Earticle

Glycans are substantially more complex than other important biological macromolecules such as nucleic acids and proteins. The linkages between individual subunits in glycans are not identical whereas nucleic acids and proteins are linear molecules in which the individual subunits are linked by identical phosphate and peptide bonds. Consequently, glycans may be linear or branched and may be covalently attached to a protein or lipid. Glycans are therefore highly efficient vehicles for information storage. Efficient chemical assembly of glycans has been a major concern in synthetic organic chemistry over the past decade due to important roles of complex glycans in many fundamental life-sustaining processes. Development of efficient and stereoselective glycosylation methodologies is crucial for the efficient synthesis of glycans. The selection of an appropriate glycosyl donor is one of the key processes for the successful glycosylation in terms of efficiency and stereoselectivity. Lecture starts with simple transformations of polyhydroxy compounds such as the selective oxidation of diols or triols and the selective protection or deprotection of polyols. Lecture also includes the synthesis of higher-carbon sugars, higher-carbon sugar nucleosides, branched-chain sugars, and inositols. Lecture continues with describing our works on development of new glycosyl donors and stereoselective glycosylations with the new glycosyl donors such as 2´-carboxybenzyl (CB) glycosides, glycosyl pentenoates/PhSeOTf, and C-1 hydroxy sugars/phthalic anhydride/Tf2O. Complexoligosaccharides, which were synthesized by our new glycosylation methods, are listed Lecture also includes the effect of remote protecting groups of glycosyl donors on glycosylation stereochemistry. Mannosylations of various acceptors with donors possessing an electron-withdrawing group at O-3, O-4, or O-6 positions were found to be b-selective except when donors had 3-O-acyl and 6-O-acetyl groups, which afforded a-mannosides as major products. Directing effect of remote protecting groups of other glycosyl donors was found to be quite different from the directing effect of those of mannosyl donors.

(재)경기과학기술진흥원 경기바이오센터는 국내 생명공학분야의 효율적 육성과 연구개발을 위해 바이오/제약기술의 산업화 지원을 수행함으로 지역산업 고도화 및 국가경쟁력 강화에 기여하고자 설립되었으며, 道 내 바이오 기업의 연구개발을 전폭적으로 지원하여 신약, 세포 치료제, 바이오 신소재 개발을 돕고 기업의 가치와 경쟁력을 높여가고 있습니다. 이를 위해 Translation Research를 통하여 기초연구 성과의 산업화에 앞장서고 산․학․연 협 력의 시너지 효과를 극대화함으로서 기초연구 성과로부터 21세기 기업이 원하는 기술을 발 굴, 재가공하여 신기술의 실용화를 지원하고 있습니다. 또한, 첨단 인프라를 구축하고 전문 분석시험, Pilot, 단백질 위탁생산, HTS/HCS 스크리닝 등 전문서비스를 통해 기업의 기술상 용화 또한 적극 지원하고 있으며, 국내외 우수 연구기관과의 기술도입 ․ 이전 네트워크를 구 축하고 활발한 기술교류협력을 추진하고 있습니다. 본 세미나에서는 이러한 경기바이오센터 의 역할과 기능에 대하여 소개하고자 합니다.

Mucin type O-glycans have been implicated to have important role in tumorigenesis. There are four different major mucin type O-glycans. Initially, both core1 and core3 structure are synthesized from Tn-antigen (GalNac-Ser/Thr) by β3 galactose transferase and β3 N-acetylglucosamintransferase 6, respectively. Core4 structure is made by single enzyme, core2 N-acetylglucosamintranseferase2 from core3 structure, but three different enzymes, core2 N-acetylglucosamintransferase1, 2, and 3 exist in synthesizing core2 structure based on core1 structure. Each core structure is expressed differentially in conjunction with the differentiation and malignant transformation of various cells and tissues (Brockhausen, 1999). Analyzing the each function of cell surface glycans is critical for evaluating of physiological roles of carbohydrate in cell migration and invasion, which is important for cancer therapy. Core3 synthase which is a unique glycosyltransferase for core3 structure, GlcNacβ1,3-GalNacɑ-Ser/Thr, was cloned through EST screening and reported to have restricted distribution, mainly to the stomach, colon, and small intestine (Iwai et al., 2002). Core3 structure was reported that it is very important cell surface glycan structure for gastric track disease such as colitis (An et al., 2007). Iwai et al. showed forced expression core3 synthase into human fibrosarcoma HT1080 FT-10 cell results in reduced tumor formation. To explain the molecular mechanisms how increased core3 structure modulate the tumorigenesis, Lee et al. (2009) analyzed the effect of core3 structure on ɑ2β1 integrin, which is an important molecule in cell migration and signaling. Core2 O-glycan structure is reported to have important functions in gastrointestinal track in vivo (Stone et al. 2009) and it has important roles in the maturation of gastrointestinal epithelial cell by regulating the cell surface expression of sucrase isomaltase and dipeptidyl peptidase IV (Lee et al 2010). Those results indicate the possibility that mucin-type O-glycans should have important roles in many biological events.

Misfolded and misassembled glycoproteins are retained in the endoplasmic reticulum (ER) where they are exposed to the protein folding machinery and protein quality control. The Unfolded Protein Response is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the ER. Eventually, misfolded and mis-assembled glycoproteins are targeted for degradation by a process called ER-associated protein degradation (ERAD). EDEM1 is an ERAD component that interacts with misfolded luminal glycoproteins and routes them for dislocation. This is followed by their ubiquitination and proteasomal degradation. Although EDEM1 was proposed to be a lectin–like protein and to react with Man8GlcNAc2 oligosaccharides of glycoproteins, still very little is known about the turnover and degradation mechanism of EDEM1 and how this relates to the fate of its substrates. Previously, we reported that EDEM1 exits the ER by a vesicular non-COPII-mediated mechanism and becomes rapidly degraded by basal autophagy. Here, we provide detailed insight into the mechanism by which EDEM1 becomes degraded. After its dislocation to the cytosol, EDEM1 is apparently making complexes with the selective autophagy receptors p62, NBR1 and Alfy. We observed co-distribution of EDEM1 and selective autophagy receptors by double and triple confocal laser scanning immunofluorescence. By quantifying the relationship of EDEM1 and the selective autophagy receptors as visualized by confocal laser scanning immunofluorescence and double immunogold electron microscopy, dramatical changes were observed in HepG2 cells. Following inhibition of autophagy by wortmannin, the number and size of cytoplasmic clusters composed of EDEM1 and the selective autophagy cargo receptors dramatically increased and this aggregate formation was independent of the activity of HDAC6. We observed that deglycosylation of EDEM1 occurred by the action of the cytosolic peptide N-glycanase since treatment with inhibitors resulted in a strong increase in the amount of glycosylated EDEM1. Inhibition of cytosolic peptide N-glycanase also inhibited wortmannin-induced aggregation of EDEM1 and its complex formation with p62. This indicates that deglycosylation of EDEM1 is a prerequisite for subsequent ubiquitination and interaction with selective autophagy receptors. This demonstrates that the ERAD component EDEM1 itself undergoes ERAD involving selective autophagy

The biological activity of many natural products and pharmaceutically used drugs are attributed by the glycosidic residues attached to it. Generally, the glycosylation determines the competence of the most of the molecules as a drug. Therefore, engineering the sugar moiety in the natural products is one of the most efficient ways of generating novel compounds with diverse pharmacological properties. The screening and engineering of flexible glycosyltransferases from different sources and applying those enzymes for the biosynthesis of novel small molecule glycosides is still significantly demanding. In this context, we have identified a novel glycosyltransferase (YjiC) from Bacillus licheniformis and applied to glycosylate small molecule natural products like, flavonoids, isoflavonoids, chalcones, stillbenes, curcuminoids etc. The substrate flexibility of thus identified glycosyltaransferase is found to have broad, which resulted to generate a number of natural product glycosides including novel compounds. The enzyme glycosylates non-regiospecifically at suitable hydroxyl groups to produce O-glucosides of compounds. Moreover, this glycosyltransferase has shown flexibility towards NDP-sugar donors. This property of the enzyme would help to engineer the sugar moiety of most of the small molecules which might exhibit potentially different biological activities. The exchange of sugar moiety might help to modulate the biological application of the parent bioactive compounds. Plenty of biologically active aglycones and their glycoside derivatives are characterized to have diverse biological activities against wide-range of diseases. The production of such pharmaceutically important glycoside compounds and their glyco-engineering to alter biological function would be noteworthy in scientific world to develop a novel drug for future use.

Using a simple fluidic device fabricated with a PVC tube, a syringe needle, and a glass capillary tube, we produced uniform microspheres from poly(ε-caprolactone) (PCL), ethyle-2-cyanoacrylate (ECA), and gelatin. Precise control over spheresize could be achieved by varying the concentration of the discontinuous phase, the flow rates for each phase, and/or dimensions of the fluidic device. We developed inverse opal scaffolds from chitosan and poly(D,L-lactide-co-glyclide) (PLGA) by using PCL and gelatin lattices as templates, respectively. The scaffolds exhibited uniform pore size and well-interconnected pore structure in three-dimensional (3D) fashion. We believe that the inverse opal scaffold could provide a promising platform for both in vitro and in vivo experiments related to 3D tissue engineering. We subcutaneously implanted four kinds of inverse opal scaffolds with different pore sizes into miceto evaluate the effect of pore size on degree of neovascularization. Histology analysis confirmed that the density and area ratio of blood vessels were directly governed by the morphology of the scaffolds. Beside the inverse opal scaffolds, uniform PLGA microbeads with a hollow interior and porous wall were prepared using a fluidic device with three-way channels. The microstructured microbeads could be potentially useful for the encapsulation of cells as well as active agents. We also successfully prepared uniform, porous PLGA beads with controllable pore sizes by employing unstable water-in-oil emulsion as the discontinuous phase, which can be useful for therapeutic cell delivery and tissue engineering. We believe that the advanced materials, including uniform microspheres, inverse opal scaffolds, uniform beads with a core and porous wall, and uniform porous beads, can be applied to most of strategies in biomedical engineering, eventually resolving significant problems that we currently encounter.

Many tumor cells rely on aerobic glycolysis for their continued proliferation and survival, which is in part due to actively inhibited mitochondrial function. Myc and HIF-1 are believed to promote such inhibition by upregulating gene expression of pyruvate dehydrogenase kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial pyruvate dehydrogenase complex (PDC). However, how oncogenic signals activate PDHK1 to regulate cancer cell metabolism remains unclear. we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia, increased oxidative phosphorylation with decreased lactate production and enhanced mitochondrial utilization of pyruvate, and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation activates PDHK1 to inhibit mitochondrial function, providing a metabolic advantage for tumor growth. Moreover, inhibition of PDHK1 attenuates tumor growth, suggesting that PDHK1 may serve as a therapeutic target in cancer treatment.

A carbohydrate chip has been suggested as a powerful tool for robust, rapid, and diverse screening of carbohydrate-protein interaction. 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 carbohydrate by reductive amination, in which reducing sugar groups were coupled with 4-(2-aminoethyl)aniline, which has amine groups at both ends. The modified carbohydrates were attached to amine-reactive NHS-activated glass surface by formation of stable amide bonds. This new method was applied for efficient construction of a carbohydrate microarray platform to analyze carbohydrate-protein interactions. This array prepared using our method can be successfully used for screening the specificity of GBPs (glycan-binding proteins) such as lectins and microbial toxins, and identification of carbohydrate-protein interactions

Anthocyanins, a class of flavonoids, are recognized for their diverse functions in plant development and beneficial effects on human health. Many of the genes encoding anthocyanin biosynthesis enzymes and the transcription factors that activate or repress them have been identified. Regulatory proteins that control anthocyanin biosynthesis by regulating the expression of different structural genes at the transcriptional and post-transcriptional levels are differentially modulated by environmental and biological factors such as light, temperature, sugar and hormones. This minireview summarizes the recent findings contributing to our understanding of the role of sugars and hormones in the modulation of the anthocyanin biosynthesis pathway with emphasis on the coordinated regulation of the critical transcriptional R2R3-MYB/bHLH/WD40 (MBW) complex in Arabidopsis.

To metastasize, tumor cells must acquire abilities to penetrate surrounding normal tissues and overcome barriers of dissemination and distant growth such as invasion, anoikis resistance, evasion of immune surveillance, extravasation, colonization, and growth in new microenvironments. Recent evidence suggests that galectin-3, a galactose binding protein, can promote metastasis by regulating detachment of cancer cells. Moreover, highly metastatic human breast cancer cells show higher levels of expression of galectin-3 and overexpression of galectin-3 enhances cell motility and invasiveness in lung cancer cells, suggesting that galectin-3 regulates cancer metastasis. However, the mechanism by which galectin-3 promotes metastasis has not been fully understood. We demonstrated here that overexpression of galectin-3 induced Epithelial-to-Mesenchymal Transition (EMT) and EMT induced by galectin-3 overexpression was remarkable in serum deprivation condition. The cell-cell adhesion molecule E-cadherin is stabilized by linking intracellularly with the actin cytoskeleton but overexpression of galectin-3 weakened the interaction of E-cadherin with cytoskeleton. Additionally, we found that galectin-3 overexpressing HT-29 cells were more resistant to anoikis stress and serum deprivation than parental HT-29 cells. Based on these findings we propose that galectin-3 is an important molecule that regulates cancer metastasis through induction of EMT and anoikis resistance.

A water-soluble polysaccharide (SPS-CF) was isolated and purified from Korean green alga Maesaengi Capsosiphon fulvescens mainly by dilute acid extraction, ethanol precipitation, and DEAE-cellulose ion exchange chromatography. The results of present study performed for monosaccharide composition of this polysaccharide using HPLC of PMP labelled sugars, HPAEC-PAD, GC-FID and TLC, clearly demonstrated that the most prominent neutral monosaccharides of SPS-CF are xylose (44–52 mol %) and rhamnose (34–45 mol %), while mannose and galactose are present at much lesser extent or in negligible amount. These extensive monosaccharide analyses and correlation NMR measurements confirmed that it is a sulphated glucuronorhamnoxylan (ulvan) type polysaccharide, whose backbone is composed of alternating sequence of 4-linked L-rhamnose-3-sulphate and D-xylose residues (ulvobiose) carrying monomeric D-glucuronic acid or D-glucuronic acid-3-sulphate on O-2 of some L-rhamnose-3-sulphate units as the side chains. The SPS-CF exhibited significant invitro anticoagulant activity by which the activated partial thromboplastin time (aPTT) and thrombin time (TT) were significantly prolonged. Also, Enzyme Linked Immunosorbent Assay showed that the SPS-CF significantly stimulates the release of the pro-inflammatory cytokines, TNF-α and IL-6, in a dose-dependent manner. RT-PCR analysis demonstrated that the SPS-CF also induced a more than two-fold increase in the expression of iNOS and COX-2, responsible for the induction of NO and PGE2, respectively, at 5 μg/ml in RAW264.7 murine macrophages. The results of this study demonstrated that the SPS-CF isolated from Korean C. fulvescens Maesaengi is an ulvan type polysaccharide and can be considered as potential anticoagulant and immunostimulating agent.

Squids and Hydroids utilize organic biopolymers as building blocks to form their load-bearing tissues. These polymeric tissues are appealing for their unusual physical and mechanical properties, including high hardness and stiffness, toughness, and low density. Here, the biochemical structure and composition of the hydroid perisarc of Aglaophenia latirostris Nutting, 1900, were investigated. Chitin, protein, and melanin, were found to represent 10, 17 and 60 wt% of the perisarc, respectively. Interestingly, similar to the adhesive and coating of marine mussel byssus, a DOPA (3, 4-dihydroxyphenylalanine) containing protein and iron were detected in the perisarc. A resonance Raman microprobe analysis of perisarc indicates the presence of DOPA-iron (III) complexes in situ, but it remains to be determined whether the DOPA-iron (III) interaction plays a cohesive role in holding the protein, chitin and melanin networks together. We also designed a method to improve the mechanical properties of chitosan in wet conditions and minimized the swelling behavior of chitosan film due to water adsorption by mimicking the sclerotization of hydroid perisarc and squid beaks, i.e., catechol-meditated cross-linking. The biomimetic chitosan composite film was prepared by mixing chitosan with L-3,4-dihydroxyphenylalanine (DOPA) as a catecholic cross-linker and sodium periodate as an oxidant. The catechol-meditated cross-linking provided a 7-fold enhancement in the stiffness in wet conditions compared to pure chitosan films and reduced the swelling behavior of the chitosan film. This strategy expands the possible applications for the use of chitosan composites as load-bearing biomaterials.

D-Tagatose, which is an isomer of D-galactose, is a natural ketohexose sweetener and rarely found in nature. Since D-tagatose has been classified as a generally recognized as safe (GRAS), it can be used in confectionary, beverage, health foods, and dietary products as a low-calorie bulk sweetener. Moreover, it can be potentially used for health functional ingredients having anti-hyperglycemic function. Initial quantities of D-tagatose have been produced commercially by a chemical reaction using calcium catalyst at Arla Foods Inc. However, since the chemical process had disadvantages on limited conversion yield and purification performance, several patented L-arabinose isomerase (AIs) have been exploited as potential resources for the commercial galactose-tagatose isomerization process. The industrially enzymatic reaction for the production of D-tagatose should be operated at high temperatures above 55oC to minimize the risk of contamination, to improve the mass transfer, and to lower the viscosity of substrates. Similar to High Fructose Corn Syrup (HFCS) process, higher yields of conversion can be also achieved by increasing the reaction temperature. AI from Thermotoganeapolitana was commercially produced and immobilized as biocatalysts for continuous semi-fluidized reactor system. The isomerization resultants containing 40~45%(w/v) of D-tagatose was purified further by ion-exchange chromatography followed by simulated moving-bed chromatography and crystallization. The final crystalline products consist of more than 99% of D-tagatose and less than 1% of others (sugar, ash, and water). In 2011, CJ Cheiljedang built up a new commercial facility for the commercial production of D-tagatose (1,800MT/yr). Since then, CJ has sold it as a novel food ingredient in South Korea and US. The operational stability and reduced process impurities (waster material) are important key properties for mass production of bulk sugar ingredient. The enzymatic isomerization process should be more unique and practical method for the large scale production of D-tagatose.

Protein O-mannosylation is evolutionarily conserved protein modification of fundamental importance, which is mediated by protein O-mannosyltransferases(Pmtproteins). The whole genome information of the thermotolerant methylotrophic yeast Hansenula polymorpha reveals the presence of five PMT homologues (HpPMT1, HpPMT2, HpPMT4, HpPMT5, and HpPMT6) encoding Pmt proteins, In this study, we carried out molecular characterization of HpPMT5 and HpPMT6, which show homologies to Saccharomyces cerevisiae PMT1 and PMT2 subfamily members, respectively. Although any detectable growth defects were not detected in the Hppmt5 and Hppmt6 single deletion mutants, they showed significantly increased sensitivity to the PMT1 inhibitor R3A-1c. As expected, the Hppmt1pmt5 and Hppmt1pmt6 double mutants became more susceptible to cell wall disturbing reagents, compared to the single Hppmt1 mutant. Furthermore, O-mannosylation of HpWsc1p and HpMid2p, the cell surface sensors of cell wall integrity-signaling, was significantly defected in the both Hppmt1pmt5 and Hppmt1pmt6 mutants. Interestingly, phosphorylaiton of Mpk1 protein, which results in the stimulation of the cell wall integrity pathway, was more markedly induced in the Hppmt1pmt5 than in Hppmt1 even under normal condition. The membrane fractionation experiment showed that all the HpPmt proteins are localized at the ER/Golgi membrane except HpPmt6p, which was mostly detected as soluble protein. By co-immunoprecipitation experiments, we confirmed the complex formation between HpPmt1p and HpPmt2p, but no interaction between HpPmt5p and HpPmt2p. Altogether, these results support that HpPmt5p and HpPmt6p have redundant functions to significantly compensate the loss of HpPmt1p in protein O-mannosylation that are essential for cell growth, cell wall integrity, and stress resistance of H. polymorpha.

Photodynamic therapy (PDT), which employs photosensitizers (PSs), a light source with appropriate wavelength, and oxygen molecules, has potential for the treatment of various tumors and non-oncological diseases due to its high efficiency in directly producing cellular death, vascular shutdown, and immune activation. For the past decade, many PSs were developed with improved optical and chemical properties. However, some weak points still remain such as low solubility and low tumor specificity by hydrophobic PSs. So, many PDT studies based on soluble polysaccharide carriers such as PSs conjugated polysaccharide nanocarrier and PSs loaded nanocarrier have been carried out in order to improve the water solubility and target specificity of PSs. In case of our group, we prepared the polysaccharide based nanocarrier system composed of cancer cell target-moiety-labeled polysaccharide-graft PS by a dialysis method to improve their targeting efficiency and water solubility. The synthesis was confirmed by 1H NMR spectroscopy, and degree of substitution was determined by UV-spectrophotometery. The size of spherical nanogels was approximately 170 nm, and its photocytotoxicity was studied by an XTT assay. While the PS grafted nanocarrier circulated in the blood, the nanocarrier’s photoactivity may be suppressed by a self-quenching effect between PS molecules in the PS grafted nanocarrier system, similar to the fluorescence resonance energy transfer (FRET) effect. Because the PS grafted nanocarrier system is thought to be internalized into cancer cells following tissue penetration, its photoactivity may be restored without the FRET effect by cleavage of its ester bonds through enzymatic attack in the extracellular matrix and cellular compartments such as lysosomes. The PS grafted nanocarrier system showed a self-quenching activity in the aqueous environment, and has the capacity of tumor tissue targeting by nano-size induced EPR. After accumulation of the PS grafted nanocarrier system in tumors, the fluorescence signals will be gradually enhanced by the release of the PS from broken PS grafted nanocarrier caused by cleavable ester linkage. The phototoxicity effects in the cells by the irradiation of external light resulted in generation of singlet oxygen, at the same time when the near-infrared fluorescence molecular imaging can be detected.

Gangliosides, play important regulatory roles in cell adhesion, survival and immunosuppressive activity. In this study, we investigated whether gangliosides can affect to immune responses in lipopolysaccharide (LPS)-stimulated porcine kidney cell line, PK15. Cell proliferation decreased, whefreas nitro oxide (NO) increased in 5 ㎍/ml of LPS-stimulated PK15. In high-performance thin layer chromatography (HPTLC) and immunofluorsence analysis showed that gangliosides GM3, GM2, GD3, GD1a and GD1b were detected, however, expression of GM3 and GM2 decreased in LPS-stimulated PK15. Furthermore, we knock- downed UDP-glucose:ceramide glucosyltransferase (Ugcg) by short hairpin (sh) RNA, which catalyzes the initial step in glycosphingolipids biosynthesis pathway. Western blot and HPTLC analyse showed that Ugcg protein and gangliosides expression decreased in Ugcg shRNA infected PK15. When we stimulated Ugcg gene knock-downed PK15 with LPS (5 ㎍/ml), cell proliferation was decreased, however, NO release increased compared to un-infected PK15. Moreover, we investigated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α), and these protein expression levels increased in LPS-stimulated Ugcg gene knock-downed PK15. These results suggest that gangliosides, specifically GM3 and GM2, may play a role in the interaction with components of the inflammatory response, and to be relevant for designing future therapeutic strategies intended to prolong xenotransplantation.

Complements, such as C1q and C3, and macrophages in the splenic marginal zone (MZMs) play pivotal roles in the efficient uptake and processing of circulating apoptotic cells. SIGN-R1, a C-type lectin that is highly expressed in a subpopulation of MZMs, regulates the complement fixation pathway by interacting with C1q, to fight blood-borne Streptococcus pneumoniae. Therefore, we examined whether the SIGN-R1-mediated classical complement pathway plays a role in apoptotic cell clearance and immune tolerance. SIGN-R1 first-bound apoptotic cells and this binding was significantly enhanced in the presence of C1q. SIGN-R1-C1q complex then immediately mediated C3 deposition on circulating apoptotic cells in the MZ, leading to the efficient clearance of them. SIGN-R1-mediated C3 deposition was completely abolished in the spleen of SIGN-R1 knockout (KO) mice. Given that SIGN-R1 is not expressed in the liver, we were struck by the finding that C3-deposited apoptotic cells were still found in the liver of wild-type mice, and dramatically reduced in the SIGN-R1 KO liver. In particular, SIGN-R1 deficiency caused delayed clearance of apoptotic cells and aberrant secretion of cytokines, such as TNF-α, IL-6, and TGF-β in the spleen as well as in the liver. In addition, anti-double- and single-stranded DNA antibody level was significantly increased in SIGN-R1-depleted mice compared with control mice. These findings suggest a novel mechanism of apoptotic cell clearance which is initiated by SIGN-R1 in the MZ and identify an integrated role of SIGN-R1 in the systemic clearance of apoptotic cells, linking the recognition of apoptotic cells, the opsonization of complements, and the induction of immune tolerance.

Methylation and glycosylation are two different but vital modification processes in plants to generate diversified secondary metabolites. Methylation increases the lipophilicity of compounds whereas glycosylation enhances the solubility. Most of the methylated and prenylated natural compounds are significantly active against cancers. 7, 8-dihydroxyflavone is a member of a flavonoid family of chemicals, which are abundant in fruits and vegetables. The compound’s selective effects suggest that it could be the founder of a new class of brain-protecting drugs as it can cross the blood-brain barrier by triggering tyrosine kinase B (TrkB) proving it could be a powerful anti-oxidant and neuroprotectant drug. In this study, we used E. coli BL21 (DE3) expressing SPOMT 2884, a Streptomyces peucetius ATCC 27952 derived O-methyltransferase, cell as a biocatalyst for the production of methylated derivative of 7, 8-Dihydroxy flavone. The supplementation of 0.2 mM of 7, 8-dihydroxyflavone in the growing induced culture of E. coli BL21 (DE3) harboring pET28-SPOMT2884 recombinant resulted in the production of a mono methylated compound which was confirmed by HPLC (Rt:17 min) and high resolution LC-QTOF-ESI/MS (m/z+ 269.08). Further, this enzymatically synthesized methylated derivative of 7, 8-DHF was purified in large amount and used as a substrate for in-vitro glycosylation by Yjic, a glycosyltransferase from Bacillus licheniformis DSM13. This reaction mixture analysis revealed the presence of glycosylated product which was analyzed by TLC and HPLC. Further structural elucidation is necessary for the confirmation of methylation and glycosylation positions. However, this study reveals a method that might be useful for the enzymatic biosynthesis of the methylated compound and subsequent modification of the same by glycosylation.

Isoflavonoids are the plant secondary metabolites biogenetically derived from 2-phenylchroman skeleton of flavonoids. They are particularly prevalent in subfamily of Leguminosae; Papilonoidae. The remarkable biological properties of isoflavonoids described as antimicrobial, antioxidant, anti-inflammatory, estrogenic and cancer chemoprotectant. To enhance the bioavailability and biological properties of isoflavonoids, their diversification via acetylation, malonylation, hydroxylation, prenylation, glycosylation are under study. Glycosylation is one of the important tools to diversify and extend such biological functions in natural products and secondary metabolites. The process is catalyzed by UDP-glycosyltransferases in the formation of glycosidic linkages by transferring sugar moiety from a donor substrate to an acceptor. In present study, we have analyzed the in vitro enzymatic reactions of isoflavonoids using YjiC, a glycosyltransferase from Bacillus licheniformis DSM 13. UDP-D-glucose was considered as a sugar donor and isoflavonoids genestein, diadzein, formononetin and biochanin A as acceptor substrates. Reaction products were analyzed by HPLC and high resolution LC-QTOF-ESI/MS which revealed the detection of two mono-glucosides and one di-glucoside in genestein and daidzein where as single mono glucoside with formononetin and biochanin A. Glycosylation at the probable positions of 4’ and 7 hydroxyl groups of the genestein and daidzein was suspected while biochanin A - 7-hydroxyl position might have been prominent. In case of formononetin having a single hydroxyl at 7th position, high resolution LC-QTOF-ESI/MS analysis conforms the exact configuration and positioning of glucose attachment. Although glycosides of the rests of isoflavonoids were detected from high resolution LC-QTOF-ESI/MS analysis, the exact configuration of sugar attachment is yet to be identified.

Glycosylation can significantly improve the water solubility, stability and thus increase bioavailability and enhance biological activity of low molecular weight organic compounds. Enzymatic glycosylation methods have several advantages over the classical methods of organic syntheses in terms of cost, efficiency and selectivity. The UDP-glucosyltransferase YjiC from Bacillus licheniformis DSM 13, a member of family 1 glycosyltransferase was expressed in Escherichia coli BL21 (DE3) as N-terminal hexahistidine-tagged fusion protein. Purified YjiC was used to glycosylate apigenin, baicalein, chrysin, diosmetin and luteolin. The products have been analyzed by high performance liquid chromatography (HPLC) and high resolution liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (HRLC-ESI-Q-TOF-MS) methods. The formation of mono and diglucosides has been reported with all the substrates. Even though glycosylation is preferred at positions other than C5 carbon of flavonoids whenever there is the presence of more than one phenolic hydroxyl groups, the promiscuity of YjiC was not observed. The exact position of glycosylation by YjiC is yet to be determined.

Signaling from the Notch receptor family is important in development, and its role in disease has assigned the Notch signaling pathway as a therapeutic target (1). Mammalian Notch receptors are single-pass transmembrane glycoproteins containing 29-36 EGF-like repeats. Notch receptors on "receiving" cells signal when they interact with Notch ligands expressed on "sending" cells. Glycosylation of some Notch EGF-like repeats modulates signaling (2). EGF repeat glycosylation includes constitutive fucosylation of some EGF repeat-localized serine/threonine residues, and elongation of fucose-initiated glycans regulated by the Fringe family of glycosyltransferases (3). We sought to determine how O-fucosylated glycans modulate Notch signaling, using cell-based Notch signaling assay by co-culturing with Notch ligand bearing cells and beads and protein-protein binding assay with surface plasmon resonance (SPR). We find that lunatic Fringe modification of Notch1 receptors enhances Dll1, Dll4, and Jag2 binding affinities and signaling, while it reduces Jag1-mediated binding affinity and signaling. SPR study indicates that Notch signaling is correlated with association constant (KA) and maximum binding capacity (Rmax) may be critical in Notch signaling from Jag2 in solid-phase Notch signaling assay.

Surface plasmon resonance (SPR) can provide a kinetic information for an interaction and rapidly monitor any dynamic process, such as adsorption or degradation, without the need for any sample preparation. Herein, we used SPR system to analyze carbohydrate-protein interaction, especially, Vibriocholeratoxin-GM1pentasaccharide. The understanding of carbohydrate contribution in carbohydrate-protein interaction may provide meaningful information about carbohydrate-related biological system. Interaction between ctxA and ctxB is firstly investigated in this study since the binding is a prerequisite for virulence mechanism. Interaction kinetics between ctxB and GM1 pentasaccharide using the our proposed saccharide immobilization method were evaluated, and the results showed similar degree of kinetics to the previous reports, therefore our direct immobilization could make SPR analysis simply for carbohydrate-related researches. And the introduction of ctxA during the interaction showed higher affinity than that of ctxB-GM1 although KD constant was 10-times lower than ctxB-GM1 binding. Comparative analyses for GM1 analogues-ctxAB were conducted in order to evaluate kinetic values from structural differences. Although these results did not show significant difference, determination of the association constants for ctxAB revealed the following order: LST-b > GM3 > LST-a > asialo GM1 and dissociation constants for ctxAB showed following order: LST-b > asialo GM1 > LST-a > GM3. These results indicate that sialic acid thumb is sufficient for recognition, and the terminal glalactose and GalNAc finger are required to stabilize the ctxAB-ganglioside GM1 interaction. Collectively, direct immobilization of carbohydrate at SPR-based analytical system can evaluate structural contribution of carbohydrate moieties in carbohydrate-protein interaction as well as provide valuable information for understanding the interactions.

Cryptococcus neoformans is an encapsulated basidiomycete causing severe diseases, mostly meningoencephalitis, in immunocompromised individuals such as AIDS patients. Secreted phospholipase B1 (PLB1) is important for infection of C. neoformans to the host lung. PLB1 are conserved in all serotypes of C. neoformans, with an overall sequence homology of more than 90%. Previous studies have shown that PLB1 is highly modified by N-linked glycosylation which is essential for the three PLB1 enzymatic activities and secretion. Noticeably, the PLB1 proteins for all cryptococcal serotypes are reported to have different molecular mass, reflecting serotype-specific N-glycans assembled on the secreted PLB1 proteins. As an effort to obtain the purified PLB1 proteins with altered N-glycan structure, which would be used to analyze the effect of N-linked glycans on its role as a virulence determinant we constructed His-tagged and glycosylphosphatidylinositol (GPI) anchorless PLB1 that can be secreted due to lack of membrane association. The GPI anchorless PLB1 protein was expressed in C. neoformans H99 wild type (serotypes A), and glycosylation mutant strains such as Cnoch1Δ, and Cnmnn2Δ, which have defects in the outer chine biosynthesis of N-linked glycans. Compared to the size of the wild type PLB1, those of secreted PLB1 in the mutant strains appeared to be reduced but converged to the same molecular mass after N-glycosidase treatment, indicating the size difference is attributed to the altered structure of N- glycan outer chines. Moreover, the results indicate that the outer chain structures of N-glycans do not crucially affect the secretion efficiency of PLB1.

Biosimilar recominant DNA products have been developing actively as medicines along with the growth of biotechnology. The difficult availability of original drugs as reference materials can be the technical barrier in biosimilar development. In this study, the applicability of international and national standards as original drug substitutes was reviewed throug mass spectometry-based characterization. We established characterization testing method using LC-MS and MALDI-TOF-MS. International/national standards and reference products of somatropin and granulocyte colony-stimulating factor(G-CSF) were used to analyze molecular weight measurement, peptide profiling, peptide mapping and disulfide bond identification by LC-MS. And N-glycans extracted from erythropoietin(EPO) included in International/national standards and reference products were used to obtain distribution information about sialyation, antennary, fucosylation, phosphorylation modificaiton, and O-acetylation modification by MALDI-TOF-MS. This study showed international and national standards can replace original drugs as reference materials in characterization of non-glycoprotein drugs, but it is difficult for glycoprotein drugs because of their glycosylation difference.

Glycan of glycoprotein drugs, which are over 70 % of recombinant DNA products, is a very important evaluation factor in product development or quality evaluation, because it was known as have important roles in both medicinal effects and stability. Especially, in case of biosimilar drugs, which are being recently actively developed, for proving similarity with their innovator, comparative analysis of glycan structure has come to the core as a fundamental evaluation factor. Therefore we desired to establish the useful scientific foundation for quality evaluation and approval evaluation of glycoprotein drugs, thereby establishing standardized glycan analysis methods for glycoprotein drugs using high-tech mass spectrometer in this study. To achieve this, we proposed glycan analysis methods and confirmed these methods, by testing human IgG as glycoprotein and on sale glycoprotein drugs(Infliximab and Trastuzumab). As a result, we established simplified sample preparation method in comparison time-consuming other researches and check method whether or not glycosylation using deglycosylation enzyme treatment and MALDI-TOF-MS. We also established glycosylation pattern and glycan sequence qualtitative analysis method using MALDI-TOF-MS, which has advantage of rapid analysis, and glycan relatively quantitative analysis method using LC-QTOF-MS, which has advantage of quantitative analysis. Established MALDI-TOF-MS method can use to check sample preparation properly and to analyze glycosylation pattern and glycan sequence qualitatively. For relatively quantitative analysis of glycan content and precisely qualitative analysis of glycosylation pattern and glycan sequence, LC-QTOF-MS is more effective than MALDI-TOF-MS. In addition, we proposed glycosylation site analysis method and confirmed the method using LC-QTOF-MS and glycan database. Through this study, we established total five SOPs(Standard Operating Procedures); ① Glycosylation ascertainment test by MALDI-MS, ② Glycan qualtitative analysis by MALDI-MS, ③ Glycan qualitative and quantitative analysis by LC-MS, ④ Glycosylation site analysis by LC-MS, ⑤ Sample preparation ascertainment test by MALDI-MS. These SOPs can be useful in characterizaion of biosimilars.

Exosomes (microvesicles, or micropaticles) are small membrane-enclosed vesicles and these are secreted by various cell types, including tumor cells. These vesicles play an important role as mediators in extracellular communication. They are composed of membrane, cellular proteins, DNA, and RNA derived from their origin cells. It is also known that exosomes are involved in tumor metastasis, angiogenesis, and antitumor immunity. These biological functions are probably due to the glycosylation on their membrane proteins. Thus, the study of glycosylation of exosomes will be another potential source of new biomarker. However, there is a little study about the glycosylation of exosomes. Here, we targeted and analyzed N-glycans of exosomes derived from five cancer cell lines (A549, PC9, PC9/ZD, MCF-7, and MDA-MB231) using nano-LC/MS. We also have compared glycans of exosomes with glycans on their origin cell membrane to examine glycosylation correlation between origin cells and exosomes on three lung cancer cell lines. Additionally, we have compared anticancer drug resistant cell line PC9/ZD (Gefitinib resistant) and untreated PC9 in terms of origin cells and exosomes glycan profiling via isomer separation. Exosomes and cell membranes were prepared from three lung cancer cell lines: A549, PC9, and PC9/ZD. And two breast cancer exosomes were prepared: MCF-7 and MDA-MB231. Glycans were released by PNGase F, then enriched by graphitized carbon solid phase extraction. Nano-LC/chip Q-TOF MS was used for overall glycan profiling and quantitation. We successfully release and profile N-glycans from exosomes. Origin cells and exosomes of lung cancer contain high-mannose glycans in abundance. Although, exosomes have less high-mannose glycans compared with origin cells. Both origin cells and exosomes, isomer separation of sialylated glycans are different between PC9 and PC9/ZD. On breast cancer exosomes, two different matastatic samples represent quite different profiling. This is the first study of comprehensive glycan profiling of exosomes using mass spectrometry.

Age-related biochemical and physiological changes have been found in all of the body's cells, tissues, and organs, affecting the function of all body systems. Glycosylation also undergoes changes in the pathophysiological process of aging. Glycosylation, which is highly sensitive to the biochemical environment, is a common post-translational modification of proteins, with over 50% of all human proteins glycosylated. Unlike N-glycans, the research of O-glycan for aging and longevity biomarkers are more challenging owing to the absence of abroad-specificity glycosidase for the release of O-glycans from glycoproteins. In this study, O-glycans in mouse skin tissues were chemically released by reductive β-elimination and analyzed by nano-LC chip/Q-TOF mass spectrometry (Nano-LC/MS). O-glycan enrichment was dramatically improved by optimized lipid removal prior to glycan release. The 20 O-glycan compositions were found in mouse skin tissues by Nano-LC/MS, and O-glycan structures were elucidated by tandem MS. Isomer-specific glycan analysis was successfully performed by accurate mass Q-TOF MS coupled with chip-based liquid chromatography. The alteration of O-glycans during aging was monitored by quantitative analysis. Furthermore, we could identify the relative contribution of neutral, sialylated and fucosylated glycan types. Sialylation was found to be highly correlated with the aging process. Further studies with larger sample sets will help confirm the initial data and lead to more confident detection of aging biomarkers.

Erythropoietin is a therapeutic glycoprotein that stimulates red blood cell production. The quality, safety, and potency of recombinant erythropoietins are determined largely by their glycosylation. Small variations in cell culture conditions can significantly affect the glycosylation, and therefore the efficacy, of recombinant erythropoietins. Thus, detailed glycomic analyses are necessary to assess biotherapeutic quality. We have developed a platform for qualitative and quantitative glycomic analysis of recombinant erythropoietins. The platform was used to profile native N-glycans from three different type erythropoietin a common form of recombinant erythropoietin. Darbepoetin alfa was found to contain an abundance of large, multi-antennary N-glycans with high levels of sialylation, O-acetylation, and dehydration. Results were verified by independent orthogonal analysis with both MALDI-TOF and nano-LC/Q-TOF MS. This platform may be applied to quality control and batch analysis of not only recombinant erythropoietin but also other complex, glycosylated biotherapeutics and biosimilars.

IgA Nephropathy (IgAN) and thin basement membrane nephropathy (TBMN) have identical hematuria symptom but different seriousness in renal function. In case of TBMN, renal function is usually normal but IgAN is the most common cause of end-stage renal failure. In general, the biopsy of the kidney is performed to distinguish between IgAN and TBMN. There are a lot of needs for clinicians to have a sensitive and specific biomarker for differentiation IgAN and TBMN. Exosomes which are small cell-derived vesicles have been suggested as potential biomarkers in diagnosis of disease because these are secreted from original cell with intracellular fluids and apical membrane. Urinary exosomes, 40-100nm vesicles secreted to urine by renal cells, may provide non-invasive diagnosis manner of kidney disease. Glycosylation is highly sensitive to the biological environment changes and considerably affected by disease states. Glycan is known as playing key roles in cancer metastasis and intracellular recognition. Therefore, the examination of glycosylation change in urinary exosome may be a new way for potential biomarkers to differentiate TBMN and IgAN. In this study, we have analyzed 18 individuals exosomes secreted into urine from renal epithelial cells. Samples were composed of three different groups, mainly TBMN, IgAN patients, and healthy volunteers. Briefly, N-glycans from urinary exosomes were released by PNGase F digestion and then enriched by solid phase extraction using a porous graphitized carbon cartridge. N-glycans were eluted with three different solution (10% ACN, 20% ACN, and 40% ACN with 0.05% TFA in H2O) based on the glycan size and polarity. Enriched glycans were analyzed for qualitative and quantitative profiling using high resolution MALDI-TOF/TOF mass spectrometry and performed tandem MS to obtain extensive structure information. We also separated isomer-specific glycans by nano-LC chip/Q-TOF mass spectrometry. We found that high mannose glycans are high in abundance in normal group. Complex, non-sialylated fucosylated tri-, and tetraantennary glycans are significantly different between normal and patients, which suggest the potential biomarker.