Earticle

Insect cell expression system using baculovirus has several benefits from high capacity, flexibility, and safety to humans and glycosylation capability. Thus, the baculovirus insect cell system has been widely used for production of recombinant protein. The HC and 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 DH10Bac. Immunoflouorescence analyses and Western blot 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 ranging (0.2, 1 and 5). HPLC analysis 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 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.

Accumulation of unfolded proteins in the lumen of endoplasmic reticulum (ER) induces ER stress and activates signaling pathway called unfolded protein response (UPR) for relief of ER stress. In yeast and mammalian cells, an ER-located transmembrane receptor protein kinase/ribonuclease called Ire1 transmits the signal to the nucleus culminating in the transcriptional activation of genes encoding an adaptive response. Plants including Arabidopsis are also containing homologous genes. However, it has been unclear whether the homologous genes are involved in similar mechanism in plants. Arabidopsis ire1 mutant did not show different phenotype compared to WT in normal and stress conditions. However, induction of ER chaperone genes including Bip3 were significantly delayed in ire1 compared to WT. To further investigate the role of AtIre1 in plant UPR signaling, we overexpressed AtIre1 under the control of CaMV 35S promoter. The result indicate that AtIre1 is possibly involved in UPR signaling in plants. [Supported by EB-NCRC & BK21 program]

Plant-based expression of recombinant proteins offers significant advantages in terms of the production of pharmaceuticals for human diseases. These include cost-effective production of recombinant proteins due to the ease of scale-up, minimal possibility of product contamination by animal pathogens and the availability of natural storage organs such as tubers, fruits and seeds. Among the many plant-based production systems that have been developed for pharmaceuticals proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume and in stable environment in which they are protected from degradation. In this respect, we overexpressed human glucocerebrosidase (GC) in Arabidopsis seeds driven by cruciferin 3 promoter. In attempt to produce mannose-terminated N-glycan of GC, the construct was introduced into Arabidopsis gnt1 mutant deficient in the activity of N-acetylglucosaminyltransferase I, the first enzyme in the complex glycan biosynthesis. Our results indicate that the human GC is highly expressed in the seed. [Supported by EB-NCRC & BK21 program]

In the endoplasmic reticulum (ER) elicit a highly conserved ER stress response called the unfolded protein response (UPR). Accumulation of misfolded proteins elicits signals by three types of ER stress sensor/transducer in mammal. Among them, ATF6 is a type II transmembrane protein normally retained in the ER. In this study, we have identified two membrane-associated basic domain/leucine zipper (bZIP) factors AtbZIP17 and AtbZIP28 in Arabidopsis thaliana as candidates for ER stress sensors/transducers similar to mammalian ATF6. AtbZIP17 and AtbZIP28 have N-terminal bZIP domain, a transmembrane segment, respectively. Truncated forms of AtbZIP17 and AtbZIP28 lacking the C-terminal domain were overexpressed in Arabidopsis. AtbZIP17 and AtbZIP28 overexpressed Arabidopsis showed upregulated the expression of ER chaperone genes in the absence of stress conditions. Interestingly, they growed bigger and more vigorously than WT in normal growth condition. Under stress conditions, AtbZIP17 and AtbZIP28 overexpression plants showed enhanced stress tolerance compare with that of WT plants. These results suggest that AtbZIP17 and AtbZIP28 function as sensor/transducer in Arabidopsis to mediate ER stress response related to UPR. [Supported by EB-NCRC & BK21 program]

N-acetylglucosaminyltransferase I (GnTI) is an enzyme that controls the formation of hybrid and complex type N-glycans. Plant produce complex N-glycans with β1,2-xylose and core α1,3-fucose residues linked to the conserved core oligosaccharide. Production of heterologous glycoprotein in plant for therapeutic purposes is limited by the presence of plant specific oligosaccharide residues that are considered to arouse immunogenic response in the recipient body. Therefore, efforts for developing humanized N-glycosylation system in plants are required for the large scale production of safe therapeutic glycoproteins. In this regard, we isolated a rice mutant lacking GnTI activity. Biochemical analyses showed that rice gnt1 mutant predominantly produced high mannose type glycans and β1,2-xylose and core α1,3-fucose were absent on the endogenous glycoproteins. However, the rice gnt1 mutant showed severe developmental defects such as retarded shoot and root development, failure in tiller formation, and finally resulted in early developmental lethality. Interestingly, callus induced from gnt1 seeds was maintainable and proliferation rate of gnt1 callus was similar with that of WT. However, gnt1 calli were relatively smaller than that of WT. [Supported by EB-NCRC & BK21 program]

The (1,6)-fucosylation of core N-glycan (core fucose) has been known to play crucial roles in various pathophysiological events such as oncogenesis and metastasis. Therefore, a fluorescent probe recognizing core fucose will be a powerful tool for elucidating its functional role and further diagnosing cancer. The fleA gene, which has been known to code Aspergillus oryzae lectin (AOL), was cloned from A. oryzae RIB40. We fused it with the either citrine or cerulean gene (improved variants of yellow or cyan fluorescent protein) to generate two expression vectors. The resulting fusion gene products (FleA-citrin & FleA–cerulean) were successfully expressed in Escherichia coli and purified homogeneously using two step affinity purification method. The fluorescent FleA fusion proteins were shown to detect core fuose in glycoproteins on the surface of cancer cells. The reduction of core fucose amount induced by the transfection of (1,6)-fucosyltransferase gene, Fut8, siRNA was also successfully detected by the FleA fusion proteins. These results show that our FleA-fluorescent fusion proteins would be employedas a valuable fluorescent probe for detecting core fucose.

Novel chimeric enzyme, lacdiNAc (GalNAcb1,4GlcNAc) synthesizing glycosyltransferase was generated by chimeric fusion of -lactalbumin and b1,4-galactosyltransferase 1. The chimera was expressed in Lec8 cells and exhibited lacdiNAc synthesizing activity in the absence of exogenous -LA as well as other glycosyltransferase activities including lactose synthesizing glycosyltransferase, and galactosyltransferase. Glycosyltransferase activities of the chimera were compared to those induced in the conventional system of co-presence of bovine galT1 and -LA, indicatingthat each domain of -LA and galT1 on the chimera is not only folding correctly but also interacting together. Furthermore, we found that the chimera were auto-lacdiNAcylated and can synthesize lacdiNAc structures in Lec8 cells.

cDNAs encoding three isoforms of OGT were expressed in Escherichia coli and the co-expression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoforms while the co-expressed target proteins were strongly and differentially O-GlcNAcylated depending on OGT isoforms. We found that sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Under these conditions, more importantly, sp1 can interact with ncOGT by which recognition of target protein byOGT is newly formulated. These results strongly suggest that sp1 may modulate OGT activity of target recognition.

Novel cDNAs of zOGT was identified in zebrafish (Danio rerio) genome and six different isoforms of transcriptional variants, designated var1 to var6 were isolated. Transcriptional variants of zOGT were differentially generated by alternative splicing and in particular, var1 and var2 were contained by a new exon-sequence, demonstrating that this form of zOGT was not found in mammals. We found that var1 and var2 were highly expressed at early phase including unfertilized egg until at 5-6 hpf whereas var3 and var4 were begins to be expressed at 5-6 hpf until late phase during embryogenesis. These results strongly indicate that var1 and va 2 are maternal transcripts. Coexpressionassay in E. coli–p62 system showed that zOGT catalytic activity of var3 and var4 was found to be active whereas those of other variants were inactive.

α-Glucosidase of Thermotoga maritima belongs to family 4 of the Glycosyl hydrolases. The enzyme α-glucosidase has the unusual property of requiring NAD+, divalent metal cations Mn2+ and reducing conditions (DTT) for activity. The gene aglS encoding 459 amino acid with 51 kDa was expressed under the control of the T7 promoter in Escherichia coli. It was found soluble form and thermo-stable up to 70oC. It showed hydrolyzing activity towards para-nitrophenyl-α-D-glucoside (pNP-α-Glc). The transglycosylation activity was tested using maltoseand sucrose as donor and cyclodextrin as acceptor. The molecular mass of cyclodextrin containing two glycosyl moieties is seen in ESI/MS when maltose and sucrose was used as donor. The further analysis is yet to be confirmed.

Antimicrobial activities of a series of chitosans with different molecular weight were investigated against a Gram-positive meticillin-resistance Staphylococcus aureus (MRSA) and a Gram-negative bacteria Pseudomonas aeruginosa to determine the effects of molecular weight and concentration on the inhibition of bacterial growth. Medium-molecular weight chitosan (P6-MMC) with average of 110-kDa was obtained by enzymatic digestion of high molecular weight of native chitosan using an immobilized chitosanase (N-174) on silica-gel. In comparison with high molecular weight of native chitosan, low-molecular weight chitiosan (LMWC) and medium-molecular weight of chitosan (MMWC), P6-MMC prepared in this study has shown the most significant antimicrobial activities in a dose-dependent manner against MRSA, and toward P. aeruginosa as well. Our present data observed in this study suggest that antimicrobial activity against antibiotic-resistance microorganisms of a series of chitosan varied depending on the concentration of chitosan, molecular weight of chitosan, and type of microorganisms.

Carbohydrate-protein interactions are involved in various biological events, including viral and bacterial infection, immune response, differentiation and development, the progression of tumor cell metastasis. Carbohydrate microarrays are new technology that has potentiated the high-throughput analysis of carbohydrate-protein interaction and contributes to significant advances of glycomics. Here, we proposed a facile, efficient, and cost-effective method whereby diverse carbohydrate types are modified in a single step and directly immobilized onto a glass surface, with retention of functional orientation. We modified various types of carbohydrates by reductive amination, in which reducing sugar groups were coupled with the linker. The modified carbohydrates were covalently attached to an amine-reactive NHS-activated glass surface by formation of stable amide bonds. This method was applied for construction of functional carbohydrate microarray platform to analyze carbohydrate-protein interactions. This array by using our method can be powerful tool for screening the specificity of GBPs (glycan-binding proteins) such as lectins, growth factors, antibody, and microbial toxins, and identification of carbohydrate-protein interactions.

Capsular polysaccharides (CPSs) of Streptococcus pneumoniae are representative T-lymphocyte independent type 2 (TI-2) antigens, causing the immune escape from the host and majority of meningitis in human adults. However, the detail mechanism of this immune escape by S .pneumoniae has been poorly understood. Serotype 14 CPS taken by SIGN-R1+ macrophages in the splenic maginal zone is regurgitated to follicular dendritic cells (FDCs). It was consistent with polysaccharide dextran, another ligand for SIGN-R1, and its regurgitation was confirmed in SIGN-R1 transfectants in vitro. While understanding the regurgitation of dextran, we found that SIGN-R1 bound to Binding immunoglobulin protein (BiP), a well-characterized endoplasmic reticulum (ER) chaperone, primarily on the cell surface. Furthermore, SIGN-R1+ macrophages in the maginal zone showed the higher expression of BiP than other macrophages in the red pulp, implying the important role of SIGN-R1 binding to BiP in vivo. Surprisingly, after the treatment of dextan in vitro or in vivo, dextran was rapidly translocated into the ER membrane and subsequently regurgitated here, depending on SIGN-R1 and complement C3. Therefore, these results demonstrated that the interaction of SIGN-R1 with Bip mediates the regurgitation of polysaccharide dextran through the ER membrane, demanding further studies to understand the reasons of the non-immunogenecity of bacterial polysaccharides.

The stereoselsctive construction of α-sialyl linkages still remains one of the most challenging tasks in synthetic carbohydrate chemistry. In this study, a new stereoselective α-sialylation method with sialyl pentenoates as sialyl donors has been developed employing PhSeOTf as a promoter. Especially sialylations of various acceptors with 5-N-trifluoroacetyl-sialyl pentenoate in acetonitrile were highly a-selective to provide exclusively or predominantly α-sialosides. This new method was applided to the synthesis of a disaccharide part of the glycosyl phosphatidyl inositol (GPI) anchor of the scrapie prion protein.

O-linked beta-N-acetylglucosamine (O-GlcNAc), one of the most important post-translational modification, regulates many cellular processes, including signaling, cell cycle, and transcription. However, the physiological function of O-GlcNAc in vivo is completely unknown. Some studies have shown that O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) modulates macronutrient storage and dauer formation in C. elegans and provide a genetic model for examining the role of O-GlcNAc in cellular signaling and insulin resistance. Here, we show that Drosophila melanogaster OGT and OGA regulates developmental growth through Insulin-IGF signaling pathway. Because not only human nucleocytoplasmic O-GlcNAc trasnferase (ncOGT) and OGT of drosophila melanogaster have highly conserved amino acid sequence but also human OGA and the hyaluronidase domain of drosophila OGA share the highly conserved sequence, our study provides insight into the mechanisms of O-GlcNAc modification in developmental growth and into the pathobiological basis for diabetes and obesity.

Previously, we reported an efficient one-pot direct glycosylation method with anomeric hydroxy sugars as glycosyl donors and phthalic anhydride and Tf₂O as activating agents. Although this method is very efficient for the selective β-mannosylation and other glycosylations, in certain cases, undesired self-condensed esters were generated as minor products. In order to suppress the generation of the self-condensed ester, 4,5-dimethoxyphthalic anhydride, 2,3-pyridinedicarboxylic anhydride, 3,4-pyridinedicarboxylic anhydride, and 4-methylphthalic anhydride were examined as activating agents instead of phthalic anhydride. 2,3-Pyridinedicarboxylic anhydride provided the best result and the improved glycosylation was carried out in three steps (ⅰ) the reaction of anomeric hydroxy sugars with 2,3-pyridinedicarboxylic anhydride in the presence of DBU at room temperature, (ⅱ) addition of Tf₂O at -40 ℃, and (ⅲ) addition of glycosyl acceptors to the reaction mixture at -40 ℃.

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification decreases. However, recent reports demonstrated increased GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine: fructose-6-phosphate midotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic athway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.

The mycolyl arabinogalactan-peptidoglycan (mAGP) complex plays a crucial role for the survival and pathogenicity of M. tuberculosis. It is important to synthesis the motifs of the mAGP complex are attractive synthetic targets, because of not only their potential as anti-TB drug but also their biological significance as substrates for enzymes involved in the biosynthesis of mycobacterial cell wall. The present study describes synthesis of compound 1, a suitably protected form of heptafuranoside, {β-D-Galf-(1->5)-β -D-Galf-(1->6)-[α-D-Araf-(1->5)]-β-D-Galf-(1->5)-β-D-Galf-(1->6)-β -D-Galf-(1->5)-β-D-Galf},from the arabinogalactan-peptidoglycan complex in the mycobacterial cell wall. Key steps include the coupling of seven monosaccharide building blocks with complete stereoselectivity by glycosylations using 2-carboxybenzyl glycosides and glycosyl fluoride as glycosyl donors.