Earticle

The objective of this study was to determine the molecular and crystalline structures of starches from diverse rice cultivars for three major food processing (cooked rice, brewing and rice cake). Rice starches were isolated from 10 different rice varieties (4 varieties for cooked rice, 2 varieties for brewing, and 4 varieties for rice cake) grown in Korea. Apparent amylose contents of rice starches from cooked rice, brewing and rice cake varieties were 20.2-25.3%, 9.87-35.6%, and 20.8-28.3%, respectively. Rice starches from cooked rice varieties showed higher pasting viscosity and final viscosity but lower pasting temperature than those from brewing and rice cake varieties. Swelling factor at 80 °C of rice starches from cooked rice, brewing and rice cake varieties was 3.7-7.2, 3.5-11.4, and 2.4-7.6 , respectively. Based on structure and physicochemical properties of rice starches extracted from different rice varieties, principal component analysis (PCA) results showed that these rice varieties could be clearly classified according to processing adaptability for cooked rice and rice cake.

N ano- scaled starch particle is one of modified starch having potential applications due to its higher specific surface area relative to that of native starch, Acid hydrolysis is one of most convenient and most investigated procedure for preparing starch nano-particles, But the traditional acid hydrolysis approach has resulted in very low recovery of starch nano-particle, because transformation of both amorphous and crystalline structures in the starch granule into soluble sugar by the end of long batch acid hydrolysis, Herein, some alternative approaches has been studied to maximize yield of starch nano-particles, Combination of limited moisture content and higher reaction temperature effectively modified both molecular and starch granular, which could produce starch nano-particle with yield of higher than 80%,

Recently, some amylolytic enzymes, designated as glycoside hydrolase family 57 ( GH5 7), are newly assigned as second a -amylase family. The heterothermophilic archaea, Thermococcales, efficiently utilizes the carbohydrates using two main GH57 enzymes, 4-a -glucanotransferase (GTase) and maltose-forming amylase (MAase), which existed in all of reported genomes of the genus Thermococcus and Pyrococcus (1). GTase is specific for a -1,4-glucan with high transferase activity, while MAase is new type amylase, exhibiting unique dual hydrolysis activity toward a -1,4- and a -1,6-glycosidic linkages and only recognizes maltose in exo-type manner (2). The crystal structure of MAase at a resolution of L8 A showed that both enzymes shared similar substrate binding sites based on GH57 core structure ( 13/ah barrel-fold despite of their distinct specificity ( 3). Structural features further demonstrate that both enzymes have an aromatic amino acid residue which is located on the entrance of substrate binding site at + 1 to + 2 in same manner, From the mutagenesis analysis, we revealed that they have distinct role in substrate binding. Aromatic ammo acid residue of GTase can accommodate the substrates at acceptor binding region, while that of MAase can control the binding of substrates linked by a -1,4- or a -1,6-glycosidic bond and limit the length of substrates.

Enzymatic syntheses of sugar fatty acid (FA) esters are important because of their wide range of applications in the food, cosmetic, and pharmaceutical industries. These molecules are commonly synthesized from F As and mono-, or di-saccharides by chemical or enzymatic esterification. Previously, sugar esters were produced using oligosaccharide, and interfacial tension values and inhibitory effects towards tumor cell lines were improved when higher DP saccharides were used. In this study, the direct esterification of maltoheptaose (G7) with vanous length of F As was investigated by the catalytic action of Candida antartica lipase B. Among the F As palmitic acid (PA) was the best substrate for the G7 ester formation. Optimal reaction conditions including solvent composition and molar ratio of G 7: P A were established for the G7-PA. The conversion yield and sugar ester structure were analyzed using various instrumental analyses. The results showed that G7-PA monoester was successfully synthesized with a molar ratio of I :20 (G7: PA) in I 0% DMSO of t-butanol at 60°C, and the conversion yield was over than 21%. The position of ester bond formation was identified that PA was attached to C-6 of the reducing end glucosyl moiety. The melting temperature of G7- PA monoester was reached at 56 °C, lower than that of pure G7 and PA. In addition, the G7- p A esters and commercial sucrose esters were compared as emulsifiers. Both type of sugar esters prevented coalescence during 7 days and showed good emulsifying properties in 0/W emulsions since the droplets diameter did not have significant change during the storage.

Protein post-translational modification (PTM) increases the functional diversity of the proteome by the covalent addition of proteins. Therefore, identifying and understanding PTMs is critical in the study of protein bioscience. Protein glycosylation is acknowledged as one of the major post -translational modifications, with significant effects on protein folding, conformation, stability and activity. The need to characterize glycoproteins continues to increase as more irrefutable examples of the essential role that covalent carbohydrates m the proteins play in biological processes and functions. The extant challenge of glycobiology and glycotechnology is to approach the extremely low level of sensitivity, used for PTM analysis over the protein identification. In this study, several biologically important proteomic samples was analyzed by shotgun proteomics method, the resulting modified peptides are separated and detected by ultrahigh -performance nano liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). We, for the first time, identified specific 0-GleN Ac modification sites of Octl, Oct4, and 0-linked glycosylation site of Tango 1 with the state-of-the-art nano LC-Orbitrap Fusion Tribrid mass spectrometer. Additionally, We developed the Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) method to label the light C2C) or heavy CuC1) glucose on glycans of plant, yeast, and fungi. we quantitatively analyzed the N-linked glycan of Oryza sativa (rice) usmg metabolic labeling with stable isotopic glucose by a mass spectrometry. We also applied metabolic isotope labeling strategy for glycan quantitation in yeast (Saccharomyces cerevisiae).

In the production of therapeutic glycoprotein using recombinant CHO(rCHO) cells, sialylation is the critical factor in terms of biological functions, Previously, it was suggested that the sialic acid of glycoproteins produced from rCHO cells can potentially be impaired by sialidase released upon cell lysis during CHO cell culture, Bcl-xL overexpression in CHO cell culture not only increased Fc-fusion glycoprotein production, but also reduced the impairment of sialylation of Fc-fusion glycoprotein by maintaining high viability during the culture, Although the stressful conditions-induced cell membrane disruption can mainly lead to impairment of glycoprotein in CHO cell culture, rCHO cells maintained with viability more than 95% under some stressful condition can also produce impaired glycoprotein not caused by the release of intracellular enzymes such as proteases, From the NanoString nCounter analysis, we successfully identified differentially expressed N-glycosylation -related genes and miRN As to improve key bioprocess -relevant characteristics in the generation of engineered rCHO cells, Taken together, a number of factors can affect the sialylation of Fc-fusion glycoprotein produced from rCHO cells,

Galectin-1, a family of carbohydrate-binding proteins with a high affinity for 0 -galactosides, is differentially expressed by various normal and tumor tissues and appears to function in tumor progression and metastasis through the intracellular and extracellular activity. Here, we demonstrated that galectin -1 is increased in gastric malignant tissues and supports cancer sternness. Silencing of galectin-1 decreased the cancer sphere formation, side population, invasion and migration, whereas overexpression of galectin-1 increased them. We monitored changes in gene expression in galectin -1 silenced cells, employing DNA microarray analysis. The expression level of SOX2 and NANOG, a sternness-regulating transcriptional factor, was significantly decreased and confirmed by RT-PCR and western blot analysis. Interestingly, we determined the interaction between galectin-1 and SOX2. however, galectin-1 couldn't interact with 0-GlcNAcylation mutant SOX2. We predicted N -acetylglucosamine binding amino acid at galectin-1, and R71 and R74 is important to recognize 0-GlcNAcylation of SOX2. Overexpression of R71A and R74A mutant galectin-1 didn't increases cancer sternness property, such as cancer sphere formation, side population. Taken together, we suggest that galectin-1 interacts with SOX2 through 0-GlcNAcylation and increases SOX2 transcriptional activation and sternness character in gastric cancer progression.

Understanding the carbohydrate-protein interactions provides useful information on cullular processes in living organisms and is also helpful for the understanding the secretion pathway with respect to stress. Ribosomal proteins are usually known as structural proteins for the synthesis of proteins when cells are under a normal condition. However, some ribosomal proteins have extra-ribosomal functions in case of stress conditions. For example, ribosomal protein S3, a multi-functional protein, is also involved in DNA repair, apoptosis, cell cycle control etc. We have discovered that this protein forms a dimer and is secreted into medium after N -glycosylation only from various cancer cell lines as well as cancer patients but not in normal cells or people who do not have cancers. We also have found that other ribosomal proteins such as S2, S20 and Rackl from human and yeast cells are also secreted under various stress conditions. The secretion pathway appears to be a standard ER -Golgi dependent pathway. We are currently investigating the glycosylation mechanism of these ribosomal proteins which could be used as useful biomarkers.

Abcertin was developed as orphan drug, a new enzyme replacement therapeutic product which is imiglucerase for Gauchers patient's therapy in Korea. Glycosylation is a one of critical quality attributes for Imiglucerase. In this slide, the useful tools and approaches are shown for glycosylation analysis for imiglucerase in aspect of therapeutic protein development. And some of case study for Structure and function relation ship for glycosylation characterization will be introduced.

Heterogeneity and complexity of the glycosylation on biotherapeutics greatly depend on expression system, process conditions, and environment of cell culture of products. In order to evaluate biosimilarity of antibody drug such as Herceptin®, Remicade®, and Remsima®, we have developed a deep learning model, Deep Y, using intact glycoprotein analysis by LC-MS. Briefly, each antibody drug was independently analyzed to identify its intact glycoprotein composition. As a result, the list of identified intact glycoprotein compositions from each MS data was merged in the intact glycoprotein database, where a total of 34 intact glycoprotein compositions was identified from all three antibody drugs. Independently, the deconvoluted masses and their abundances of antibody drugs generated by Mass Hunter were used as data sets such as training, validation, and test set, for development of a deep learning model using convolutional and fully connected neural network. The accuracy was 100%, 90% and 85% at training, validation and test set, respectively, where the experiments of more than 100 ng/ml were predicted as 100% of accuracy at test set. The DeepY could predict the biosimilarity and distinguish the low quality of mass spectra from all antibody drugs. We will further test the antibody drugs in batch to batch, expand to other original antibody drugs and their biosimilars.

In order to assess similarity of biotherapeutics, many regulatory agencies recommend a stepwise approach for demonstrating biosimilarity between a proposed biosimilar product and an innovative (reference) biological product. In particular, glycosylation critical quality attributes (gCQA) of biosimilar can significantly affect drug's quality, safety, and efficacy. Additionally, the glycosylation is highly heterogeneous because it is primarily influenced by external conditions such as host cell lines and culture conditions. Thus, assessment of gCQA are necessary for evaluation similarity of biosimilars now commg to market. Here, we discuss the state of the art in analytical technologies to assess biosimilarity regarding glycosylation from regulatory perspective.

Sialylation modulates pharmacokinetic properties of recombinant therapeutic glycoproteins affecting their in VIVO half-life. N-glycan branching on glycoproteins provides the potential attachment sites for sialic acid. Here, we introduce a new approach for increasing the sialylation of recombinant human erythropoietin (rhEPO) produced m CHO cells by modulating poly-N-acetyllactosamine (poly-LacN Ac) biosynthesis. Initially, we found that repressiOn of the f33gnt2 increases tri- and tetra- N-glycan structures. We did not observe an mcrease in rhEPO sialylation, however, until the feedback inhibition by intracellular cytidinemonophosphate-N-acetylneuraminic acid ( CMP-N eu5Ac), which is a limiting factor for sialylation, was released. Thus, we found that a combined approach inhibiting poly-LacN Ac biosynthesis and releasing CMP-N eu5Ac feedback inhibition produces the most significant increase in rhEPO sialylation in metabolically engineered CHO cells. Furthermore, a detailed analysis of the resulting N -glycan structures using LC/MS revealed increased tri- and tetra- sialylated N-glycan structures accompanied by a reduction of di-sialylated N-glycan structures. These results validate our new approach for glycosylation engineering, and we expect this approach will be useful in future efforts to enhance the efficacy of other therapeutic glycoproteins.

Glycosylation ts one of the most frequent post-translational modification, playing an essential role m the normal development and physiology of cells. Altered expression of glycans and glycoconjugates has been associated with numerous pathologies, including congenital disorders and cancer. To achieve early diagnosis, cancer biomarkers have been sought for several purposes preferably in blood and pinpointing cancer cells-derived aberrant glycoproteins would be a well-grounded approach to cancer biomarker discovery. One of the glycosyltransferases responsible for aberrant glycosylation m cancer lS N-acetylglucosaminyltransferase v (GnT-V), which catalyzes an addition of betal ,6-N-acetylglucosamine (GicNAc) to the core N-glycan, and many lines of evidence have demonstrated the role of N-acetylglucosaminyltransferase V (GnT-V) in cancer development. Tissue inhibitor of metalloproteinase-1 (TIMP-1) and protein tyrosine phosphatase kappa (PTPk) were suggested to be involved in cancer malignancy upon aberrantly glycosylation by GnT-V. Although cancer biomarker candidates were drived with many lectins and mass spectrometry, an efficient validation method based on a sensitive and multiplexed platform was hampered. Although ELISA-based analytical methods are very powerful for detecting specific proteins, the lectin/antibody sandwich method, which simultaneously recognizes proteins and glycans, is difficult to apply because the antibodies themselves are glycoproteins. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity, but there are difficulties in cost and reproducibility due to the many steps. Ultimately, in order to overcome this, we have made a mouse that produces a non-glycosylated antibody through a genome editing method, and a recent study on a glycan humanized mouse is also introduced -

X -linked inhibitor of apoptosis (XIAP), an inhibitor of apoptotic cell death, has a RING domain and functions as an E3 ligase, catalysing the ubiquitination of various substrates such as apoptosis-inducing factor, TGF- [3-activated kinase 1, and MEK kinase 2. Here, we identified 0-linked N-acetylglucosamine ( 0-GlcNAc) transferase ( OGT) as a substrate of XIAP. We showed that OGT catalyses the 0-GlcNAc modification of XIAP at serine 406, and this modification regulates the E3 ligase activity of XIAP towards OGT. Substitution of XIAP Ser406 by alanine decreased its E3 ligase activity toward OGT but not toward other substrates. Stable overexpression of XIAP in HCT116 human colorectal carcinoma cells decreased OGT protein levels and inhibited cancer cell growth and invasion. These results suggest that XIAP requires 0-GlcNAcylation in the regulation of its E3 ligase activity toward OGT, which leads to OGT degradation and the inhibition of cancer cell growth.

Recently, many polysaccharides and oligosaccharides derived from marme seaweeds, plants, mushrooms, microorganisms as well as animals, have been attracting great attention due to their diverse range of beneficial effects on human health, such as anti-inflammatory, anti-viral, anti-coagulant, anti-tumor, and anti-metastatic, anti-oxidant, immuno-stimulating, anti-osteoporotic, anti-angiogenesis activities, etc. Among natural polysaccharides, sulfated polysaccharides (SPS) that contain hemi-ester sulfate groups in their sugar residues are commonly found in marine algae and have been target materials of numerous researches. Apart from industrial uses, these algal polysaccharides have emerged as a rich and important source of bioactive natural compounds, due to their diverse pharmacological activities and their availability in a relatively large amount from the raw source materials. On the other hand, although ginseng marc is a by-product obtained during manufacturing of various commercial ginseng products and has been routinely discarded as a waste, it still contains considerable amounts of polysaccharides. For these reasons, it would be worth evaluating various bioactivities and applications of these compounds for the development of new therapeutic agents or more value-added functional ingredients of health beneficial foods. However, it has been generally recognized that the innate structural variations and complexity, high molecular weights, and viscous nature of polysaccharides may limit their successful applications as health-beneficial food materials or therapeutic agents, suggesting that smaller-sized oligosaccharides would overcome these problems. Indeed, significant efforts have focused on preparing bioactive oligosaccharides from high-molecular weight polysaccharides (HMP), mmmg especially for their pharmaceutical usage. Therefore, a reliable polysaccharide-degrading enzyme preparation would be highly desirable to obtain lower-sized oligosaccharides and thereby help overcome these problems. In this context, we have isolated and purified polysaccharides from natural resources including Korean seaweeds and Korean ginseng roots and characterized their structures and biological activities such as immunomodulating, anti-viral, anti-coagulant, anti-cancer, anti-inflammatory, anti-obesity, anti-oxidant, anti-osteoporotic activities, etc. In addition, we also isolated and identified some enzyme activities that can degrade HMP, producing lower-sized oligosaccharides. This presentation will discuss about the structures and bioactivities, such as immunostimulating, anti-cancer, and anti-viral activities, of these polysaccharides and oligosaccharides derived from Korean seaweeds and gmseng. Their action mechanisms involved in their respective bioactivity will be discussed as well.

Post-biosynthesis modifications of natural products (NPs) provides opportunity to bring chemical diversity to the parent molecule. Such modifications usually play vital roles in executing biological activities of the molecules. Thus, engineering of molecules by diverse post-modifications is increasingly becoming a tool to design or produce novel biologically potent biologics. To develop the rapid and sustainable system for the production of different flavonoid glycosides, central metabolic pathway for the production of pool of UDP-0-glucose, UDP-0-xylose, TDP-L -rhamnose, TDP-0-viosamine, TOP 4-amino 4,6-dideoxy-D-galactose, and TOP 3-amino 3,6-dideoxy-D-galactose was engineered in E. coli BL21 (DE3) cells. Different glycosyltransferases were engaged to transfer sugar moieties to aglycones. Several flavonoids and isoflavonoid glycosides including natural and non-natural o- and C- glycosides were produced by microbial cell fermentation. In a different approach of enzymatic biosynthesis, a number of glycodiversified flavonoids were generated using several NDP-sugars and GTs in vitro. As a result, glycodiversified resveratrol, flavonol, epothilone A, mupirocin conjugated with glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugars were produced. Multiple glycosides of other flavonoids, isoflavonoids, chalcones, stilbenes, xanthonoids, anthraquinones, anthracyclines, and terpenoids were also generated with significantly high yield. Some of the selected glycosides exhibited promising anticancer, antibacterial, immunomodulatory, anti-inflammatory, antioxidant, and anti-asthmatic activities in in vitro and in vivo mouse models. This approach of microbial and enzymatic synthesis of novel glycosides derivatives of NPs using highly flexible and promiscuous enzymes from diverse sources opened up possibility of development of new molecules with better stability, bioavailability, and novel biological activity.

Immune tolerance at feto-maternal interfaces is a complex phenomenon. Although maternal decidual macrophages are well-known immune cells, little is known about fetal-derived macrophages (Hofbauer cells) within chorionic villi. Preeclampsia (PE) is a major cause of maternal mortality in the field of obstetrics, and the innate immunological role of maternal decidual macrophages is well known. In this study, we assessed the differential phenotypes and marker expressiOn m fetal macrophages, known as dendritic cell- specific ICAM -grabbing nonintegrin (DC-SIGN) -positive Hofbauer cells. We compared Hofbauer cell properties between normal and PE placenta chorionic villi and performed sequential staining of DC-SIGN, CD14, and CD68 to evaluate the existence of Hofbauer cells. Furthermore, to evaluate the immunological function of these cells, we stained the cells for CD163, a marker of immunoregulatory type 2 (M2) macrophages. Additionally, we examined the expression of the immunosuppressive cytokine interleukin (IL) -10, which is known to be produced by M2 macrophages. DCSIGN +/CD14+, DC-SIGN +/CD68+, and CD163+/DC-SIGN + cells were quantified based on photomicrographs. The results showed that CD14, CD163, DC-SIGN, and IL-10 levels were significantly downregulated in PE compared with normal. Additionally, CD 163 +/DC -SIGN+ Hofbauer cells were significantly less frequent in PE than in normal. DC-SIGN Hofbauer cells produced IL-10 at lower levels in the PE than in the normal. Thus, we speculate that fetal-derived Hofbauer cells may play an important role in normal pregnancy with immunosuppressive effects based on their M2 macrophage characteristics to maintain immune tolerance during pregnancy. Additionally, in PE, these functions were defective, supporting the roles of these macrophages in PE development.

Dihydroxybenzenes exhibit biological functions such as anti- cancer, anti- inflammation, and antioxidants. They are also widely used in the cosmetics industry and as a treatment of skin related disease. In spite of these functions, they are easily oxidized by light in aqueous solutions and are limited by toxicity and side effects. The transglycosylation for these compounds have been shown to resolve these problems of solubility, stability and toxicity. Amylosucrase from Deinococcus geothermalis (DGAS) has been selected as the enzyme to be used for the transglycosylation. This enzyme is industrially useful because it supplies glucose using a relatively inexpensive donor substrate (sucrose). In this study, amylosucrase gene ( dgas) was codon optimized according to E. coli BL21 (DE3) codon usage. Sucrose was used as a source of glucose and it was used to produce three dihydroxybenzene (Hydroquinone, Resorcinol, Catechol) glucosides derivatives. Optimization of temperature, pH, donor (sucrose) and acceptor substrates concentrations increased glucoside derivative productivity. In addition, amyloglucosidase was used to reduced gluco-oligosaccharides and increased the production of mono-glucoside derivatives. Finally, the DgAS enzyme was immobilized on AMICOGEN LKZ118 beads. Immobilized enzyme could be used repeatedly up to 35 ~ 40 cycle on average. The percent yield of the total glycosides of hydroquinone and catechol varied from 85% to 90% until in immobilized system, however, in case of resorcinol the yield was in between 65% to 70%. It showed production of hydroquinone-mono-glucoside 13.3 g (35 cycle, 175mL), resorcinol-mono- glucoside 5.9 g ( 40 cycle, 200mL) and catechol-mono- glucoside 11.5g ( 40 cycle, 200mL) using a immobilized enzyme. The immobilized DgAS 1s cost reduction due to the separation and repeated use of the enzyme, to be effective for industrial application.

Minor ginsenosides, those have low content in white or red ginseng, are proved to have strong pharmaceutical activities in literature. There are many minor ginsenosides can be formed during transformation in various pathways. In which, the biotransformation pathway of Rb2→Rd→F2→C-K or Rc, Rbl, Rb2→Rg3→Rh2 by using various bacteria, yeast, and fungus was well-known. However, the other pathway that converts Rb2→C-0→C-Y→C-K or Rc→C-Mc→C-K were seldom occurred. In this study, utilization of red ginseng residue (RGR), a byproduct of red ginseng extraction, for minor ginsenoside production by using enzyme cellulose, hemicellulase, and lactic acid fermentation was performed. Biotransformation of ginsenosides was analyzed by UPLC-UV /EIS-QQQ mass spectrometry. During enzymatic reaction and lactic acid fermentation, the relative abundance of ginsenoside Rbl, Rg3, Rb2, and Rc were decreased while a significant increase in rare ginsenoside F2, Rh2 , C-K, C-0, and C-Mc were obtained. Our results suggested that combination of cell wall degrading enzyme and metabolism of lactic acid bacteria (LPlantarium and LLactis) could remove glucosyl moieties from ginsenosides. Particularly, C-0, C-Mc could be obtained during enzymatic treatment and lactic acid fermentation. This method could be applied for production of C-0, C-Mc, and C-K from RGR. For further research, this result may open a new direction toward RGR utilization for minor ginsenosides.

A functional glycan chip combined with on-chip enzymatic glycosylation was developed to prepare complex glycan sources and to apply glycan -involved applications simultaneously. Here, GM3 trisaccharide, GM2 tetrasaccharide, and GM1 pentasaccharide were successfully direct biosynthesized on lactose-immobilized surface through consecutive three glycosyltransferase reactions. Biosynthesized GM1-related complex glycans were demonstrated to provide the information on substrate specificity of whole cholera toxin. Thus, the proposed on-chip glycan biosynthesis system can provide a new direction toward obtaining complex glycan sources and complex glycan -involved applications such as glycan -protein interaction analysis and glycan biomarker-employed diagnosis.

Alginate has been widely used in hydrogel- based tissue engineering area as an excellent carbohydrate biomaterial due to its good biocompatibility and easy gelation by cation induction such as calcium ions. 3D bioprinting has been regarded as a powerful tool for mimicking natural structure, because it can fabricate on-demanded hierarchical artificial tissues. Due to several advantages of alginate, it has been the most frequently used biomaterial in inkjet-based bioprinting area. However, this ionically crosslinked alginate IS noticeably impoverished under a physiological, aqueous conditions due to easy loss of calcium ions. Thus, the low structural integrity and poor mechanical stability limited long-term cell culture on alginate-based bioprinted structures. While barium or copper IOns can increase its stability, these also increase cytotoxic effects. In this work, we proposed a new visible light- activated crosslinkable alginate-based bioink, showing fast and non -cytotoxic gelation. We constructed 3D cell-laden structures using this novel alginate material and various cell types. The light/ionic double crosslinked alginate bioink showed excellent biocompatibility, increased mechanical properties, and improved dimensional stability. Further development of this novel alginate- based 3D inkjet bioink may facilitate preparation of transplantable human tissues.

Glycosylation plays important roles m a therapeutic glycoprotein such as stability, efficiency, and pharmacokinetics. Especially, sialic acid expressed as an outer terminal unit on the glycoprotein is involved in half-life in serum. In particular, Neu5Gc, a non-human sialic acid, is often observed in therapeutic glycoproteins produced from mammalian cell lines. And exogenous Neu5Gc can be an immunogenic antigen in human cells. Therefore, the screening and quantitation of Neu5Gc in a therapeutic glycoprotein is highly required in the manufacturing and development processes. In this study, we developed an analytical method for selectively identifying and quantifying Neu5Gc with high sensitvity usmg mass spectrometry. The amount of Neu5Gc isolated from various mAb-based therapeutics such as infliximab and trastuzumab was correctly determined. Briefly, sialic acids were liberated from each therapeutic glycoprotein by chmical hydrolysis and further enriched using solid phases extraction with a PGC cartridge. The Neu5Gc was chromatographically separated on a PGC column, then analyzed by MRM-MS. The concentration of Neu5Gc from mAb-based therapeutics was determined at low nano mole levels with high reproducibility ( CV < 6%). Batch-to-batch variations of Neu5Gc from a therapeutic glycoprotein were quatitatively determined. This method will be a valuable platform for not only QbD processes of biotherapeutics but also QA/QC of products to assess impurites and immunogenecities

Aberrant protein glycosylation IS frequently observed in vanous disease states including cancer, thereby making efforts to utilize a specific glycoprotein of a protein as a disease biomarker feasible. The immunoassay using biomarker-specific antibodies is one of the most reliable, robust and convenient methods. Conceptually, the combination of an antibody and a lectin (or glycan- specific antibody) may enable detection of a specific glycoform of a biomarker when applied to the sandwich ELISA platform. However, the glycans m immunoglobulin G interfere with the interactions between an antigen and a lectin, thereby making such immunoassay fundamentally unfeasible. Several efforts have been made to overcome this problem including deglycosylation using PNGase-F, enzymicdigestion of an antibody using pepsin, and chemical modifications of glycans of antibodies. However, such efforts rendered unsatisfactory analytical outcomes. To resolve this matter fundamentally, we genome-engineered mice by mutating the N-glycosylation consensus site (asparagine-coding codon) into non-asparagine coding sequences. We found that aglycosylated antibodies were produced in the genome-engineered mice following immunization with antigens. The stability of the aglycosylated antibodies produced from the engineered mice was equivalent to that of conventional antibodies. Moreover, the aglycosylated antibody in combination with a lectin was reliably applicable to the ELISA platform to quantify a specific glycoform with diagnostic validity. The genome-engineed mice can also be used as a host for generation of an aglycosylated antibody to measure a specific glycoform of biomarkers.

An epithelial cell adhesion molecule (EpCAM) is a type I transmembrane protein expressed in the majority of normal epithelial tissues and overexpressed in the human epithelial cancers including breast cancer. Treatment with plant-derived anti-EpCAM mAb (mAbr COK) and Raw264. 7 cells inhibited cell growth in the human breast cancer cell line MCF -7. In MCF-7 treated with mAbr COK and Raw264.7 cells, expression of p53 and p21 increased, whereas the expression of G1 phase-related protein, cyclin D1, CDK4, cyclin E and CDK2 decreased. In addition of treatment with mAbP COK and Raw264. 7 cells decreased the expressiOn of anti-apoptotic protein Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-u, caspase-3, caspase-6 and caspase-9, increased. Moreover, cells treated with mAbr COK and Raw264. 7 cells expressed metastasis-related gangliosides GM1 and GD1a. These results suggest that mAbr COK has an effect on anti-cancer activity.

Glycosylation plays important roles m a therapeutic glycoprotein such as stability, efficiency, and pharmacokinetics. Especially, sialic acid expressed as an outer terminal unit on the glycoprotein is involved in half-life m serum. In particular, Neu5Gc, a non-human sialic acid, is often observed in therapeutic glycoproteins produced from mammalian cell lines. And exogenous Neu5Gc can be an immunogenic antigen in human cells. Therefore, the screening and quantitation of Neu5Gc in a therapeutic glycoprotein is highly required in the manufacturing and development processes. In this study, we developed an analytical method for selectively identifying and quantifying Neu5Gc with high sensitvity usmg mass spectrometry. The amount of Neu5Gc isolated from various mAb-based therapeutics such as infliximab and trastuzumab was correctly determined. Briefly, sialic acids were liberated from each therapeutic glycoprotein by chmical hydrolysis and further enriched using solid phases extraction with a PGC cartridge. The Neu5Gc was chromatographically separated on a PGC column, then analyzed by MRM-MS. The concentration of Neu5Gc from mAb-based therapeutics was determined at low nano mole levels with high reproducibility ( CV < 6%). Batch-to-batch variations of Neu5Gc from a therapeutic glycoprotein were quatitatively determined. This method will be a valuable platform for not only QbD processes of biotherapeutics but also QA/QC of products to assess impurites and immunogenecities

Gangliosides are amomc glycosphingolipids containing one to several sialic acid residues. Although they play an important role in neuro-biological functions including synaptic plasticity and memory formation, there are only few studies in ganglioside due to their structural complexity and lack of effective analytical methods. In particular, isomeric investigation of gangliosides is necessarily required in order to understand the ganglioside biology with the consideration of its biosynthesis however, isomer separation based on reversed-phase (RP) chromatography is highly challengeable. In this study, for the purpose of construction of localized mouse brain ganglioside library, we examined ganglioside from anatomically dissected nine mouse brain regions using UHPLC-QTOF MS and MS/MS. Briefly, gangliosides were extracted by modified Folch method with chloroform and methanol, followed by purification and enrichment by Cl8-SPE. Then they were identified and quantified by UHPLC (Cl8 column) QTOF MS. Ganglioside isomers were completely separated depending on their glycan traits on a Cl8 column by addition of formic acid in the mobile phase. Furthermore, tandem MS analysis was performed to confirm the identification based on diagnostic fragment ions. Through LC-MS/MS based isomeric investigation of gangliosides, region- specific mouse brain ganglioside biosynthetic pathway including 0-, a-, b- and c- series could be suggested. Interestingly, major ganglioside were distributed with distinguished qualitative and quantitative pattern for each nine brain regions. In the future, the constructed library of mouse brain ganglioside will be used for monitoring alteration in KO mouse models including glycosylation transferase KO mice.