Table Of ContentExperimental Glycoscience
Glycochemistry
N. Taniguchi, A. Suzuki, Y. Ito, H. Narimatsu,
T. Kawasaki, S. Hase (Eds.)
Experimental Glycoscience
Glycochemistry
Naoyuki Taniguchi, M.D., Ph.D.
Professor, Department of Disease Glycomics, Research Institute for Microbial
Diseases, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan;
Systems Glycobiology Group, Advanced Science Institute, RIKEN, Wako,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Akemi Suzuki, M.D., Ph.D.
Professor, Institute of Glycotechnology, Future Science and Technology Joint Research
Center, Tokai University, Hiratsuka, Kanagawa 259-1292, Japan
Yukishige Ito, Ph.D.
Chief Researcher, RIKEN Discovery Research Institute, Synthetic Cellular Chemistry
Laboratory, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Hisashi Narimatsu, M.D., Ph.D.
Research Center for Medical Glycoscience, Advanced Industrial Science and
Technology, AIST Tsukuba, Central 2, Tsukuba, Ibaraki 305-8568, Japan
Toshisuke Kawasaki, Ph.D.
Professor, Research Center for Glycobiotechnology, Ritsumeikan University, 1-1-1
Noji-Higashi, Kusatsu, Shiga 525-8577, Japan
Sumihiro Hase, Ph.D.
Professor, Graduate School of Science, Osaka University, 1-1 Machikaneyama,
Toyonaka, Osaka 560-0043, Japan
ISBN: 978-4-431-77923-0 Tokyo Berlin Heidelberg New York
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Cover: A ribbon diagram of a legume lectin (Lathyrus ochrus isolectin II) complexed with a biantennary glycan
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Preface
There is growing interest in glycoscience, which is known to be one of the most important
research areas in the medical and life sciences. It is now clear that glycans are impli-
cated in various diseases such as cancer, immune diseases, infectious diseases, muscle-
degenerative diseases, neurodegenerative diseases, and diabetes. Glycans also play a
pivotal role in fertilization, growth and development, regeneration, and the aging process.
Moreover, glycans are likely targets for the discovery of biomarkers, vaccines, and drugs
for cancer and infl ammatory diseases.
For these reasons, scientists in every fi eld of research have realized that the application
of glycoscience contributes to the development of their research projects. They also have
a feeling, however, that glycoscience is technically diffi cult because of the heterogeneity
and complexity of glycans. It is evident that many researchers from different disciplines
who are not familiar with the specialized techniques used in glycoscience require detailed
advice.
Newly developed analytical techniques—namely, the development of mass spectrom-
etry, capillary electrophoresis, and high-performance liquid chromatography—and the
accumulated resources of cloned glycosyltransferase genes, lectins, chemically and/or
enzymatically synthesized compounds, bioinformatics, and KO mice or gene-targeting
animals have opened new avenues for glycoscience.
The Japan Consortium for Glycobiology and Glycotechnology (JCGG) published the
Japanese monograph entitled Glycoscience: A Door to Open the Future in 2006 written
by more than 150 glycoscientists to summarize recent advances in this fi eld in Japan and
to offer readers a broad introduction to glycoscience. This present book is the English
version but is largely modifi ed and updated. Many of the contributors are experts in their
respective fi elds and have made an effort to present their material in a manner that is
understandable to those with general knowledge but with a different background in the
biological sciences. The book is presented in three ways: 1) as a “cookbook” with which
researchers will be able to prepare the solutions described and follow “recipes” to com-
plete their experiments without needing to consult original research papers; 2) by
“concept,” which will allow researchers to search out the discipline, research equipment,
research device, or methodology required; and 3) as a “table” providing an overview of
the research data, among other information. For those who wish to delve into these topics,
we included several references at the end of each chapter for further reading. This book
will therefore be of interest to many scientists both in glycoscience and in the broader
fi elds of biology, chemistry, and medicine, as well as to postdoctoral fellows, students,
and young scientists.
I would like to take this opportunity to introduce the JCGG which was launched 5
years ago (Yoshitaka Nagai, JCGG president, professor emeritus, The University of
Tokyo). The organization of this consortium is slightly different from that of the Con-
sortium for Functional Glycomics (CFG) in the United States. Instead of receiving offi -
cial support from the Japanese government for its operation, scientists took the initiative
for creating and supporting the consortium by using their existing government research
grants. Scientists who have been funded by different ministries of the Japanese govern-
ment, such as the Ministry of Education, Culture, Sports, Science and Technology
V
VI Preface
(MEXT); the Japan Society for the Promotion of Science (JSPS); the Japan Science and
Technology Agency (JST); the Ministry of Economy, Trade and Industry (METI); the
New Energy and Industrial Technology Development Organization (NEDO); and the
Ministry of Health, Labour and Welfare, are involved in this effort. They have joined
together to form this consortium and to provide support through their individual research
grants.
The JCGG aims to facilitate the exchange of scientifi c information; the sharing of
resources, equipment, and facilities; the fostering of young scientists; and the construc-
tion of a database and infrastructures, among other goals. It also aims to launch national
research centers in Japan, such as a Systems Glycobiology Center, where glycobiology
can merge with such areas as nanotechnology, bioinformatics, and chemical biology to
pursue the goals mentioned above, including medical applications. The JCGG holds an
annual symposium in which more than 600 people usually participate.
On behalf of the editors, I gratefully acknowledge all those who contributed to this
volume despite their busy schedules. Special thanks are due to Mr. Keiichi Yoshida, the
secretary general of JCGG, for his skillful assistance in editing. I also thank Kinpodo,
Kyoto, who published the original Japanese version, for agreeing to publication of the
English version.
Thanks also go to the editorial staff members at Springer Japan, for their constant
support, patience regarding the deadline for the manuscript, and skill in directing the
production of this book.
I hope this publication will provide an impetus for future research.
On behalf of the editors,
Naoyuki Taniguchi, MD, PhD
February 10, 2008
Editorial Board
Editors:
Naoyuki Taniguchi, M.D., Ph.D.
Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka
University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan;
Systems Glycobiology Group, Advanced Science Institute, RIKEN, Wako, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
Akemi Suzuki, M.D., Ph.D.
Institute of Glycotechnology, Future Science and Technology Joint Research Center,
Tokai University, Hiratsuka, Kanagawa 259-1292, Japan
Yukishige Ito, Ph.D.
RIKEN Discovery Research Institute, Synthetic Cellular Chemistry Laboratory,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Hisashi Narimatsu, M.D., Ph.D.
Research Center for Medical Glycoscience, Advanced Industrial Science and Technol-
ogy, AIST Tsukuba, Central 2, Tsukuba, Ibaraki 305-8568, Japan
Toshisuke Kawasaki, Ph.D.
Research Center for Glycobiotechnology, Ritsumeikan University, 1-1-1 Noji-Higashi,
Kusatsu, Shiga 525-8577, Japan
Sumihiro Hase, Ph.D.
Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka
560-0043, Japan
Co-editors:
Koichi Furukawa, M.D., Ph.D., School of Medicine, Nagoya University, Japan
Makoto Kiso, Ph.D., Faculty of Applied Biological Sciences, Gifu University, Japan
Tomoya Ogawa, Ph.D., RIKEN Yokohama Institute, Japan
Shoichi Kusumoto, Ph.D., Suntory Institute For Bioorganic Research, Japan
VII
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
Editorial Board . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII
Contents of Experimental Glycoscince: Glycobiology. . . . . . . . . . . . . . . . . . . . . . XII
List of Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII
Part 1 Structural Analysis of Sugar Chains
Section I Release of Sugar Chains and Labeling
Chemical Liberation of N-linked Oligosaccharides from
Glycoproteins
S. Nakakita . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Release of N-glycans by Enzymatic Methods
N. Takahashi, H. Yagi, K. Kato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Release of O-glycans by Chemical Methods
S. Natsuka . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Release of O-glycans by Enzymatic Methods
H. Iwase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Ceramidase and Related Enzymes
M. Ito . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Labeling of Oligosaccharides
S. Nakakita . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Chemical Labeling of Sialyloligo/polymer
C. Sato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Separation of Oligosaccharides by 2D HPLC
S. Natsuka . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Analysis of Oligosaccharides by Capillary Electrophoresis
M. Kinoshita, K. Kakehi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Section II Sequence Analysis
Energy-Resolved Mass Spectrometry (ERMS) of Oligosaccharide
A. Kurimoto, S. Daikoku, O. Kanie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
LC/MS of N-Linked Oligosaccharides
N. Kawasaki, S. Itoh, T. Yamaguchi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
VIII
Contents IX
Convenient Structural Characterization of Intact Glycosphingolipids by
MALDI-TOF Mass Spectrometry with Increased Laser Power and
Cooling Gas Flow
M. Suzuki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Structural Analyses of Glycoconjugates by NMR
K. Kato, Y. Yamaguchi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Sugar Chain Analysis by Enzymatic Digestion and 2D Mapping
by HPLC
M. Maeda, Y. Kimura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Analysis of Binding Sites of Sugar Chains by Methylation Analysis
S. Hase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Glycan Profi ling
J. Hirabayashi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Monoclonal Antibody as a Clue to Structural Analysis of Bioactive
Functional Glycoconjugates
R. Kannagi, N. Kimura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Microsequencing of Functional Chondroitin Sulfate Oligosaccharides
K. Sugahara, S. Yamada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Structural Characterization of PA-oligosaccharide Isomers Derived from
Glycosphingolipids by MALDI-TOF Mass Spectrometry
M. Suzuki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Structural Analysis of Phospho-Glycosphingolipids in Lower Animals
S. Itonori, M. Sugita . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Structural Analysis of Polysialic Acid
C. Sato, K. Kitajima . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Milk Oligosaccharides: Structural Characterization and Future Aspects
T. Urashima . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Section III Sugar Chain Analysis by Mass Spectrometry
An XML Description of Carbohydrate Structures
N. Kikuchi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Construction of a Diagnostic Library for Glycans Using Multistage
Tandem Mass Spectrometry (MSn)
A. Kameyama . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
A Method for Large-Scale Analysis for N-linked Glycoproteins by the
Glycosylation Site-Specifi c Stable Isotope-Labeling and LC/MS
Shotgun Technology
H. Kaji, T. Isobe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Mass Spectrometry of Glycopeptides
Y. Wada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
X Contents
Development of MS Method for the Analysis of Sugar Peptide
K. Takahashi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Calculations for Saccharides by the Use of MS Data
K. Fukui . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Section IV Analysis of the Three-dimensional Structure of
Sugar Chains
Molecular Modeling of Oligosugar Structures
M. Ishiguro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Prediction of Sugar-Binding Sites on Proteins
T. Shirai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Section V Analysis of Sugar–Protein Interactions
Frontal Affi nity Chromatography: An Effective Analytical Tool for
Protein–Sugar Interaction
K. Kasai . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Analyses of Sugar–Protein Interactions by NMR
Y. Yamaguchi, K. Kato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Surface Plasmon Resonance and Sugar Chip Analysis for Sugar Chain–
Protein Interactions
Y. Suda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Analysis of Interactions Between Carbohydrates and Proteins Using
Capillary Affi nity Electrophoresis
M. Kinoshita, K. Kakehi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Equilibrium Dialysis
S. Natsuka . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Interaction Assay of Oligosaccharide with Protein Using Fluorescence
Polarization (FP) and Fluorescence Correlation Spectroscopy (FCS)
T. Inazu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Development of Neoglycoconjugate Probes and Detection of Lectins
H. Ogawa, K. Nakagawa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Part 2 Chemical Synthesis of Sugar Chains
Section VI Chemical Synthesis of Sugar Chains
Synthesis of Glycolipid and Its Application
H. Ishida, M. Kiso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Chemo-Enzymatic Synthesis of Glycoconjugates
T. Inazu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Contents XI
Construction of O-Linked Glycopeptide Library Using Human
Glycosyltransferases
H. Ito . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Alteration of N-Linked Oligosaccharides
Y. Kajihara . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Stationary Solid-Phase Reaction (SSPR) for Oligosaccharide Synthesis
T. Ako, O. Kanie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Sugar Chain Synthesis by the Use of Cell Functions
T. Sato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Glyco-Chemistry Cycle System Based on Glycosidases
S. Shoda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Endoglycosidases (Glycosaminoglycans)
I. Kakizaki, K. Takagaki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Oligosaccharide Synthesis Based on Combinatorial Chemistry and
Labo Automation
H. Tanaka, T. Takahashi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Endoglycosidases (Glycoproteins)
K. Takegawa, K. Yamamoto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Glycosaminoglycans
J. Tamura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Recent Advances in the Production of Mammalian-Type Sugar Chains
in Yeast
Y. Chiba, Y. Jigami . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Solid-Phase Synthesis of Glycopeptides
Y. Nakahara, H. Hojo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Effi cient Synthesis of Oligosaccharides and Synthesis of Pathogen-
Associated Molecular Patterns for Their Biofunctional Studies
K. Fukase, Y. Fujimoto, K. Tanaka . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Sugar Polymers (Dendrimers and Pendant-Type Linear Polymers)
K. Matsuoka . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Rapid Synthesis of Oligosaccharides: Resin Capture–Release Strategy
Y. Ito, S. Hanashima . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
