This group of antibiotics includes at least eight drugs, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, and tobramycin. One of them, we study the biosynthesis of UDP-kanosamine. UDP-kanosamine(UDP-3-amino-3-deoxyglucose) is a sugar moiety of kanamycin that was highly inhibitory to growth of plant-pathogenic oomycetes and moderately inhibitory to certain fungi and inhibited few bacterial species tested. Biosynthesis of UDP-kanosamine starts with UDP-glucose via UDP-3-keto-glucose. KanC converts UDP-glucose to UDP-3-keto-glucose by dehydrogenation and KanD converts UDP-3-keto-glucose to UDP-kanosamine by aminotransferation. Here we have cloned KanC and KanD in expression vector pET32a+ and expression was carried out. The soluble protein of KanC and KanD were used for enzymatic assay to synthesize UDP-kanosamine and further analysis was carried out.

Poly(lactic-co-glycolide) (PLGA) microspheres have been investigated for a long time as a protein/peptide delivery depot, and some products were already commercialized. However, recent publications have report about their disadvantages in the long term delivery of protein/peptide drugs such as the denaturation, aggregation, and deamidation of protein/peptide drug. The phenomena are induced by proton generated from the hydrolysis of ester linkages in PLGA. It is well known that an acidic condition facilitates the denaturation, aggregation, and deamidation. Therefore, the development of new protein/peptide delivery depot is necessary. In this study, pullulan acetate microsphere (PAM) was inspected as a depot for long term delivery of exendin-4 which is a drug for type II diabetes. The mean particle size of PAM ranged from 35 to 110 mm, as determined by SEM. Their shapes were regular and spherical, as visualized via SEM photographs. The encapsulation efficinecy of exenatide in PAMs was 69.1%, 80.4%, and 90.3% in PAM1, PAM2, and PAM3, respectively. And the exenatide release from PAMs was shown a sustained release profile for 21 days. We identified a biocompatibility of PAM through the tissue reaction by H&E staining. On the basis of these results, the microspheres may give an idea for development of new protein/peptide depot in long term delivery.

Various seaweed polysaccharides are getting great attention for intensive research due to their diverse biological activities, including effects on the immune system and cancer. Capsosiphon fulvescens, a green algae 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, with an yield of approximately 2.49% in mass. It was further purified by DEAE-cellulose column chromatography. The purified polysaccharide was examined for its immunostimulating activity on Raw 264.7 cells and shown to stimulate the production of TNF-α in a dose-dependent manner. The primary structure of this polysaccharide was analyzed by High Performance Anion Exchange Chromatography and Fourier Transform Infrared (FT-IR) spectrometry. From the HPAEC-PAD analysis, the monosaccharide composition of this purified polysaccharide was shown to be mannose(58%), rhamnose(33%), galactose(3.8%), xylose(3.3%), fucose(0.76%), and arabinose(0.67%). The IR spectrum showed a strong absorption band at 1256 cm-1, indicating the presence of sulfate esters. Absorption bands at 845 cm-1and 1060 cm-1 characteristic for galactose-4-sulfate and Sulfur trioxide, respectively, were also observed. From HPLC analysis molecular mass of this polysaccharide was determined to be approximately 385 kDa.

The crushed fruiting body of Alaskan Phellinus pini (Brot. ex Fr.) Ames was 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. Whereas ES did not show any detectable level of antiviral activity, EP showed significant dose-dependent inhibition of plaque formation by coxackievirus B3 (CVB3) on HeLa cells, with an EC50 (50% effective concentration) of 0.45 mg/mL, and strongly inhibited plaque formation up to 94.9% against herpes simplex virus (HSV-1) on Vero cells at 5 μg/mL. EP also effectively inhibited neuraminidase activity in a dose-dependent manner, showing up to 80% inhibition at 1.7 mg/mL. From the EP preparation, two major carbohydrate-positive fractions, named EP-AV1 and EP-AV2, were purified by mainly Sepharose CL-4B column chromatography with apparent molecular mass of approximately 1,006 kDa and 100 kDa, respectively. Both EP-AV1 and EP-AV2 inhibited the plaque formation by CVB3 on HeLa cells by 32% and 84%, respectively, at 1 mg/mL. 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 TLC analysis after laminarinase digestion showed that both polysaccharides contain, at least partly, β-1,3-linked glucose residues and β-1,6-linked glucose residues, suggesting that these polysaccharides are a β-(1,3)(1,6)-glucan.

In this study, alterations in glycan chains, especially in sialylation, of the glycoproteins in brain cortex tissues during early post-natal development of E18 and post-natal rats (1-day, 4-week and 8-week-old) were investigated. The lectin histochemistry using sialic acid specific lectins, SNA and MAA, lectin blotting of total proteins, and total N-glycan structure analysis showed that both α2,3- and α2,6-linked sialic acid residues of the several proteins on the surface of cortex tissues are significantly expressed in cortex tissues of 1-day-old rats and that, as the rats grow up to young adult (8-week-old), their expression is markedly down-regulated. The activity of sialidase, which catalyzes the cleavage of terminal sialic acids of glycan chain of glycoproteins, significantly increased as rats grow up. Interestingly, however, western blotting analysis of sialidase and α2,3- and α2,6-sialyltransferase showed that there was no significant difference in sialidase protein level in between E18 and 8-week-old young adult rat brains, whereas the protein levels of α2,3- and α2,6-sialyltransferases gradually decreased. These results strongly suggest that the decreased level of α2,3- and α2,6- sialylation on N-glycans would correlate with the up-regulation of enzyme activity, not the protein level of the sialidase, and also with the down-regulation of both α2,3- and α2,6-sialyltranferase protein level. The results suggest that the observation on the developmental stage-dependent decrease in α2,3 and α2,6 sialylation on glycoproteins could be attributed to a certain post-translational regulation of sialo-enzymes. Elucidating the mechanism and function of these regulation events would be very important for better understanding the normal development of brain tissues and also to effectively treat neurodegenerative diseases. Furthermore, the specific roles of those glycoproteins, observed in this study, which showed significant degree of age-dependent alterations in glycosylation and sialylation remain to be elucidated and these sialoglycoproteins may provide new, even specific, regulatory molecules in the early post-natal development of rat brain nervous system.

Complex of polyelectrolytes have been investigated to keep protein stability in poly(lactide-co-glycolide) (PLGA) microsphere because the system protects protein denaturation induced by changing in environment conditions. A potential of chodroitin sulfate (CSA) as a polymer additive to form ionic complex with positively charged proteins in long-term protein delivery was reported [1]. However, some protein/peptides having lower pI value can not make a complex with CSA in neutral pH. Thus, the polymer applied at low pH (2-3) to make complex with protein having lower pI value such as insulin and exenatide. Here, insulin (pI=5.30-5.35) was employed as a model protein. Zeta potential result showed that above 1:0.5 feeding ratio of insulin and CsA, net charge on the surface of the complex is approached at 0. Based on this preliminary study, PLGA microspheres including Ins/CsA complexes was prepared by a multi-emulsion method. Protein stability in the PLGA microspheres was preserved during both microsphere preparation and protein release. The profiles of insulin released from PLGA microspheres revealed nearly zero-order kinetics. From the result, we concluded that complex system is useful for long term delivery of protein (having a lower pI value) at lower pH.

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, we designed the insect cell expression system 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 transfered into a parent bacmid in E.coli (DH10Bac). The bacmid was transfected to Sf9 insect cells to generate the baculovirus expressing mAb CO17-1A (CO17-1A-Bac virus). Western blot and immunoflouorescence confocal analyses confirmed the mAb CO17-1A expression in CO17-1A-Bac virus infected insect cells. The optimum conditions for mAb expression were validated at 24, 48 and 72h 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 72h after transfection with the MOI 1. HPLC chromatography revealed that the mAb CO17-1A expressed in the insect cell 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.