Earticle

Our center, Daejeon-KRIBB-FHCRC Research Cooperation Center, was established to develop ‘cancer biomarkers’ in collaboration with Fred Hutchinson Cancer Research Center (FHCRC) in Feb. 2005 with supports from the Daejeon Metropolitan City. Cancer is often difficult to achieve early diagnosis, which is, however, a decisive requisite for favorable outcome in cancer treatments. In order to develop the cancer biomarkers, we’ve tried to pinpoint cancer cells-originating aberrant glycoproteins which would be a well-grounded approach to cancer biomarker discovery ultimately using blood. One of the glycosyltransferases responsible for aberrant glycosylation in cancer is N-acetylglucosaminyltransferase V (GnT-V), which catalyzes an addition of b1,6-N-acetylglucosamine (GlcNAc) 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 good models involved in cancer malignancy upon aberrantly glycosylation. Basically by adopting multi-lectins enrichment strategy, candidate proteins showing high sensitivity and specificity during the discovery phase were selected and the panel of biomarker candidates is currently under in-depth analyses for validation. Validation is a time-consuming step for biomarker developments requiring confirmation of the biomarker candidates through multiple pairs of clinical samples. That is why a sensitive, multiplexing validation method is necessary. To address this challenge, we are developing as a DNA-tagged antibody-based ‘New’ validation method, enabling a multiplexed validation of biomarkers. Furthermore, the collaboration with the FHCRC leads to an expansion of the ‘epigenetic’ studies to ‘glycogenome’, requiring incessant collaborations. Recently, Daejeon Metropolitan City became a ‘sister city’ for Sapporo City in Japan. It’s noteworthy that these relationships prompt to establish a ‘north pacific network of cancer biomarker development’ among the KRIBB, FHCRC and Sapporo Medical University.

The use of glycan changes in their structure and function play pivotal roles in novel biomarker discovery and disease therapeutics. For example, regarding cancer biomarkers, fucosylated AFP and fucosylated haptoglobin are promising cancer biomarkers for hepatocarcinoma and pancreatic cancer, respectively. Deletion of core fucose from IgG1 molecule results in 100 fold activation of ADCC (antibody dependent cellular cytotoxicity) activity in antibody therapy. We have recently proposed the concept of the “glycan cycle” as a functional unit of glycans that will permit the integration of glycan functions in relation to diseases For example, glucose enters the cell via a glucose transporter and is converted to UDP-GlcNAc via the hexosamine pathway. UDP-GlcNAc plays a key role in the cytosol and is then incorporated into the Golgi via the UDPGlcNAc transporter and serves as a donor substrate for various GlcNAc-transferases from GnT-I to GnT-VI. UDP-GlcNAc also serves as a donor for O-GlcNAc-transferase in the cytosol. These enzymes function to modify target proteins such as TGF-beta, the EGFR, and the glucose transporter. These receptors are localized on the cell surface and bind to lectins such as galectin-3. When the affinity of lectin binding is decreased due to changes in receptor glycosylation, the receptors are endocytosed and degraded in acidic endosome/lysosome, and the free monosaccharide is probably recycled. This conceptual “functional unit of the glycan cycle,” such as just described for GlcNAc, is intended to help developing the understanding of the integrative and dynamic analysis of glycan functions.

Colorectal cancer arises as the consequence of the accumulation of genetic alterations (e.g. gene mutations, gene amplification, etc.) and epigenetic alterations (e.g. aberrant DNA methylation, chromatin modifications, etc.) that transform colonic epithelial cells into colon adenocarcinoma cells. The loss of genomic and epigenomic stability and resulting gene alterations appears to be a key molecular and pathogenic step that occurs early in the tumorigenesis process and permits the acquisition of a sufficient number of alterations in tumor suppressor genes and oncogenes in a clone of cells to result in their ultimate transformation into cancer. It has also become clear that epigenetic alterations are common in many cancers and affect the formation and behavior of the tumors. With regards to DNA methylation, it is present normally throughout the majority of the genome and is maintained in relatively stable patterns that are established during development1. In humans, approximately 70% of CpG dinucleotides carry this epigenetic modification. However, there are regions that are enriched for CpG dinucleotides, called CpG islands, that are present in the 5’ region of approximately 50-60% of genes and are normally maintained in an unmethylated state. In cancers, many of these CpG islands become aberrantly methylated, and this aberrant methylation can be accompanied by transcriptional repression2,3. The significance of these epigenetic alterations in the pathogenesis of cancer has been a point of significant controversy4,5. Nonetheless, there is sufficient data to demonstrate that the aberrant methylation of at least some of these genes, such as MLH1, can be pathogenetic in cancer6-8. The aberrant methylation of MLH1 occurs in approximately 80% of sporadic MSI colorectal cancers, and the restoration of MLH1 expression and function by demethylating the MLH1 promoter in MSI colorectal cancer cell lines, strongly supports the idea that such aberrant methylation is a cause rather than a consequence of colorectal carcinogenesis6,7,9. Furthermore, the epigenetic inactivation of MLH1 appears to proceed the onset of mutations in genes with coding region microsatellite repeats, such as TGFBR2, suggesting epigenetic events can predispose tumor cells to mutations that drive the tumorigenesis process. Indeed, aberrantly methylated genes HLTF SLC5A8, MGMT, MINT1, and MINT31 can be found in aberrant crypt foci, demonstrating that aberrant promoter methylation occurs early in the adenoma sequence, although it does not confirm that the aberrant methylation is a primary rather than a secondary event in the tumorigenesis process10-12. The aberrant methylation of genes affects genes that are commonly targets of mutational inactivation in colon cancers and contributes to the deregulation of signaling pathways that are known to be important in these tumors13,14. Finally, a subset of colorectal cancers that hypermethylate genes belong to a distinct subclass of colorectal cancers, termed the CpG island methylator phenotype (CIMP) has been identified and appear to have a worse prognosis15-19. These aberrantly methylated genes have been shown to be early detection markers and prognostic markers for a variety of cancers14,20, and some methylated genes are already being used in clinically available assays in the United States21,22. The potential for methylated genes to be used as risk stratification markers, early detection markers, and predictive markers is high, and it is anticipated that they will move into common clinical use in the future. Finally, therapies directed at these genetic and epigenetic alterations are under active development and hold the promise to improve the treatment of colorectal cancer.

ErbB signaling is implicated in the pathology of various types of carcinoma. Ligand binding to the extracellular domain of ErbB induces conformational changes that lead to homo- or heterodimerization of the receptors and subsequent signaling. It has been reported that N-glycans of the ErbB family regulate its function. We previously reported that N-glycans of EGFR are involved in receptor dimerization and endocytosis. N-glycans of ErbB3 are also involved in receptor dimerization and activation status; deletion of the N-glycan on Asn418 of ErbB3 leads to both spontaneous homodimerization of ErbB3 and heterodimerization of ErbB2-ErbB3. The promoted heterodimerization with ErbB2 leads to upregulation of receptor tyrosine phosphorylation, downstream signalings, and transforming activity. We recently found that N-glycans of ErbB4 also regulate ligand-induced signaling. Thus, N-glycans of the ErbB family seem to play an important role in receptor activation, especially in receptor dimerization. To examine the role of N-glycans of the ErbB in detail, we prepared a wild type and an N-glycan deleted mutant of sErbB3 (domains I, II, III and IV) and compared their properties. When added to culture cells expressing ErbB2 and ErbB3, the sErbB3 N418Q mutant downregulated the heregulin b signaling more effectively than the wild type sErbB3. Through mass spectrometry analysis, we were able to determine the structure of the N-glycan on Asn418 of sErbB3. This suggests that the specific N-glycan of sErbB3 regulates the binding status of sErbB3 to ligands or receptors. We consider that the sErbB3 N418Q mutant is a potent inhibitor of transforming activity of ErbB, and may have therapeutic applications in cancer.

Recently, a great deal of interest has been developed to isolate novel bioactive compounds from natural resources due to their numerous health beneficial effects. Among them, the polysaccharides are widely distributed in the nature and their molecular structures have caused to their various biological activities. Chitosan is a naturally abundant β-1,4-linked copolymer of N-acetyl-2-amino-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-Dglucopyranose units and is a naturally occuring, biodegradable, non-allergenic deacetylated derivative of chitin, found abundantly in nature. Even though chitosan is known to have important functional activities, poor solubility makes them difficult to use in the food and biomedicinal applications. Unlike chitosan, its hydrolyzed products as chitosan oligosaccharides (COS) are readily soluble in water due to their shorter chain lengths and free amino groups in D-glucosamine units. The bioactive properties of chitosan and COS depend on their degrees of deacetylation and molecular weight. Until now, there are very less information available on bioactive properties based on both degrees of deacetylation and their molecular size. It has been reported that novel COS derivatives can be used as physiologically bioactive materials since they possess various health beneficial biological activities, such as antioxidative, antitumor, antimicrobial, anti-hypertensive, antidiabetic, calcium absorption enhancing hypo-cholesterolemic activities and immune enhancing effects. Therefore, it is needful to prepare various types of COS derivatives, such as N-acetyl COS, Amino ethyl-COS (AE-COS), Demethyl amino ethyl-COS (DMAE-COs) and sulfated COS with different degrees of deacetylation and molecular weight. furthermore, the determination of their uninvestigated biological activities such as enzyme inhibitory (β-secretase, MMP, AchE), intra-cellular free radical scavenging, and anti-HIV activities are useful to expand the health of human being. The use of these novel COS derivatives as nutraceuticals, functional foods, pharmaceuticals and cosmeceutical promissing.

Mainly, chemical and physical methods have been used for immobilization of bioactive materials. However, there are some of drawbacks with those methods. For example, chemical method may produce potential toxic by-product and, in case of physical method, low efficiency of immobilized bioactive material is observed. To solve these problems, recently, the immobilization by photoreaction has been researched widely. The advantages of photo-immobilization are 1) high selectivity of chemical reactions or processes under mild conditions (ambient temperature of also much below), 2) typically no need for added catalysts or special solvents, 3) spatially addressable effects (2D and 3D structuring possible) and 4) applicable to very small and (relatively) large scales. To use for photo-immobilization, various natural polymers reacted by irradiation to ultra violet and visible light can be synthesized. It is considered that these photoreactive natural polymers can be applied for medical area to increase biocompatibility and functionality, for example, coating agent for bioinnert devices like stent and implant, anti-adhesive agent, wound dressing and bio-adhesive.

Recently, coaxial electrospinning have gained much attention to fabricate a continuous double layer of nanofibers. Using this technology, some difficult-to-process polymer solutions could be co-electrospun to form an ultra-fine core within the shell of other polymer materials. This core/sheath nanofiber would be valuable for some specific applications, such as maintaining some agent for a certain period of time, postponing drug or growth factors release to a later stage, or achieving different degradation rates in different periods of usage. Current emphasis of research is to exploit such properties and focus on determining appropriate conditions for electrospinning various polymers for eventual applications. In our study, biodegradable core/shell non-woven mats of chitosan (CS) and poly (lactic acid) (PLA) were fabricated by coaxial electrospinning. In first case, PLA formed the core of the fibers and chitosan was deposited on the surface of the PLA fibers. High molecular weight of chitosan nanofibers were not feasible to be fabricated by single electrospinning due to highly viscous solution and its polycationic possession. However, it was found that the fabrication of CS nanofiber was facilitated by coaxial electrospinning method. The PLA/CS core/shell nanofibers can be used as antibacterial materials in fields such as active and bioactive package, in biomedical and filtration areas. In second case, CS/PLA core/shell non-woven mats were prepared in which CS acted as core and PLA acted as shell. This non-woven mats have potential in drug release application. In both case, measurement of contact angle using iodine methane and water confirmed the core/shell structure of nanofibers in which the shell completely covered the core. Furthermore, the contribution of PLA and CS in the core/shell nanofibers was evaluated by tensile test and different scanning calorimetry (DSC).

Detection of lymph node (LN) metastasis by magnetic resonance imaging (MRI) has obtained clinical significance for treating cancer patients. LN metastasis often happens through regional lymphatic system, leading to distal tumor formation invluding lungs, liver and bones. Successful imaging of small and microscopic LN metastasis provides the helpful information in deciding the therapeutic option of cancer. Mannan is a water-soluble polysaccharide having high content of D-mannose residues which can be recognized by mannose receptors on activated macrophages and dendritic cells. Mannan-coated superparamagnetic iron oxide nanoparticles (Mannan-SPION) were developed to be specifically delivered to macrophages in lymph node by receptor-mediated endocytosis. Mannan-SPION was proven to be suitable for MR imaging due to small size, excellent stability in ferrofluid, and low cytotoxicity. In addition, mannan-SPION exhibited enhanced targeted delivery efficiency to macrophages in lymph nodes in vivo compared with PVA-SPION. Especially, LN enhancement of Mannan-SPION on MRI was dramatically increased at the later stage after intravenous injection compared with PVA-SPION control, indicative of the potential to successfully detect micrometastasis in LN.

Corynebacterium diphtheriae is a Gram-positive pathogenic bacterium that causes diphtheria. It can decorate its cell surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using its surface-localized sialidase related enzymes. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. C. diphtheriae sialidase NanH can cleave α(2,3)- and α(2,6)-linked sialic acid from sialic acid-containing substrates. Even though the efficiency was low, the sialidase was able to catalyse the transfer of sialic acid using several sialoconjugates as donor. In addition, using the reversible reaction mechanism of C. diphtheriae NanH, we also constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. C. diphtheriae nanH gene was cloned into the yeast surface display vector pYD1 based on the Aga1p-Aga2p platform to immobilize the enzyme on the surface of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids from pNP- α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage. The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis of N-glycans containing α (2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling.

Mannosylphosphorylation of N- and O-glycans, which confers negative charges on the surfaces of cells, requires the functions of both MNN4 and MNN6 in Saccharomyces cerevisiae. To identify genes relevant to mannosylphosphorylation in the dimorphic yeast Yarrowia lipolytica, the molecular functions of five Y. lipolytica genes showing significant sequence homology with S. cerevisiae MNN4 and MNN6 were investigated. A set of mutant strains in which Y. lipolytica MNN4 and MNN6 homologues were deleted underwent glycan structure analysis. In contrast to ScMNN4, the Y. lipolytica MNN4 homologue, YlMPO1, encodes a protein that lacks the long KKKKEEEE repeat domain at its C-terminus. Moreover, just a single disruption of YlMPO1 resulted in complete disappearance of the acidic sugar moiety in both the Nand O-linked glycan profiles. In contrast, even quadruple disruption of all ScMNN6 homologues, designated YlKTR1, YlKTR2, YlKTR3, and YlKTR4, resulted in no apparent reduction in acidic sugar moieties. These findings strongly indicate that YlMpo1p performs a significant role in mannosylphosphorylation in Y. lipolytica with no involvement of the Mnn6p homologues. Mutant strains harboring the YlMPO1 gene disruption may serve as useful platforms for engineering Y. lipolytica glycosylation pathways for humanized glycans without any yeast-specific acidic modifications.

Dynamic protein modification, called post-translational modification (PTM), is the chemical modification of a protein and an important biological regulatory mechanism to understand cellular function and interactions. Glycosylation is one of the most common post-translational modifications of proteins and is involved in many physiological functions and biological pathways. Analysis of glycoproteins including site mapping and glycan characterization is an essential sub-field of proteomics using high throughput techniques by mass spectrometry. Recent improvements in mass spectrometry provide sensitive methodologies by liquid chromatography tandem mass spectrometry (LC-MS/MS) as an innovative analytical technology applicable to a wide number of analyses in glycoproteins. In addition to mapping sites of N-linked glycosylation, mapping sites of O-linked glycosylation are another current challenge in proteomics. Here we demonstrate that the methodologies allow for analyses of glycoproteins and their mapping sites in various biological species and also discuss the effective uses of mass spectrometry for glycomic analysis and modern techniques of biomolecular mass spectrometry with the glycome from glycoproteins.

Many plants have long been known to have medicinal effect therefore have been used as medicines especially in traditional medicines. Even today, about 75% of the world population still relies on plant, plant extracts and other tools of traditional medicines in basic health need. Because of the circumstances such as the increase of chronic disorders of endogenous nature and the severe adverse effects of modern synthetic medicines, traditional medicines herbs have began to attract worldwide attention. Herbal extracts contain substances with both low and high molecular weight. Although biologically active substances of the former compounds in medicinal herbs have been studied, they can’t account for all of the clinical effects achieved. Pharmacological activities have been observed in fractions with high molecular weights from boiled water extracts of the medicinal herbs. Among the high molecular weight substances, pectins have been shown to possess a variety of pharmacological activities such as several immune-stimulating activities, anti-metastasis activity, anti-ulcer activity, cholesterol decreasing effect and so on. Pectins are also applicable for drug delivery and as the vaccine for typhoid fever. Pharmacological activities which were observed in pectins and pectic polysaccharides seem to be dependent on the fine carbohydrate structures. Many pharmacological activities have been appeared in the ramified region (rhamnogalacturonan I), but some high molecular weight rhamnogalacturonan II also showed immuno-modulating activities such as Fc-receptor up-regulation for macrophages, NK-cell activation and cytokine production enhancing activity. Even if natural pectin had no activity, chemical and enzymatic modifications may provide useful product for health care. Present observations suggest that application of pectin on health care brings many possibilities of benefits for human being.

최근 사용되고 있는 항암성 화학요법제 및 항생물질은 정상세포에 대한 독성, 암세포의 내성획득, 인체내에서의 신속한 분해와 배설 등의 결점과 임파구 및 세포 등을 파괴한다. 이런 화학물질의 결점을 보완하는 대안으로 약용식물과 더불어 담자 균류인 버섯을 대상으로 많은 연구가 진행되고 있다. 일반적으로 담자균류 버섯으로 부터 생산되는 활성물질은 주로 다당체인 β-glucan으로 밝혀져 있으며, 약리적인 특 성은 면역증대, 항암, 암전이억제, 항산화, 항당뇨, 항균, 상처치유능, 항염증, 항혈전 등 여러 효능이 보고되고 있다. 이러한 다당체의 생리활성은 분자적 구조특성인 3차 원 입체구조, 분자량, side chain의 분지정도, 구성성분 등에 따라 차이를 나타내고 있다. 버섯유래 다당체를 이용하여 질병을 치료하는 것은 획기적인 새로운 분야이다. 바이오 산업은 크게 의료융복합 및 산업융복합 분야로 분류되며 향후 산업발전에 중 추적인 역할을 담당할 것으로 기대된다.

Algae and mushrooms are rich sources of bioactive natural components. Among these compounds, polysaccharides are interesting due to their immunomodulating, antitumor, antiviral, coagulating and other properties. Polysaccharides play an outstanding role in the construction of algal and mushroom cell walls. First step of the strategy of polysaccharide isolation from such raw material is fractionation of individual cell wall components by subsequent extractions. The second step is rigorous purification of obtained fractions by using of preparative chromatography, chemical reagents and specific enzymes. Finally, individual polysaccharides within a fraction can be separated from each other by procedures based on the difference in their solubility in various media, sensitivity to chemical or enzymatic destruction, affinity to specific molecules and complexation with metal cations. Structural analysis of isolated algal or mushroom polysaccharides is based on combination of separation, chemical and spectroscopic methods. Total hydrolysis followed by HPLC or GC analysis gives the information about the monosaccharide composition. Gel chromatography equipped with various detectors is used for the molecular mass estimation. FTIR spectroscopy is effective for detection of functional groups, estimation of fraction purity as well as type of polysaccharide. Correlation NMR spectroscopy together with sugar linkage analysis is a powerful tool for detail analysis of polysaccharide structure (types of linkage and substitution, branching, anomeric configuration etc.). Polysaccharide fractions isolated from fruiting bodies of medicinal mushrooms Pleurotus ostreatus, Pleurotus eryngii, Agaricus blazei and Piptoporus betulinus were defined mainly as specific glucans. Branched 1,3-1,6-β-glucan and amylose-like 1,4-α-glucan were found in water soluble fractions, while linear 1,3-α-glucan in alkali soluble fractions. Effective deproteinisation and separation of α- and β-glucans was achieved by the treatment with phenolic reagent. Insoluble fractions contained some amount of chitin as a component of cell wall chitin–glucan complex. Fruiting bodies of wood decay fungi of genera Phellinus and Inonotus contains polyphenols integrated with cell wall polysaccharides. Most of polyphenols, which are interesting due to their strong antioxidative activity, were successfully removed from raw material by extraction with acidic ethanol. Soluble and insoluble polysaccharide fractions isolated from these mushrooms still contained colored products of polyphenol degradation. Polysaccharides were purified by the treatment with acidic or alkali solutions of hydrogen peroxide and defined mainly as branched β-glucans (soluble fractions) and chitin–glucan complex (insoluble part). Korean seaweeds Undaria pinattifida and Capsosiphon fulvescens are sources of bioactive polysaccharides, which were isolated and defined respectively as sulfated/acetylated galactofucan (fucoidan) and sulfated glucuronorhamnoxylan. These polysaccharides have shown various biological activities which make them interesting compounds for medicinal applications. This work was supported by Czech Science Foundation (Project No. 525050273), Ministry of Education of the Czech Republic (Projects No. CEZ: MSM6046137305) and Gyeonggi-do province, Korea (GRRC program of the Catholic University of Korea)

In order to obtain feasibility data regarding the possibility of using chondroitin sulfate (CS) in drug delivery system, CS was chemically modified by a one-step process with acetylation. Although 3 polymers with different degrees of acetylation were synthesized, only the sample with the highest degree of acetylation (AC-CS3) was tested as a nanogel because the others (AC-CS1 and 2) dissolved in distilled water (DW) in the test range. The AC-CS3 nanocarrier was characterized by fluorescence probe and dynamic light scattering (DLS) techniques. Its critical aggregation concentration (CAC) was < 2.0 × 10−2 mg/ml at 25 °C. The partition equilibrium constant, Kv, of the nanoparticle (7.88 × 105) was similar to that of polymeric micelles, which means that the acetyl group may act as a hydrophobic core controlling pharmacokinetic behavior. The higher surface charge value in the nanoparticle, above -40 due to carboxyl and sulfate groups in CS, explains its good stability. The anticancer drug doxorubicin (DOX) loading efficiency of the AC-CS3 nanoparticle was also superior, at above 90%. Changes in the size of the polydispersion index (PDI) of nanoparticle loaded with DOX over a 3-week period were negligible. The nanoparticle interacted with HeLa cells and were internalized together with the entrapped drug within the cytoplasm, probably via an endocytic mechanism exploited by sugar receptors. Based on these results, the AC-CS3 nanogel is expected to prove useful as an anti-cancer drug carrier for chemotherapy.

Photodynamic therapy (PDT) is typically harmless without light, tumor site treatment can be precisely targeted by selective illumination, limiting damage to surrounding healthy tissues. However, there are some difficulties with the use of photosensitizers(PSs), such as water-insolubility and low tumor-selectivity, which often limit its broader clinical applications in PDT. For overcome of these problems, we have improved their targeting efficiency and solubility through conjugate with target moiety labeled polysaccharides(1-3). During circulation in the blood, nanogel photoactivity may be suppressed by a self-quenching effect between PS molecules grafted to the target moiety labeled biocompatible polymer backbone similar to the fluorescence resonance energy transfer (FRET) effect(4). As the nanogel penetrates tissue and internalizes in cancer cells, photoactivity may be restored due to loss of the FRET effect by cleavage of the target moiety labeled biocompatible polymer backbone and the ester bond in the PS graft through enzymatic attack in the extracellular matrix and cellular compartments such as lysosomes. The target moiety conjugated with biocompatible polymer via DCC- and DMAP-mediated ester formation and dialyzed against distilled water for 2 days using a dialysis membrane. PS conjugate with target moiety labeled biocompatible polymer by the formation of an ester linkage, similar to the method of target moiety/biocompatible polymer conjugation the chemical conjugation was confirmed by 1HNMR spectroscopy and the degree of substitution was determined by UV–vis spectrophotometry. The conjugates was dissolved in 10 mL DMSO and dialyzed against excess deionized water . In 3 days, dialysates were collected and the volume of them was adjusted to 100 mL . The size distribution was measured by DLS and its photocytotoxicity was studied by XTT assay(5). The resulting nanogel are spherical, stable with a average size of 170nm and show self-quenching in aqueous system and have the capacity for tumor tissue targeting for nano-size induced EPR. After accumulated in tumor, the fluorescence signal will enhance gradually by the release of the dye because of broken down of nanogel as the exit of cleavable ester linkage .The irradiation with light of suitable wavelength of the photosensitizing drug uptaken by the cells resulted in generation of singlet oxygen for photodynamic therapy, simultaneity the near-infrared fluorescence molecular imaging can be detected

Carbohydrate-protein recognition events play key roles in physiological and pathological processes. The understanding of these biomolecular interactions provides deep insight into glycan-mediated biological processes and enables the development of more effective therapeutic agents and diagnostic tools. Carbohydrate microarrays, which are composed of diverse glycans orderly and densely attached to a single chip substrate, have been extensively developed as reliable and efficient tools for the rapid analysis of carbohydrate-based biomolecular interactions. The most general method for construction of these microarrays involves site-specific and covalent immobilization of chemically modified glycans to properly derivatized surfaces. This immobilization strategy requires the use of properly functionalized sugars, which are typically prepared by multi-step synthetic sequences. In order to avoid the need for functionalized glycans, immobilization strategies that employ unmodified sugars have been developed. In recent investigations directed at this goal, we have developed a novel, direct, site-specific technique for immobilization of unmodified carbohydrates, including simple carbohydrates, oligosaccharides and polysaccharides, on hydrazide-derivatized surfaces. This method eliminates the need for laborious and tedious glycan derivatization. To demonstrate the scope and applicability of this approach, carbohydrate microarrays containing a variety of glycans (fifty eight glycans) have been constructed by using one-step, direct attachment of free carbohydrates to a hydrazide-coated surface. These microarrays have been employed for analysis of sugar binding specificities of lectins, antibodies and bacterial cells. In addition, this microarray format has been applied to the determination of binding affinities between proteins and glycans.

Modification of nuclear and cytosolic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is ubiquitous in cells. The in vivo function of the protein O-GlcNAcylation, however, is not well understood. Here, we genetically manipulated the cellular O-GlcNAcylation level in Drosophila and found that it promotes developmental growth by enhancing insulin signaling. This alteration of growth is due to cell growth and apoptosis and not to cell proliferation, and is mediated, at least in part, through O-GlcNAcylation of Akt. These results indicate that O-GlcNAcylation is one of the crucial mechanisms involved in control of insulin signaling in normal Drosophila development.

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification is one of the post-translational modifications. Protein O-GlcNAcylation is reversibly regulated by O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT). More than 800 proteins as diverse as transcription factors, enzymes, cytoskeletal proteins, ribosomal proteins and chaperones have been identified to be modified with O-GlcNAc. Thus, protein O-GlcNAcylation appears to be involved in many different cellular activities regulating in transcription, translation, protein degradation, protein localization and protein-protein interaction. Moreover, O-GlcNAc modification has important and global effects on various cellular signal pathways because of the reciprocal relationship between O-GlcNAc and O-phosphate. Recently, O-GlcNAc modification, as a sensor of cellular states, has been implicated in human diseases including diabetes, neurodegenerative diseases and cancer. We found that O-GlcNAcylation pattern is changed during all-trans retinoic acid (tRA)-induced neutrite outgrowth in the MN9D cell line. We identified several O-GlcNAcylated proteins including alpha- and beta-tubulin and detected O-GlcNAc modified regions between residues 173 and 185 of tubulin and between residues 216 and 238 of beta-tubulin by using mass spectrometry. Also, We found that overexpressing OGT or introducing OGA inhibitor such as NButGT, PUGNAc, increases O-GlcNAcylation on alpha-tubulin, which ultimately leads to inhibition of tubulin polymerization. Accordingly, Our data indicate that the O-GlcNAcylation of tubulin negatively regulates microtubule formation

b-O-linked N-acetylglucosamine (O-GlcNAc) is dynamic post-translational modification in nucleus and cytosol. O-GlcNAc transferase(OGT) and O-GlcNAcase are involved in O-GlcNAc modification. Many proteins such as transcription factors, cytoskeletal proteins and proteins of variable signal transduction pathway are modified with O-GlcNAc at their Ser/Thr residues and their functions are affected by O-GlcNAc. The hexosamine biosynthesis pathway (HBP) consumes about 5% of glucose in total glucose flux, and it’s final product is UDP-GlcNAc, which is source of O-GlcNAc modification. It is reported that O-GlcNAc modification is involved in skeletal muscle metabolism and development process. We studied whether O-GlcNAc modification is involved in differentiation in myoblast C2C12 cell lines. Myoblast C2C12 is differentiated to myotube in differentiation media for 4-5 days. We observed that total O-GlcNAc modification level was dynamically changed during myogenesis. During first 24 hours after induction of myogenesis, total O-GlcNAc modification level decrease and increase again. Moreover, O-GlcNAcase activity increased and total cellular UDP-GlcNAc level decreased at the same time period. Also, we treated NAG-thiazoline derivative, specific O-GlcNAcase inhibitors and observed the block of the decrement of total O-GlcNAc modification and inhibition of myotube formation and expression of myogenic markers. Especially, after treatment NAG-thiazoline derivative, we observed that myogenin expression level decreased. So, we checked the myogenin protein stability using MG132 and mRNA level of myogenin using RT PCR. Even after treatment of MG132, the result of the experiment is same as previous experiment, thus myogenin protein stability is not involved with decrease of myogenin expression after treatment NAG-thiazoline derivative. And using RT PCR, we found that mRNA level of myogenin decrease after treatment NAG-thiazoline derivative. So we conclude that O-GlcNAc modification affect the transcription of myogenin.

Protein O-phosphorylation often occurs reciprocally with O-GlcNAc modification and represents a regulatory principle for proteins. O-phosphorylation of serine by glycogen synthase kinase-3β on Snail1, a transcriptional repressor of E-cadherin and a key regulator of the epithelial-mesenchymal transition (EMT) programme, results in its proteasomal degradation. We show that by suppressing O-phosphorylation-mediated degradation, O-GlcNAc at serine112 stabilizes Snail1 and thus increases its repressor function, which in turn attenuates E-cadherin mRNA expression. Hyperglycaemic condition enhances O-GlcNAc modification and initiates EMT by transcriptional suppression of E-cadherin through Snail1. Thus, dynamic reciprocal O-phosphorylation and O-GlcNAc modification of Snail1 constitute a molecular link between cellular glucose metabolism and the control of EMT.

In general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases when cellular glucose concentrations fall below normal levels. However, recent reports demonstrated that O-GlcNAcylation was increased 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 cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was not changed, it contained less O-GlcNAc and the activity was increased. Also, the activity of O-GlcNAcase was reduced. The studied glycogen containing cells, and we show that its degradation by 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 amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. 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 it may be useful for cancer cells to survive.

The epithelial cell adhesion molecule (EpCAM) was initially described the human colorectal carcinoma-associated GA733 antigen as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti-cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that GD1a play important roles in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. Immunohistochemical analysis revealed that anti-EpCAM mAb at a dose of 100 mg/mouse inhibited tumor cell growth. The expression of pro-apoptotic proteins and GD1a were increased by treatment with anti-EpCAM mAb. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

Gangliosides are complex glycosphingolipids that contain one or more sialic acids, the major components of cytoplasmic cell membranes. These are thought to play a role in the control of biological process including cell surface interaction, cell differentiation and transmemebrane signaling. In this study, we demonstrated that change of gangliosides pattern and glycosyltransferase gene in mouse induced pluripotent stem cells (miPSCs). The miPSCs were derived from OG2 mouse embryonic fibroblasts (mEFs) using retrovirus-mediated delivery and expression of Oct-4, Sox-2, Klf-4 and c-Myc. These cells were expressed alkaline phosphatase (AP), stage specific embryonic antigen-1 (SSEA-1) and mRNA such as Oct-4, Sox-2 and Nanog. In HPTLC and immunochemistry analysis, ganglioside GM3 and GD1a were expressed in mEFs, miPSCs and mESCs. However, only GM2 and GM1 were expressed in mEFs and mESCs. Ganglioside GM3 and GD3 expression level was low in miPSCs. Moreover, A2B5 antigen, c-series gangliosides such as GT3 and GQ1c, was only expressed in mESCs. To provide a better understanding of the metabolic basis of glycosyltransferase genes expressions, we analyzed expression of glycosyltransferase genes in mEFs, miPSCs and mESCs, and found that expression of sialyltransferase-I (ST-I), ST-II, ST-III, N-acetylgalactosamine transferase (GalNAcT), galactosyltransferase-II (Gal-II), ST-IV and ST-V. However, ST-II was not only expressed in mEFs. These results suggest that ganglioside GM3 and GD1a unique and powerful cell-surface biomarker to identify of mESCs and miPSCs, and change of ganglioside expression could be affected by gangliosides synthase activation. Therefore, gangliosides may play a role in the cell differentiation process.

Gangliosides are ubiquitous component of the membranes of mammalian cells that have been suggested to play important roles in various cell functions such as cell-cell interaction, adhesion, cell differentiation, growth control and signaling. However, the role that gangliosides play in immune rejection response by xenotransplantation is not yet clearly understood. In this study, regulatory roles of human leukocyte in the ganglioside expression of the primary cultured micro-pig aortic endothelial cells (PAECs) were investigated. To study whether human leukocyte are related to expressional changes of gangliosides in PAECs we performed high-performance thin layer chromatography (HPTLC) in PAECs incubated with fetal bovine serum (FBS), as control, or with recombinant human tumor necrosis factor-α (TNF-α), or with human serum and FBS contained with human leukocyte. Both HPTLC and immunohistochemistry analyses revealed that PAECs incubated with FBS are contained GM3, GM1 and GD3 as major gangliosides. However, ganglioside GM1 significantly decreased in PAECs incubated for 5hrs with TNF-α (10 ng/ml), or with 10% human serum and FBS contained with human leukocytes. Taken together, these results suggest that human TNF-α secreted by human leukocyte against PAECs and possibly happened expressional pattern changes of ganglioside GM1 in PAECs by secreted TNF-α. The finding that human leukocyte promotes expressional pattern changes of ganglioside in PAECs depending on TNF-α secreation may be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

Syndecan-4, a transmembrane heparan sulfate proteoglycan, plays a critical role in cell adhesion as adhesion receptor. Both transmembrane and cytoplasmic domain of syndecan-4 are known to be involved in the regulation of syndecan-4 functions. In this study, we further investigated the domain hierarchy of syndecan-4. Fluorescence resonance energy transfer (FRET)-based assays showed that expression of syndecan-4 enhanced RhoA activation. RhoA activation was much lower in rat embryonic fibroblasts (REFs) plated on fibronectin-fragment lacking the heparin binding domain, than that of cells on fibronectin. Syndecan-4 transmembrane domain mutants that were defective in transmembrane domain-induced oligomerization diminished RhoA activation and RhoA-related functions such as cell adhesion and spreading. On the other hand, syndecan-4 phosphorylation-mimicking cytoplasmic domain mutants showed SDS-resistant dimer formation and the similar inhibitory effects as transmembrane domain mutants, but to a less extent, suggesting the existence of domain hierarchy of syndecan-4. Consistently, syndecan-4 transmembrane domain mutant inhibited syndecan-4-mediated protein kinase C activation more efficiently than that of the cytoplasmic domain mutant, and the cytoplasmic domain activation failed to overcome inhibition of transmembrane domain mutants. Taken together, these data suggest that the domain hierarchy of syndecan-4 plays a role in the regulation of syndecan-4 functions.

A novel sialic acid-specific lectin (MCsialec) was detected from an expressed sequenced tag (EST) sequence from manila clam hemocytes infected with Perkinsus olseni (Kang et al. 2006, Dev. Comp. Immunol. 30, 1119–1131). The cDNA of the lectin was cloned using gene-specific primers based on a previously determined EST and characterized. The full-length cDNA of MCsialec is 603bp in length and encodes a polypeptide of 200 amino acids with a calculated molecular mass of 21.928 kDa. Sequence alignment and protein motif analyses showed that MCsialec shares identity with sialic acid-specific invertebrate lectins from Cepaea hortensis, Helix pomatia, and Haliotis discus discus. The lectin was expressed in E.coli M15cells and purified using a Ni-NTA His-binding resin matrix for antibody production. The presence of the lectin in various tissues of Perkinsus-infected and -uninfected manila clams was analyzed by both PCR and immunohistochemical localization assays. MCsialec was detected in each tissue of the clams; however, upon infection, the level of expression of the lectin increased in each tissue. Vibrio tapetis infection also induced high-level expression of MCsialec in the hemocytes. These data suggest that MCsialec plays a crucial role in the immune system of the manila clam during pathogenic infection.

Protein O-mannosylation is evolutionarily conserved essential protein modification from bacteria to humans, which is initiated in the ER by protein O-mannosyltransferases (PMTs) that catalyze the transfer of mannose from Dol-P-Man to Ser/Thr residues of secretory proteins. We identified and characterized five PMT subfamily genes, PMT1.1, PMT1.2, PMT2, PMT4 and PMT6 in the thermotolerant methylotrophic yeast Hansenula polymorpha. The hydropathy profile analysis predicts all H. polymorpha Pmt proteins to be integral membrane proteins with multiple transmembrane domains. For confirmation of subcellular localization of HpPmt proteins and characterization of PMT complexes, we constructed a set of HA or FLAG epitope-tagged versions of H. polymorpha Pmt proteins. All the C-terminal epitope-tagged HpPmt proteins were shown to be fully functional in vivo and to localize at the ER/Golgi membrane, except for Pmt6p. The significant sensitivity of Hppmt1.2 strain to the PMT1 inhibitor R3A-1c suggested that HpPMT1.2 might have a minor function redundant with HpPMT1.1. However, co-immunoprecipitation experiments using monoclonal anti-HA or anti-Flag bead revealed the complex formation between HpPmt1.1p and HpPmt2p, but no interaction between HpPmt1.2p and HpPmt2p even in the absence of HpPmt1.1p. The results strongly support the notion that interaction with HpPmt2p is required for the full mannosyltransferase activity of HpPmt1.1p, which plays a major role in O-mannosylation essential for cell wall integrity in H. polymorpha.

Dynamic protein modification, called post-translational modification (PTM), is the chemical modification of a protein and an important biological regulatory mechanism to understand cellular function and interactions. Glycosylation is one of the most common post-translational modifications of proteins and is involved in many physiological functions and biological pathways. Analysis of glycoproteins including site mapping and glycan characterization is an essential sub-field of proteomics using high throughput techniques by mass spectrometry. Recent improvements in mass spectrometry provide sensitive methodologies by liquid chromatography tandem mass spectrometry (LC-MS/MS) as an innovative analytical technology applicable to a wide number of analyses in glycoproteins. In addition to mapping sites of N-linked glycosylation, mapping sites of O-linked glycosylation including are another current challenge in proteomics. Here we demonstrate that the methodologies allow for analyses of glycoproteins and their mapping sites in various biological species and also discuss the effective uses of mass spectrometry for glycomic analysis and modern techniques of biomolecular mass spectrometry with the glycome from glycoproteins.