Earticle

Mechanisms underlying disease pathogenesis are not well understood in the context of common etiological factors such as microbial infection, inflammation, malignancy or tissue breakdown. Such processes may be elucidated by identifying disease-related molecular markers, such as acute phase proteins, cytokines, cytoskeletal fragments and autoantigens. In an attempt to identify such markers, we have developed a novel platform, namely multiple lectin affinity chromatography (M-LAC) to study the plasma glycoproteome in patients and healthy donors in a series of studies ranging from cancer to autoimmune disease. The M-LAC platform allowed the evaluation of changes in concentration of glycoproteins, and to comprehensively survey the plasma proteome. We then used a second method, intact peptidomics, to assess changes in endogenous proteolytic activity by analyzing the low molecular weight (LMW) component of blood. The integrated proteomic and peptidomic analysis of plasma samples identified a number of cytoskeletal and Ca2+-binding proteins and their proteolytic fragments in the disease samples. The measurements were compared to healthy donors and several of the observed differential quantitations were independently verified by ELISA. The identified changes in plasma proteome and peptidome, and the underlying changes in glycosylation together with altered endogenous protease activity may result in the generation of novel autoantigens. We and others have confirmed this hypothesis by the observation of autoantibodies in patients and upon extension of these studies to larger populations of patient; we may gain additional understanding of the role of etiological factors in different disease pathways.

Glycoproteins are characterized by their complexity and diversity. To clarify their functions, synthetic approaches are considered to be promising. Synthesis of biomacromolecules is an important subject in organic synthesis. Establishment of sophisticated technologies in peptide and nucleic acid synthesis has played key roles in molecular, cellular, and structural biology. Chemical synthesis of oligosaccharides has been more elusive. Development of synthetic methodologies useful for efficient and facile preparation of glycoprotein glycan is a focal issue in carbohydrate chemistry. In light of their structural diversity, practical strategy to speed-up the synthesis of oligosaccharide is expected to be highly valuable. A major portion of our research has activities has been focused on stereoselective synthesis of glycoconjugate oligosaccharides, which resulted in developments of novel methodologies toward -sialoside and several types of 1,2-cis glycosides.1-3 Using these methodologies, syntheses of several types of novel glycoprotein glycans were conducted. Recently, a systematic strategy to synthesize glycoprotein glycans that play important roles in glycoprotein quality control, was established. With structurally defined glycans and in hand, analyses of various enzymes, lectins, and chaperones, which are involved in glycoprotein processing in the ER, were conducted, revealing their specificities in a quantitative manner. 4-8

Recent studies have shown the potential therapeutic uses of chondroitinase to recover loss of function in spinal cord injuries by regenerating neuronal growth. Upon injury to the spinal cord there is an up-regulation of chondroitin sulfate proteoglycans (CSPG). These CSPG help create an intertwined matrix within the glial scar, which can inhibit the advancement of neuronal growth between the sites of injury. Chondroitinase is able to degrade the chondroitin sulfate (CS) chains on the proteoglycans, thus eliminating obstacles for efficient neuron growth. In various animal models, the use of chondroitinase within injury sites allows the animal to regain the ability to walk. However, due to the instability of this enzyme, administration requires continuous, invasive, injections over a long period of time, which can further aggravate the injured site. Immobilization of chondroitinase onto a nano-solid support system could eliminate some of the problems currently faced, by increasing thermal stability, reducing immune responses, and limiting the migration of the enzyme to other parts of the body. Various methods of enzyme immobilization are used and have been shown to increase the activity of the enzymes in the literature. This technology could eventually be used for efficient treatment of spinal cord injury to recover the loss of function. Carbon nanotubes (cellulose-coated) and gold nanoparticles (citrate-reduced) were used for immobilization of chondroitinase ABC (Proteus vulgaris and Bacteroides thetaitaomicron) in the current study. While performing the immobilization process, we noticed a difference in the action pattern of both Proteus and Bacteroides enzymes. LC-ESI-MS to study the action pattern of these enzymes will be also discussed.

Saponins are glycosides of steroids and triterpenes, which exist widely and usually in considerable amount in terrestrial plants and some marine species.1 Numerous saponin extracts from herbal medicinal plants have been applied to treat various human diseases with surprisingly frequent precision, especially in China. Continuous isolation and bioassay of the components have led to the disclosure of many homogeneous saponins possessing significant biological activities. However, little has been known on the structure-activity relationships and the mechanism of action of saponins due to the poor accessibility to homogeneous saponins of various structures in appreciable amounts. This situation calls for chemical synthesis of saponins. The synthesis of saponins requires incorporation of two disparate disciplines of the synthetic chemistry, the synthetic chemistry of steroids/triterpenes and carbohydrates.2 Both disciplines have been the intensive research themes since very early years, however, it was not until 1990s the synthesis of the complex natural saponins became a feasible object and started to attract attention. Here I shall present an account on the total synthesis of natural saponins with potent biological activities. A major concern is the applicability of the contemporary glycosylation and protecting group manipulation protocols on the sophisticated scaffold of steroids and triterpenes.

Malaria continues to claim over 2 million people annually and is considered a reemerging fatal disease due to the spread of drug-resistant parasites and insecticide-resistant mosquitoes. Though vaccines have been one of the most cost- effective and easily administered means of controlling infectious diseases, no viable vaccine candidate has been developed for malaria. Glycosylphosphatidylinositol (GPI) anchors are a class of naturally occurring glycolipids that link proteins and glycoproteins via C-terminus to cell membranes. GPI originating from the malarial parasite Plasmodium falciparum has the properties predicted of a toxin, indicating that it is involved in the malarial pathogenesis. The initial efforts demonstrated that anti-GPI antibodies produced against the synthetic GPI glycan, might recognize the native GPI -containing toxin and neutralize proinflammatory activity by P. falciparum, revealing that the synthetic GPI is a prototype carbohydrate anti-toxin vaccine against malaria. In this presentation, a convergent synthesis of a series of oligosaccharides comprised of the malaria GPI glycan, a promising antitoxin malaria vaccine candidate currently in preclinical trials and related deletion sequences as well as a serological study using a GPI glycan microarray will be described.

The expression of glycan chains and hydrophobic structures of glycolipids is precisely regulated in a time- and space-dependent manner. We focus our research on two genes, Gsl5 (glycosphingolipid regulatory gene 5) and Des2 (degenerative spermatocyte 2). Gsl5 regulates the expression of GlcNAc　1-6(Gal　1-3)GalNAc containing glycolipids and glycoproteins through controlling the activity of 　6GlcNAc transferase in mouse kidney. Des2 encodes a hydroxylase which hydroxylates dihydroceramide:sphinganine at C4 position and is responsible for the expression of glycolipids containing C4 hydroxysphinganine (phytosphingosine) in the small intestine and kidney of mice. We found that Gsl5 locates at about 5.5 kb upstream of exon 1 of 6GlcNAc T1, consists of eight pairs of two GT-repeats, and controls 6GlcNAc T1 mRNA expression in kidney proximal tubular cellspecific manner. 　6GlcNAc transferase transfers GlcNAc on the GalNAc of Gal　1-3GalNAc　 and 　 of glycolipids and glycoproteins, respectively. One of glycoproteins modified by the GlcNAc transferase in the proximal tubular epithelial cells is megalin, which binds and reabsorbs small molecular weight proteins from urine produced by the blood filtration at glomeruli. We purified two types of megalin, one carries Gal　1-4(Fuc　1-3)GlcNAc　1-6(Gal　1- 3)GalNAc　1-Ser/Thr (Lewis X-core 2 structure) and the other the structure without the Gal　1-4(Fuc　1-3)GlcNAc　1-6 branch from mouse kidneys. Ligand binding capacity of the megalin with Lewis Xcore 2 structure was much higher than that without the branch structure. Des2 hydroxylase requires NADH, NADH-dependent cytochrome b5 reductase, and cytochrome b5 for producing the activity. mRNA expression of Des2 is regulated in a tissue-specific manner, highest in the small intestine and high in the kidney. We are in the process of establishing Des2 knock out mice and are going to analyze phenotypes. We consider that C4-hydroxylated sphinganine containing glycolipids play a significant role for maintaining functions of nutrient absorption.

Carbohydrate binding protein, lectin, is considered to have various functions such as adhesion molecules, signaling molecules, immune related molecules in vertebrates. However, its function in invertebrate has not been elucidated in detail yet. Using manila clam as a model system, we investigated possible roles of lectin in host defense against protozoan and microbial infection. We find Manila clam (Ruditapes philippinarum) synthesizes a lectin termed Manila clam lectin (MCL) only after infection with the protozoan parasite Perkinsus olseni. MCL is synthesized in hemocytes as a ~74 kDa precursor and secreted into hemolymph where it is converted to 30 kDa and 34 kDa polypeptides. The synthesis of MCL in hemocytes is stimulated by a factor(s) in Perkinsus-infected hemolymph, but not directly by Perkinsus itself. MCL can bind to the surfaces of purified hypnospores and zoospores of the parasite and this binding was inhibited by EDTA and GalNAc. Fluorescent beads coated with purified MCL were actively phagocytosed by hemocytes from the clam. Immunohistochemistry showed that secreted MCL localized inside of cyst-like structure. To investigate the patterns of genes expressed in Manila clams (Ruditapes philippinarum) infected with the protozoan parasite Perkinsus olseni, we constructed a cDNA library and generated 1,850 clones (expressed sequence tags). The 1,850 expressed sequence tags (ESTs) were compared to sequences in the GenBank database. Of these, 1,151 clones (62.2%) were unknown and are likely to represent newly described genes, while 699 clones (37.8%) were identified based on matches to sequences in the database. Lectins were the largest group of immune-function ESTs found in the Manila clams (6 different lectin were expressed). The expressions of lectins in the hemocytes were analyzed by RT-PCR using gene specific primers. Hemocyte from Perkinsus-infected clam expressed different set of lectins from that of Vibrio-infected one. Among these lectins we cloned MCL3, MCgalectin, and sialic acid binding lectin and characterized. It showed that has an open reading frame of 918 nucleotide and composed of 309 amino acid residues with a predicted molecular weight of 33.9kDa. Like other galectins, it did not contain signal peptide or transmembrane domain. It contained tandem repeated carbohydrate recognition domains (CRDs), in which the typical motifs important for carbohydrate recognition were conserved. Gemonic DNA analysis revealed MCGal is intronless. The carbohydrate recognition of MCGal analyzed by heptan inhibition of hemagglutination revealed that rMCGal showed a general feature of the galectin family, i.e. significant affinity for galactose and GalNAc. The expression of MCGal mRNA mainly detected in the tissues of heart, mantle, foot, adductor, palp and siphon. Immunohistochemistry using anti-MCGal antibody also conformed expression of MCGal in those tissues and hemocytes. The temporal expression of MCGal mRNA in manila clam challenged by perkinsus or vibrio was both up-regulated compared with non-challenged healthy manila clam. The recombinant MCGal agglutinated Vibrio tapitus and agglutination was inhibited by incubation with α-lactose. The rMCGal also bind surface of Perkinsus. We also find a specific protease associated with manila clam lectin (MCL). Protease was appeared in clams that were infected with Perkinsus sp, whereas uninfected clams did not contain protease. We suggest that in clam, which does not have adaptive immunity, lectins might act as strong defense molecules and their production is very inducible.

Mass spectrometry (MS) is the single most powerful enabling tool for its sensitivity and precision in glycomic mapping. It is well suited for systematic exploratory and discovery investigations since it alone is not pre-defined by availability of biological probes raised against already known molecular entities. In practice though, it is limited by the need to resolve a great depth of isomeric variation at a wide range of abundance, particularly those of larger size extended by polylactosaminoglycans, and those carrying multiple sialic acids and sulfates. Glycomic mapping, as most often employed to define glycosylation differences between genetically manipulated cell types or onco-developmental stages, therefore entails multi-staged MS analyses at increasing levels of sophistications and technical demands, in conjunction with chemo-enzymatic derivatization and chromatographic separation. A most effective analysis that is of true glycobiology significance is one that aims to address specifically if a particular glycotope of interest is expressed at what level on which glycan and protein carriers. Thus following a first-screen MALDI MS mapping and selected MALDI-MS/MS glycan sequencing to define the overall profile, further rounds of nanoESI-based analysis aiming at both comprehensiveness and selectivity in glycomic coverage are implemented. Along this line, our concerted workflows in glycomics and proteomics, as employed in recent case studies, will be presented to drive home this conceptual framework.

Cancer is often difficult to achieve early diagnosis, which is however a crucial factor for good outcome in cancer treatments. Cancer biomarkers have been sought for several purposes preferably in blood and pinpointing cancer cells-derived aberrant glycoproteins would be a well-grounded approach to cancer biomarker discovery. One of the glycosyltransferases responsible for aberrant glycosylation in cancer is N- acetylglucosaminyltransferase V (GnT-V), which catalyzes an addition of β1,6-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. To identify target proteins for GnT-V as cancer biomarker candidates in sera, we developed a protocol in which L-PHA, a lectin recognizing β1,6-GlcNAc, is conjugated to avidin-agarose complex to capture β1,6-N-acetylglucosaminylated glycoproteins from immuno-depleted sera, and the captured glycoproteins were tryptic-digested for sequence determination in an LTQ-FTICR mass spectrometer. Candidate proteins showing high sensitivity and specificity during the discovery phase were selected and the panel of biomarker candidates will be subjected to in-depth analyses for validation.

Peptidoglycan (PG) recognition protein (PGRP)-SA and Gram-negative bacteria binding proteins (GNBPs) are suggested to function as lectin-like molecules during Drosophila Toll signaling pathway. However, the molecular mechanisms of how bacterial PG and fungal beta-1,3-glucan recognition signals triggered by PGRP-SA and GNBP molecules are transferred to downstream factors are not clearly determined yet. The elegant Drosophila genetic studies have been and are very powerful for characterizing and arranging the components of the Drosophila Toll pathway. There is a limit with only genetic studies since the mechanisms involved in regulating and controlling this proteolytic cascade have done using a biochemical approach. To identify all essential components necessary for the transferring PGRP-SA or GNBP-mediated recognition signaling pathway, the biochemical approach was performed using the hemolymph of a large beetle, Tenebrio molitor larvae. For the activation of lysine (Lys)-type PG recognition signaling pathway, Lys-type PG/PGRP-SA/GNBP1/modular serine protease (MSP)/ Spätzle processing enzyme activating enzyme (SAE)/Spätzle processing enzyme (SPE)/Spätzle (Spz) cascade is an essential unit that triggers the Lys-type PG recognition signaling pathway in response to Gram-positive bacteria infection. For triggering fungal beta-1,3-glucan recognition signal, beta-1,3-glucan/GNBP3/MSP/SAE/SPE/Spz cascade is enough for transferring beta-1,3-glucan recognition signal in response to fungal infection.. The activation mechanisms of how the PGRP-SA-mediated Lys-type PG recognition signal and of how GNBP3-mediated beta-1,3-glucan recognition signal are transferred to Spz, leading to antimicrobial activity in vivo, is provided. Finally, we purified and determined the whole sequences of the induced antimicrobial peptides after injection of Lys-PG, beta-1,3-glucan, SAE and Spz to the Tenebrio larvae. Our data provides the first biochemical evidences of how the PGRP-SA-mediated Lys -type PG recognition signal and GNBP3-mediated beta-1,3-glucan recognition signal are transferred to Spätzle via PGRP-SA and GNBPs, leading to antimicrobial activities in vivo.

Oligo- or polysaccharides produced by environmental microbes such as Rhizobium meliloti, Xanthomonas oryzae and Zoogloea ramigera have shown physico-chemically novel functions such as solubilizer, chiral selector, catalyst, and morphology-directing agent. To investigate these functions, the microbial carbohydrates were purified with some column chromatographies and the purities of the saccharides were checked with nuclear magnetic resonance (NMR) and/or Infrared (IR) spectroscopy. Cyclic oligosaccharides produced by R. meliloti, cyclic -1,2-glucans (cyclosophoraoses), acted as a solubility enhancer for poorly soluble molecules, a chiral solvating agent for many enantiomers in NMR and capillary electrophoresis (CE), and a morphology-directing agent for the synthesis of Se nanowires in water. □-Cyclosophorohexadecaose (□-C16) produced by X. oryzae and Rhizobial succinoglycan octasaccharides having acetyl, pyruvyl, and/or succinyl groups as substituents, acted as catalysts for the multicomponent Strecker reaction using trimethylsilylcyanide (TMSCN) in the mixture solutions of methanol and water. Also, Rhizobial cyclosophoraose and an extracellular polysaccharide produced by Z. ramigera, zooglan, acted as catalysts for the methanolysis of oxazolone compounds and phospholipids. These functions could be due to complexing capacities or strong hydrogen bonding abilities of the microbial carbohydrates.

The glycosides are increasingly implicated in biological recognition and signalling mechanisms, thus further elevating their potential as therapeutics. This prominence creates a strong need for efficient and versatile methods for stereo- and regioselective glycoside synthesis. The enzymatic synthesis of glycosidic linkages with carbohydrate-active enzymes has been widely investigated to address this issue and is attractive in their abilities to circumvent often long-winded protection regimes. Glycosyltransferases have proven to be highly efficient in the synthesis of glycosidic linkages. However, the expense of the nucleotide sugar substrates, tight substrate specificity, and low enzyme availability limits their application. Glycosidases area attractive for large-scale application since they are more abundant, relatively inexpensive, exhibit broader acceptor-substrate specificity and use simpler substrates. However, stringent donor specificities and low yield can limit their use. In terms of reaction mechanism, transglycosidases and glycosidases belong to the same group. In this presentation we will focus on the use of transglycosidase and glycosidase for the synthesis of bioactive glycosides. The enzymes we chosen are levansucrase (transglycosidase) and β-glucosidase (glycosidase), respectively. The potential applications of newly made glycosides as well as the biochemical characteristics, substrate specificities, and transglycosylation properties of both enzymes can be discussed.

Endo-dextranases (EC 3.2.1.11), which hydrolyze -1,6-glucosidic linkage of dextran at random, are classified into glycoside hydrolase (GH) family 49 and 66 based on the amino-acid sequence similarity. Cycloisomaltodextrin glucanotransferase (CITase) is also classified into GH family 66. CITase is an interesting enzyme that catalyzes the conversion of dextran to cycloisomaltodextrins (CIs) by intramolecular transglycosylation. CI is of importance by preventing the dental caries since it strongly inhibits insoluble glucan formation by mutansucrase. Although endo-dextranase and CITase belong to GH family 66, their products from dextran are largely different in structure, such as cyclic- or linear-isomaltooligosaccharides, respectively. In this study, it was found that Paenibacillus sp. dextranase, a member of GH family 66, showed both of endo-dextranase activity and CITase activity, allowing us to classify the GH family 66 enzymes to three groups for the first time. These results may contribute to understanding of the relationship between the structure and function of GH family 66 enzymes. Recently, several other Bacillus and Paenibacillus CI-producing bacterial strains were found and the larger CI molecules up to CI-17 were isolated from their culture supernatants. In order to produce CIs for commercial scale, a high dextran producing strain Leuconostoc sp. S-51 was obtained and the B. circulans T-3040 strain was mutated to produce CITase with about 110 times the activity of the wild type strain. All CIs well inhibit glucosyltransferase of mutans streptococci and especially CI-10 and larger molecules of CIs have strong inclusion-forming activity such as preservation of Victoria blue color or solubilization of isoflavones. There have been no problems arising from safety tests of CIs in rats and humans. The fact that CIs are present in brown sugar is indicative if their safety as natural oligosaccharides.

Thioglycoligases are mutant enzymes derived from retaining glycosidases in which the acid/base carboxylic acid residue has been replaced by an amino acid that has no negative charge. The engineered glycosidase mutants catalyze the formation of a thio-glycosidic linkage with substrates bearing a good leaving group, such as dinitrophenol or fluoride, and sugar acceptors bearing a suitably-positioned thiol. The glycosidase-resistant thioglycoside analogues of the original oligosaccharides are attractive candidates as potential therapeutics. Here, a collection of galactosyl-thio-β -glycosides was prepared by a thioglycoligase derived from a β-galactosidase from Xanthomonas manihotis (BgaX), and a potent inhibitor against human lysosomal β- galactosidase. The identity of the acid/base catalyst of BgaX has been confirmed as Glu184 by kinetic analysis of mutant modified at that position. The Glu184Ala mutant of BgaX is shown to function as an efficient thioglycoligase, which synthesises thiogalactosides with linkages to the 3 and 4 positions of glucoside and galactoside in high yields. Amongst five galactosyl-thio-β-glycosides synthesized by BgaX-E184A, pNP-β-galactosyl-β- 1,3-N-acetyl-glucosamine was expected to be the best inhibitor against human lysosomal β-galactosidase based on its substrate specificity. However, pNP-β- galactosyl-β-1,3-glucoside was the best inhibitor with a reasonable potency (Ki = 8 μM) against the human enzyme. Interestingly, the inhibitor did not inhibit bacterial β- galactosidases which not only belongs to the same glycosidase family but also possess similar substrate specificity to that of the human enzyme. Test of the compound as an inhibitor toward β-galactosidases belonging to other two families of such enzyme revealed that β-galactosidase from E. coli (LacZ) showed a similar order of affinity, but β-galactosidase from B. subtilis was not inhibited. In conclusion, our result opens the interesting possibility of finding novel, and unpredicted inhibitors of enzymes of interest through this relatively simple strategy of library generation in which aglycone-diverse thioglycosides are created by thioglycoligases. From a modest set of thiosugar acceptors it is reasonable to envisage the assembly of a substantial library of thio-disaccharides for testing as inhibitors of glycosidases or carbohydrate binding proteins of interest.

For these 7 years, we have been developing new technologies for Glycomics with the sponsorship of the New Energy and Industrial Technology Development Organization (NEDO). We discovered new glyco-genes and constructed human glyco-gene library consisting of 184 genes. Knowledge of the substrate specificities of these gene products allowed us to better understand the molecular basis of human glycosylation. Taking full advantage of our glyco-gene library, we developed a glycan library that was then used as standards to develop instruments for glycan structural analysis, such as mass spectrometer-based glycan sequencer and lectin microarray-based glycan profiler. Association of aberrant glycosylation with cancer has long been recognized. However, much of the discovery still depends on serendipity, and biological meanings of these cancer-related glycosylation patterns are only gradually uncovered. In 2006, we launched a new project termed Medical Glycomics (MG) project. Our aims in the project are two-folds: (1) development of discovery systems for cancer-related glyco -biomarkers, and (2) functional analysis of the cancer-associated glycosylation. We present many candidates for cancer-glyco-biomarker which would be useful for diagnosis.

Pilus-mediated motility is essential for the optimization of photosynthesis and environmental adaptation in the cyanobacterium Synechocystis sp. PCC 6803 (Syn6803). To identify the genes required for pilus-mediated motility in Syn6803, we applied a forward genetic approach using a Tn5 mutant library and reverse genetics using interposon mutagenesis. One of the identified genes, sll0899, bears sequence similarity to acyltransferases and nucleotidyltransferases. The sll0899 gene product is not involved in the transcription or translation of pilA1, which encodes pilin, the major component of pili. Instead, the sll0899::Cmr mutant produced pilins with increased molecular mass, suggesting the existence of different post-translational modifications. Using mass spectrometry (MS), we found that the wild-type (WT) and mutant pilins were glycosylated between amino acids 67 and 75; however, the glycan of the mutant pilin was elongated with deoxyhexoses, and this was confirmed by quantitative MS using isotope-coded protein labeling of tryptic peptides. Analysis by high-pH anion exchange chromatography revealed that the glycan in WT pilin is composed of xylose and fucose, whereas an additional sugar, rhamnose, was found in the glycan of mutant pilin. Our findings suggest that an alteration in the O-linked glycan of pilin is responsible for the loss of pilus-mediated motility in sll0899::Cmr.

Biopharmaceuticals are medical drugs produced using biotechnology. They are proteins (including antibodies) and nucleic acids (DNA, RNA or antisense oligonucleotides) used for therapeutic or in vivo diagnostic purposes. These so called large molecule pharmaceuticals can be produced from recombinant microbial cells, mammalian cell lines or plant cell cultures. Biopharmaceuticals are typically large molecules, requires complex post translational modification and glycosylation processes. Its glycosylation pattern affects biological activity, function, clearance from blood circulation, and most importantly it could also elicit immune response in patients. Due to that reasons, bacteria, yeasts, fungi, insects and plants are generally not suitable for the expression of therapeutic glycoproteins such as antibodies. The worldwide market for therapeutic and diagnostic monoclonal antibodies is expected to reach USD$26 billion by 2010. First Antibody for pharmaceutical was approved in 1986 and currently there are more than 23 therapeutic antibodies approved by US FDA in the market. The numbers are still growing. With hundreds of product candidates in the pipeline getting into clinical stage phase I, II and III, there is a huge demand for contract manufacturing sector as well. InnoBio operates as a contract manufacturer (CMO) and is positioning itself as one of the player for biopharmaceutical production. Established in 2003, the services include all stages of the production of mammalian cell-based therapeutic proteins and monoclonal antibodies, from DNA recombination work and bioprocess development to cGMP manufacturing. In this talk, the experience in setting up a cGMP manufacturing plant and manpower development together with support facility will be discussed.

The hepatitis B virus (HBV) is the major cause of liver disease worldwide. The World Health Organization estimates that 2 billion people (one-third of the world's population) have been infected by HBV. Of these, approximately 400 million people have chronic infections that put them at high risk of ultimately developing cirrhosis and cancer of the liver and the death of liver tissue. In China alone, more than 100 million people have chronic HBV infections. The currently available HBV vaccine needs some improvement to induce protection among the nonresponders and also to apply the immune therapy for the chronic carriers. To improve immunogenicity of the HBV vaccine antigen, we have successfully developed a CHO cell line that can express all three types of envelope proteins (L protein, M protein and S protein) in the form of virus-like particles which are observed in the blood of HBV chronic patients. The preS antigens are exposed at the external surface of the particles composed of S antigens and the virus-like particles are properly glycosylated. Using this antigen, we have developed a strong HBV vaccine which can break immune tolerance and induce strong immune responses in a transgenic mouse model.

Patients with Fabry disease have a defect in the gene for the lysosomal enzyme α- galactosidase A (α-GAL). This defect results in an inability or diminished ability to catabolize lipids with terminal α-galactosyl residues. In the absence of lysosomal enzyme α-galactosidase A, these lipids, particularly globotriaosylceramide (GL-3), accumulate progressively in the lysosomes of many cell types throughout the body. GL-3 accumulation in renal endothelial cells may play a role in renal failure. Enzyme-replacement therapy (ERT) is an established means of treating lysosomal storage diseases such as Fabry disease. Infused therapeutic enzymes are targeted to lysosomes of affected cells by interactions with cell-surface receptors that recognize carbohydrate moieties, such as mannose and mannose 6-phosphate, on the enzymes. Production of effective mannose-6-phosphate-targeted ERTs for some disorders is difficult, because lysosomal enzymes have a short half-life when injected into the bloodstream because of rapid clearance in the liver by other carbohydrate-recognizing receptors, particularly the mannose receptor that is highly abundant on Kupffer cells. In this presentation, we can discuss the approach to control carbohydrate moieties to improve the efficacy of therapeutic enzyme.

The anti-CD20 antibody, Rituximab has been used for the treatment of B-cell non- Hodgkin's lymphoma (NHL). However, resistance or no responsiveness of some patients has evoked lots of endeavors to improve antibody efficacy through antibody engineering. The therapeutic efficacy of monoclonal antibody generally depends on the carbohydrate moiety linked to the Fc region. Especially, antibody dependent cell- mediated cytotoxicity (ADCC) has been known to be dramatically enhanced by fucose reduction. In this study we have specifically modulated the fucose residue of antibody by expressing therapeutic antibody in CHO cell that contains mutant forms of alpha- 1,6-fucosyltransferase (FUT8) and/or alpha-L-fucosidases. The modified CHO clones with these mutants were selected on the basis of FACS using LCA lectin assay. Compared to unmodified antibody therapeutics, glyco-engineered antibodies showed around 30% reduced fucose content based on the glycan analysis. As a result, these molecules had about 4-fold increase in FcrRIIIa binding activity than that of the original antibody, and up to 5-fold in ADCC using PBMC and B-lymphoma cell system with no effect on complement dependent cytotoxicity (CDC). Therapeutic efficacy of glycol- engineered antibody in B cell lymphoma mouse model could be improved at least over 2-fold when compared to Rituximab. Taken together all these results, a new strategy using FUT8 and/or fucosidase mutants could be applied to enhance therapeutic efficacy of monoclonal antibody.

Rapid and reliable quantitative plant N-glycan analysis is highly required to explore complex glycobiological research and develop plant expression systems for factory to produce therapeutic glycoproteins with human compatible glycans. We present here the high throughput quantitative analytical method for plant N-glycan based on DNA sequencing equipment. All steps including peptide N-glycosidase (PNGase) A treatment, glycan preparation and exoglycosidase digestion were optimized for high throughput applications with 96 well format procedures and automatic analysis on DNA sequencer. The glycans of horseradish peroxidase with plant-specific core α(1,3)- fucose can be discriminated by the comparison of glycan profiles obtained by PNGase A and F treatments. The peaks of glycans with (91%) and without (1.2%) α(1, 3)- fucose were easily quantified on a DNA sequencer analysis. In addition, both of them have been shown to harbor bisecting β(1,2)-xylose by simultaneous treatment of α (1,3)-mannosidase and β(1,2)-xylosidase. This technique was also applied to analyze N-glycan of plant-derived anti-rabies monoclonal antibody of which heavy chain was fused to C-terminal Lys-Asp-Glu-Leu (KDEL) sequences for the retention at endoplasmic reticulum. It revealed that most of its N-glycan (more than 80%) belongs to oligomannose type. The identities of glycans generated by DNA sequencer analysis were independently confirmed by mass spectrometry. These results suggest that the DNA sequencer- based method provides quantitative information for plant specific N-glycan analysis in a high throughput manner, which has not been achieved by glycan profiling based on mass spectrometry.

Synthesis of dibenzyl (6-O-naphtylmethyl-2,3,5-tri-O-benzoyl-β-D-galactofuranosyl)- (1→5)-(2,3-di-O-benzoyl-6-O-benzyl-β-D-galactofuranosyl)-(1→4)-(3-O-benzyl-2- O-pivaloyl-α-L-rhamnopyranosyl)-(1→3)-2-acetamido-2-deoxy-4,6-di-O-benzoyl-α -D-glucopyranosyl phosphate (A), a protected form of the tetrasaccharide phosphate of the linkage region of the arabinogalactan-peptidoglycan complex in mycobacterial cell wall, has been accomplished. Key steps include the coupling of four monosaccharide building blocks with complete stereoselectivity by glycosylations employing thioglycosides, 2'–carboxybenzyl glycosides, and glycosyl fluorides as glycosyl donors. The α-glycosyl phosphate linkage was also stereoselectively elaborated by reaction of a tetrasaccharide hemiacetal with tetrabenzyl pyrophosphate in the presence of a base.

A great deal of effort has been devoted to the elucidation of the cell wall structure of Mycobacteriumtuberculosis, the causal agent of human tuberculosis, in recent years owing to the appearance of its multi-drug resistant strains. One of the major structural components of the cell wall of is the complex of mycolic acid, arabinogalactan (AB), and peptidoglycan, which plays a extremely important role for the survival and pathogenicity of M. tuberculosis. Novel cyclicgalactooligosaccharides 1, 2, and 3 were identified amongst the degradation products of AG by extracellular enzymes isolated from Cellulomonas sp. Herein we report the synthesis of cyclic tetra-, hexa- and octasaccharides 1, 2, and 3 including the alternating (1→5)-β-, and (1→6)-β- galactofuranosyl linkages by the intramolecular cycloglycosylation of corresponding linear sugars and by the cylclooligomerization of 1,5-linked and 1,6-linked disaccharides, 4 and 5. Notably, the cyclooligomerization of (1→6)-β-galactofuranosyl disaccharide 5 presented an effective way to secure all three cyclic sugars in one operation.

Quality control and assurance of glycan profiles of a recombinant glycoprotein from lot to lot is a critical issue in the pharmaceutical industry. To develop an easy and simple quantitative and qualitative glycan profile method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), the modification with Girard’s reagent T (GT) was exploited. Since GT-derivatized quantification of oligosaccharides using MALDI-TOF MS is only possible with neutral glycans, sialylated glycans are not subjected to quantitative analysis with MALDI-TOF MS. To solve this problem, mild methyl esterification and subsequent GT derivatization were employed, which enabled us to perform rapid qualitative and quantitative analysis of sialylated and neutral N-linked oligosaccharides using MALDI-TOF MS. This modified method was utilized in the comparative quantification of N-glycans from the recombinant therapeutic glycoprotein expressed in two different kinds of chinese hamster ovary (CHO) cell line. The percentages of sialylated N-glycans to total were 22.5% and 5.2% in CHO-I cell and CHO-II, respectively, which resulted in significant difference in the biological activity of the recombinant glycoprotein.

Streptococcus pneumoniae is a causative agent for high morbidity and mortality. Although sugar moieties have been recognized as a ligand for initial contact with the host, only a few exoglycosidaseshave been reported in S. pneumoniae. In this study, a putative -galactosidase, encoded by the bgaC gene of S. pneumoniae, was characterized for its enzymatic activity and virulence. The recombinant BgaC protein, expressed and purified from Escherichia coli, was found to have a highly regiospecific and sugar specific hydrolysis activity for the Gal1-3-GlcNAc moiety of oligosaccharide. Interestingly, the BgaC hydrolysis activity was localized at the cell surface of S. pneumoniae, indicating that BgaC is expressed as a surface protein although it does not have a typical signal sequenceor membrane anchorage motif. The surface localization of BgaC was further supported by immunofluorescence microscopy analysis using an antibody raised against BgaC. Although the bgaC deletion mutation did not significantly attenuate the virulence of S. pneumoniae in vivo, the bgaC mutant strain showed relatively lower viable cell numbers compared to the wild type after 24 h infection in vivo, whereas it showed higher adherence and invasion at 6 and 12 h post- infection in vivo. Our data strongly indicate for the first time that S. pneumoniae bgaC encodes a surface β-galactosidase with high substrate specificity that is significantly associated with the infection activity of pneumococci.

The α-viniferin was reported to show anti-inflammatory effects. Cyclooxygenase-2 (COX-2) expression was detected in most adenomas and aberrant crypt foci (ACF). Glycosphingolipids are involved which cell proliferation, differentiation and cellular interactions in the plasma membrane remain to be established. In the present study, experiments were designed to assess the potentialchemopreventive properties of α- viniferin on azoxymethane (AOM)-induced colorectal carcinogenesis. Administration of α-viniferin at dose 0.05 and 0.1mg/mouse during AOM-treatment for 5 weeks resulted in a dose-dependent reduction of ACF formation, being 75% and 64% (P<0.05) inhibition of the control value. Treatment of α-viniferin at dose 0.05 and 0.1mg/mouse for 28 weeks resulted in a dose-dependent suppress of colorectal carcinogenesis, being 80.2% and 49% (P<0.05) inhibition of the control. COX-2 expression were decreased on ACF tissue and colorectal tumor tissue treated with α-viniferin. In vitro, we demonstrated that α-viniferin play the roles such as activity of apoptosis, increase of caspase-3 and inhibition of COX-2 expression in Caco-2 colon cancer cells. High -performance thin-layer chromatography (HPTLC) shows gangliosides GM3, GM2 and GT1b increasein the Caco-2 cells are treated with α-viniferin. Immunofluorescence stain results show GT1b and GM3 expression significantly increase in theCaco-2 cells by α-viniferin. The results indicated that suppression of colorectal carcinogenesis was related to the activityof apoptosis through caspases-3 activity, COX-2 inhibition and gangliosides expression by α-viniferin. These results suggested that α-viniferin may play roles of chemopreventiveproperty in carcinogens-induced colorectal carcinogenesis of mice.

Epithelial-mesenchymal transition(EMT) is a program of developing cells characterized by loss of cell adhesion and increased cell mobility. EMT is required in many developmental processes, such as gastrulation and neural crest migration. In the pathological aspect, deregulation of EMT can induce tumor progression in cancer cells. Snail is transcriptional repressor of E-cadherin, which regulates cell adhesion in epithelial cells, and the repression of E-cadherin is thought to play a major role in the abnormal manifestation of EMT. O-GlcNAc modification is a post-translational modification, which is occurredin nucleus and cytosol. Many proteins, including transcription factors, cytoskeletal proteins and various enzymes, are known to be O-GlcNAcylated. Here we firstly report that snail is an O-GlcNAcylated protein and O-GlcNAc modification on snail increases the stability of snail. In the hyperglycemic conditions, snail protein was accumulated, but mRNA level did not increase. Snail is known to be down-regulated by phosphorylation which is a signal for ubiquitin-proteasome protein degradation pathway. When O-GlcNAcylation of snail was increased, ubiquitination on snail was decreased by inhibition of phosphorylation.

Combinatorial biosynthesis is an alternative way for accessing naturally unavailable natural products or improving activity of already existing biomolecules by their modification. Biosynthesis of different deoxy-aminosugar and its attachment to the same or different anthracyclinone aglycones in vivo would lead to the formation of novel anthracycline compounds. Unlike doxorubicin, anthracyclines with N-alkylated sugar moieties were weakly mutagenic or not mutagenic at all in both bacterial and mammalian cells. Disruption of glycosyltransferase gene, dnrS, involved in the biosynthesis of the doxorubicin from Streptomyces peucetius ATCC 27952 led to accumulation of a non-glycosylated intermediate ε-rhodomycinone. Complementation experiment was carried out to introduce L-rhodosamine sugar for the production of rhodosaminyl-doxorubicin. Chromosomal integration of desVI encoding N,N-dimethyltransferase from Streptomyces venezuelae and aknS encoding glycosyltransferase along with its auxiliary gene aknT from Streptomyces galilaeus in the dnrS disruptant of S. peucetius led to formation of rhodosaminyl-doxorubicin.

Vancomycin is one of the antibiotics of last resort in the treatment of life-threatening infections by Gram-positive bacteria. Vancomycin was tried to modify glycone to overcome resistance mechanism specially. Vancomycin was modified by sialydation. Vancomycin and pseudo-vancomycin were glycosylated with UDP-galactose at 4'-C of glucose to synthesize galacto-vancomycin (KSY16-80) and galacto- pseudovancomycin (KSY0703-2). The galacto-vancomycin (KSY16-80) and galacto- pseudovancomycin (KSY0703-2) were sialylated with CMP-neuraminic aicd at 3''-C of galctose to synthesize sialyl-vancomycin (KSY16-91) and sialyl-pseudovancomycin (KSY0718-7). The sialyl-vancomycin and derivatives were tested by antibiotic resistance bacteria for MIC test. The sialyl-vancomycin (KSY16-91) shows less biological activity than vancomycin but galacto-vancomycin (KSY16-80) has the same activity.