ANNIVERSARY
COMM
25thP

anel Discussion

Research trends onglycoproteins in the last five years

K en Kitajima

Bioscience and Biotechnology Center,Nagoya University, JapanProfessor Ken Kitajima received his BSc and MSc degrees in biophysics and biochemistry at the University of Tokyo, Japan in 1982 and 1984, respectively, and obtained his Ph.D. from the University of Tokyo in 1987, where he determined overall
structures of tandem-repeated peptides with highly polysialylated glycans for rainbow trout polysialoglycoprotein and discovered its processing enzyme that are activated concomitantly with cortical vesicular exocytosis at fertilization. From 19881989, he was a postdoctoral fellow at the University of Tokyo with Professor Yasuo
Inoue, and in 1989, became the Assistant Professor at the University of Tokyo. In1996, he moved to Nagoya University as Associate Professor of Graduate School of
Bioagricultural Sciences. In 2000 he became the Associate Professor of the Bioscience and Biotechnology Center, Nagoya University, and since 2004, has been theProfessor of the same center. He is currently a member of the Editorial Boards ofGlycobiology, Glycoconjugate Journal, and the Journal of Biological Chemistry. Hewas also a National Representative of the International Glycoconjugate Organization
until 2017. He is a Board of Trustees of the Japanese Society of Carbohydrate Research since 2007. In his laboratory, three lines of research work have been going on:(I) Sialic acid metabolisms in health and disease, currently focusing on biosynthetic
mechanisms of deaminoneuraminic acid (Kdn) and sulfated sialic acid and on significance of sialic acid metabolism-related enzymes at animal level using medaka fish;(II) Structural diversity and novel functions of polysialic acids in vertebrate brains andsea urchin gametes; (III) Biological significance of membrane microdomains as a siteof the glycan-mediated interactions.

Omics in glycoscience
Biochemical and molecular biological approaches have beensuccessful in describing mechanisms for various biological phenomena with a focus on specific molecules. On the other hand, forthe last decade, methodology and concept of life science have agreat deal changed. As an extension of conventional approaches,a different discipline of research has been lately focused on, i.e.,systems biology, which aims at understanding a whole animal on
the basis of integrated information from various omics, includingtranscriptomics, proteomics, and metabolomics. Irrespective of thecurrent state of successful integration of various omics in systems
biology, each omics is practically used for identification of important molecules in specific stages or organisms. In research fields

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the 25th Anniversary of Mizutani Foundation for Glycoscienceof glycobiology, glycomics is now acknowledged as an ordinary
methodology, as is the case with proteomics in life science 1). However, for the last few years, glycoproteomic analysis, which clarifies
the glycoform of every glycosylation site on specific proteins, hasbeen developed, and is increasingly used now, because it gives
much more useful information than simple glycomics 2). Metabolomics has been established for describing whole metabolites ina specific state of cell or an individual organism. One of important
metabolites in glycobiology is a group of nucleotide sugars that areused as donor substrates for various glycosyltranferases 3). The hexosamine biosynthetic pathway, through which UDP-GlcNAc is synthesized from Fructose 6-phosphate, a metabolite of the glycolysis,
and glutamine, a protein metabolite, has been successfully studied

MEMORATIVE SYMPOSIUM
for biosynthetic regulation of N- and O-glycans of proteins and OGlcNAcylation of regulartory proteins in cytosol and nucleus 4).
Genome wide association study in glycomedicine
In addition, recent technical innovation of nucleotide sequencing
has provided us with the whole genome sequence of any organism in several hours, and genome sequences from an extremely
large number of individual samples are now available. The genome wide association study (GWAS) for the relationship between
the frequency of the single nucleotide polymorphisms (SNPs) andcertain diseases using those data has been performed lately andis still expanding now. GWAS data can be used in various ways,
and is especially powerful in quantitative trait loci (QTL) analysis.
A significant relationship between schizophrenia and the polysialyltransferase ST8SIA2/STX gene was reported 5), and it has beenshown that impaired properties of resultant polysialic acid structures on the neural cell adhesion molecule (NCAM) raise a risk fordeveloping schizophrenia 6). So called “big data” arisen from omics
and GWAS data need to be efficiently used. Therefore, it is a naturalconsequence that significance of bioinformatics is more and more
recognized. Many efforts are now being made by many researchers of informatics, including bioinformatics of glycobiology 7).
Genome editing technologies and glycoscience
Advances in genetic modification technologies have brought
about drastic changes of research style in life science. Through thetechnologies, we can pursue a role of a specific gene in a specifictime and space. RNA interference and morpholino antisence oligonucleotide technologies are currently used as a standard method.
More recently, genome editing technologies such as the transcription activator-like effector nuclease (TALEN) and the clusteredregularly interspaced short palindromic repeats (CRISPR)-CRISPR
associated protein 9 (CRISPR-Cas9) has emerged and has been
more frequently used for a few years. Using CRISPR-Cas9 technology, we can obtain cells or animals with the disrupted target gene.
It is prominent that these technologies enable us to make genetic
modification in basically any organisms. Using not only mouse but
also small fish, significance of unusual glycosylation of dystroglycan has been confirmed.
Discoveries of new glycan structures
Finally, most important features of glycobiology, in my opinion,

are those discoveries of new glycan structures, characterization ofhighly ordered glyco-complex with proteins and lipids, such as lipid rafts and exosomes, and understanding of complex biosystems
such as microbial flora, which are not directly related to the up-todate style of life science described above. With regard to protein
glycosylation, extremely novel glycan structures of α-dystroglycan
have been demonstrated, in which the Xyl/GlcA repeat, (-3Xylα13GlcAβ1-)n, and CoreM3 structure, GalNAcβ1-3GlcNAcβ1-4 (phosphate-6) Manα1-Ser/Thr, are linked by a novel tandem Rbo5P(ribitol 5-phosphate) structure 8). This type of O-mannosyl glycan
on α-dystroglycan is required for its binding to extracellular matrix
components. Recently, a novel O-Man modification on E-cadherin
was reported in mammals, and has been shown to be independentof known protein O-mannosyltransferases 1 and 2 that modify
α-dystroglycan 9). A novel O-mannosylation process on the largecadherin superfamily is predicted in mammalian cells. O-GlcNAcylation of cytosolic and nuclear proteins is recognized as the common protein glycosylation that occurs inside the cell. In contrast,
O-GlcNAc modification on extracellular protein domains has recently been found as a novel example for protein glycosylation 10),which regulates Notch signaling and vascular development.
Future perspectives
Current tendency to use glycoproteomics rather than glycomics will
be continued, and GWAS for the relationship between SNPs and disease glycoproteomics will be frequently carried out. Use of CRISPRCas9 technology to examine function of a specific gene at cellularand animal levels will be popular in the glycobiology field, although,at the same time, we have to carefully think about the situation thatthe technology of gene manipulation is expanding, irrespective of
the ethical problems. Those up-to-date technologies will enable us
to understand in detail the relationship between a specific gene andphenotypes related glycan chains, and will be able to identify unknown glycosyltransferases and their regulatory proteins not only inmodel animals, but also in various animals other than experimentalanimals. We should continuously make efforts to find novel formsof glycosylation, to understand mechanisms underlying biologicalphenomena by characterizing functional complex of glycans, proteins, and lipids, and to understand roles of various microbial flora
in terms of human health and disease. There are still more subjects
than we can solve, as has been the case so far. Diversity must be
more and more important in research subject of glycobiology.

References1)	Zoldoš V, Horvat T, Lauc G. Glycomics meets genomics, epigenomics and other high throughput omics for system biology studies. Curr Opin Chem Biol. 17: 34-40,
2013.
2)	Sawanobori A, Moriwaki K, Takamatsu S, Kamada Y, Miyoshi E. A glycoproteomic approach to identify novel glycomarkers for cancer stem cells. Proteomics. 16:3073-3080, 20163)	Nakajima K, Ito E, Ohtsubo K, Shirato K, Takamiya R, Kitazume S, Angata T, Taniguchi N. Mass isotopomer analysis of metabolically labeled nucleotide sugars andN- and O-glycans for tracing nucleotide sugar metabolisms. Mol Cell Proteomics. 12: 2468-2480, 2013.
4)	Peterson SB, Hart GW. New insights: A role for O-GlcNAcylation in diabetic complications. Crit Rev Biochem Mol Biol. 51: 150-161, 2016.
5)	Arai M, Yamada K, Toyota T, Obata N, Haga S, Yoshida Y, Nakamura K, Minabe Y, Ujike H, Sora I, Ikeda K, Mori N, Yoshikawa T, Itokawa M. Association between
polymorphisms in the promoter region of the sialyltransferase 8B (SIAT8B) gene and schizophrenia. Biol Psychiatry. 59: 652-659, 2006.
6)	Sato C, Hane M, Kitajima K. Relationship between ST8SIA2, polysialic acid and its binding molecules, and psychiatric disorders. Biochim Biophys Acta. 1860: 17391752, 2016.
7)	Aoki-Kinoshita K, Agravat S, Aoki NP, Arpinar S, Cummings RD, Fujita A, Fujita N, Hart GM, Haslam SM, Kawasaki T, Matsubara M, Moreman KW, Okuda S, Pierce
M, Ranzinger R, Shikanai T, Shinmachi D, Solovieva E, Suzuki Y, Tsuchiya S, Yamada I, York WS, Zaia J, Narimatsu H. GlyTouCan 1.0--The international glycan
structure repository. Nucleic Acids Res. 44: D1237-1242, 2016.
8)	Kanagawa M, Kobayashi K, Tajiri M, Manya H, Kuga A, Yamaguchi Y, Akasaka-Manya K, Furukawa J, Mizuno M, Kawakami H, Shinohara Y, Wada Y, Endo T, Toda
T. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy. Cell Rep. 14: 2209-2223, 20169)	Larsen ISB, Narimatsu Y, Joshi HJ, Yang Z, Harrison OJ, Brasch J, Shapiro L, Honig B, Vakhrushev SY, Clausen H, Halim A. Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2. J Biol Chem. 292:11586-11598, 2017.
10)	Sakaidani Y, Nomura T, Matsuura A, Ito M, Suzuki E, Murakami K, Nadano D, Matsuda T, Furukawa K, Okajima T. O-linked-N-acetylglucosamine on extracellular
protein domains mediates epithelial cell-matrix interactions. Nat Commun. 2:583, 2011

Commemorative Symposium

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