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Research paper : Development of basic tools for glycoscience and their application to cancer diagnosis (H. Narimatsu)−205−Synthesiology - English edition Vol.5 No.3 (2012) It is inferior in the following: (1) Absolute structure determination is impossible. (2) Lectins may not always be readily available.(4) Functions and biomarkers:In the Medical Glycomics (MG) Project, effects of the change of glycan structure on the glycoprotein functions and cell phenotypes were determined in vivo using the above technologies (1-3). As we went through, “synthesis”, “structure”, and “functions and biomarkers” have been the three main themes and interactively developed. Their application can be seen as “synthesis” in the production of functional oligosaccharides and glycan-modified glycoproteins, “structure” in the quality management of glycoprotein pharmaceuticals and ES cells, and “functions and biomarkers” in the commercial and medical applications as shown in diagnosis technologies. 4 Development of elemental technologies for basic technology tools Figure 3 shows a list of elemental technologies developed for glycoscience. The following is the summary of each element.Elemental technology 1: Identification of glycosyltransferases in human genome databases by bioinformatics technology We started from the comprehensive identification of candidate genes for glycosyltransferases by full application of the bioinformatics technology. Kikuchi, a member of our project team assigned from Mitsui Knowledge Industry Co., Ltd., developed new software to identify the candidates from the available genome databases. This software is capable of searching not only amino acid homologues but also characteristic glycogenes such as (1) glycogenes possessing a membrane binding site to hydrophobic amino acids at proximal to the N-terminus, and their lengths being about 18 to 22 amino acid residues, slightly shorter than cell membrane binding proteins, (2) glycogenes with a subsequent main structure that is rich in proline and has many serine and threonine, (3) glycogenes subsequent to the enzyme activity domain, and have active domains consisting of 300-400 amino acids and containing several cysteine and 3-amino acids of the DXD motif binding to bivalent cations. About 100 glycogenes with such characteristics were identified and their cDNAs were generated mainly based on the RNA of human cultured cells. All the candidate genes coding the entire enzyme were cloned by PCR. Elemental technology 2: Recombination of glycosyltransferases into expression vectors and substrate specificity analysis of recombinant enzymesFor the development of this technology, many original members of the Research Center for Glycobiotechnology (predecessor of Research Center for Medical Glycoscience [RCMG]) of AIST (Togayachi, Sato, Goto, Kudo, Tachibana, Cho, Kubota, Sawaki, and more) greatly contributed. Glycosyltransferases are membrane proteins binding to the Golgi membranes and endoplasmic reticulum membranes. To activate the glycosyltransferases as recombinant enzymes for in vitro synthesis of glycans, they have to be in the form of Fig. 3 Representative basic technology tools developed or utilized in RCMGGlyco-catch method/IGOT (glycoproteomics) Frontal affinity chromatography Lectin microarray Mass spectrometryGlycogene qPCR array Glycogene-modified cells/mice(KO, Tg mice)Glyco-Pathology DDS technology SMME methodGlycan synthesis (glycan, glycoprotein, glycopeptide) SE methodBioinformatics Database (JCGGDB) Glycan libraryBy GlycosyltransferaseBy YeastGlycogeneGlycan modification Functional analysisNarimatsu, H. et al. (2004) Glycoconj J. 21, 17 Review.Iwai, T. et al. (2005) PNAS. 102, 4572Sato, T. (2006) Glycobiology. 1194Bioinformatics technologyKikuchi, N. et al. (2006) Biochim Biophys Acta. 1760, 578Glycan structure analysisGlycan structure profilingGlycogene analysisAnalysis of glycan-recognizing proteinGlycoproteomicsMedical application related technologyTogayachi, A. et al. (2007) PNAS. 104, 15829Togayachi, A. et al. (2010) PNAS. 107, 11900Sato, T. et al. (2010) submitted.Ito, H. et al. (2005) Angew Chem Int Ed Engl. 44, 4547Ito, H. et al. (2007) Nat Methods. 577Amano, K. et al. (2008) PNAS. 105, 3232Kameyama, A. et al. (2005) Anal Chem. 77, 4719Kameyama, A. et al. (2006) J Proteome Res. 5, 808Ito, H. et al. (2009) Methods Mol Biol. 534, 283Kuno, A., Uchiyama, N., et al. (2005) Nature Methods 2,851Uchiyama, N. (2006) Methods Enzymol. 415, 341Kato, Y. et al. (2006) BBRC 349, 1301Ito, H. Kuno, A. Sawaki, H. et al. (2009) J Proteome Res. 8, 1358Hirabayashi, J. et al. (2002) Biochim. Biophys. Acta 1572, 232Hirabayashi, J. et al. (2003) Methods Enzymol. 362, 353Tateno, H. et al (2007) Nature Protocols 2, 2529Hirabayashi, J. et al. (2002) J. Biochem. 132, 103Kaji, H. et al. (2003) Nature Biotechnol. 21, 667Ikehara Y. et al. (2006) Cancer Res. 66, 8740Ikehara Y. et al. (2008) Cancer Lett. 260, 137Glycogene Library

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