November 29
PROGRAM

15

Glycoscience : diversity and integration

Conformational dynamics andinteractions of glycoconjugates oftherapeutic interest
Koichi Kato, Ph.D.
Professor, Okazaki Institute for Integrative
Bioscience & Institute for Molecular Science
National Institutes of Natural Sciences, JAPANKoichi Kato
ProfileKoichi Kato received his Ph.D. in 1991 at
Graduate School of Pharmaceutical Sciences, the University of Tokyo (Prof. YojiArata), and continued his research as anAssistant Professor and then as a Lecturer
in the same group. In 2000, he received theAward for Young Scientists of the Pharmaceutical Society of Japan and moved as aProfessor to Nagoya City University. Since
2005, he has been a Director of GLYENCE,a company to exploit glycotechnology ofhis group. Since 2006, he has also been aVisiting Professor at the Glycoscience Institute, Ochanomizu University. Since 2008,
He has been a Professor at Okazaki Institute for Integrative Bioscience, National
Institutes of Natural Sciences, holding theProfessorship concurrently at Nagoya CityUniversity. In 2011, he received the Award
for Divisional Scientific Promotions ofthe Pharmaceutical Society of Japan andthe 48th Baelz Prize. His research interests
include development and application ofstructural glycomics methods for characterizing conformations, dynamics, andinteractions of glycoconjugates.

The sugar chains covalently modifying
proteins and lipids are involved in a variety
of molecular recognition events, thereby
mediating a broad range of physiologicaland pathological processes on cell surfaces
as well as in cells. Therefore, these carbohydrate-protein interaction systems could
be potential therapeutic targets for various
diseases, including viral infections, autoimmune diseases and neurodegenerative
disorders1). In addition, protein glycosylation is currently considered to be one of
the most important factors in the designand development of biopharmaceuticals
typified by antibody medicines because
the carbohydrate moieties affect the physical and biological properties of proteins,
e.g. solubility, thermostability, serum halflife, and functional protein-protein interactions.
To facilitate the design of drugs targetingcarbohydrate recognition systems, deeper
insights into the structural basis of carbohydrate-protein interactions are essential.
We have developed a systematic method
for characterizing structures, dynamics,
and interactions of glycoconjugates atatomic level 2) 3). Our methodology is constituted by 1) GALAXY-based glycosylation profiling, 2) comprehensive analysisof lectin-sugar interactions using a sugar
library, and 3) structural analyses by stable-isotope-assisted NMR spectroscopy inconjunction with X-ray crystallography. In
this presentation, I will illustrate several
examples of our structural studies of the
carbohydrate-protein interaction systems
as potential therapeutic targets (Figure 1).
1. N-glycan-dependent determinationof glycoprotein fates in cells
N-linked oligosaccharides operate as tagsfor protein quality control, consigning glycoproteins to different fates, i.e. folding in
the endoplasmic reticulum (ER), vesiculartransport between the ER and the Golgi
complex, and ER-associated degradation48

the 20th Anniversary of the Mizutani Foundation for Glycoscienceof glycoproteins, by interacting with apanel of intracellular lectins in the earlysecretory pathway 4). Our frontal affinity
chromatography data demonstrated thatthe intracellular lectins exhibit distinct
sugar-binding specificity profiles 5) 6). The
glycotopes recognized by these lectins
as fate determinants are embedded in
the triantennary structures of the highmannose-type oligosaccharides and areexposed upon trimming of the outer glucose and mannose residues during theN-glycan processing pathway. Furthermore, structural basis has been provided
for the functional interplay between theL-type lectin ERGIC-53 and the EF-hand
Ca2+-binding protein MCFD2 in the intracellular transport of the coagulation factors V and VIII 7).
2. N-glycan-dependent effector functionsof immunoglobulin G
The effector functions of immunoglobulin G (IgG) critically depend on N-glycosylation of its Fc region. It is well known
that removal of the fucose residue fromthe N-glycans of the Fc portion of IgG
results in a dramatic enhancement ofantibody-dependent cellular cytotoxicity(ADCC) through improved affinity forFcγ receptor IIIa (FcγRIIIa). Recently, we
determined the crystal structure of thecomplex formed between non-fucosylated
IgG1-Fc and a soluble form of FcγRIIIa(sFcγRIIIa) having two N-glycosylationsites 8). The crystal structure demonstrates
that one of the two N-glycans of sFcγRIIIa
mediates the interaction with the N-glycanof non-fucosylated Fc, thereby stabilizing
the complex. However, fucosylation of
the Fc N-glycans impairs this interaction
because of steric hindrance. On the otherhand, our NMR data demonstrated thatTyr296 of the non-fucosylated Fc glycoform exhibits conformational multiplicity in its uncomplexed state, suggestingthat conformational selection is governedby the presence or absence of the fucose

residue of the Fc N-glycan. Fucose
depletion increases the incidenceof the active conformation ofthis tyrosine residue, and thereby
accelerates the formation of thehigh-affinity complex. These findings offer a structural basis for
improvement in ADCC of therapeutic antibodies by defucosylation.
3. Ganglioside clusters as platforms for pathological molecular events
Gangliosides are known to play avariety of physiological and pathological roles at the cell surface.
For instance, GM1 gangliosideacts as a target of cholera toxin,polyoma virus, growth-regulatory
galectin-1 and autoantibodies
associated with Guillain-Barré
syndrome. Recently, growing
evidence has indicated that gangliosides interact with amyloid β(Aβ) and promote its assembly,
which is considered to be a cruFigure 1. Structural basis for the molecular recognition events involving the glycoconjugate of therapeucial step in Alzheime’s disease. Totic interest.
better understand the underlying(A) Functional coordination between ERGIC-53 and MCFD forming a cargo receptor complex thatmechanisms of this pathologicaltransports coagulation factors V and VIII.
molecular event, it is highly desir(B) Interaction between the non-fucosylated glycoform of human IgG1-Fc and bis-glycosylated sFcγRIIIa.
(C) Aβ(1-40) accommodated on the hydrophilic/hydrophobic interface of GM1 cluster.
able to obtain detailed structural
information of these interaction
systems. Our NMR data indicatethat the ganglioside clusters provide aintermolecular interactions leading to between the GD1a and GD1b gangliosides,
unique platform at their hydrophobic/
providing clues to the structural basis of
the formation of their pathogenic aggrehydrophilic interface for binding coupledgates 9)10). In addition, by NMR approach epitope recognition by the autoantibodies
with conformational transition of Aβ
using mixed micelle system, we success- in Guillain-Barré syndrome.
molecules, rendering their spatial rearfully identified the atomic contact of therangements restricted to promote specificcarbohydrate-carbohydrate interaction
References1) 	Kamiya Y, Yagi-Utsumi M, Yagi H, Kato K. Structural and molecular basisof carbohydrate–protein interaction systems as potential therapeutic targets.
Curr. Pharm. Des. (17): 1672-1684, 2011.
2) 	Kato K, Sasakawa H, Kamiya Y, Utsumi M, Nakano M, Takahashi N, Yamaguchi Y. 920 MHz ultra-high field NMR approaches to structural glycobiology. Biochim. Biophys. Acta-General Subjects (1780): 619-625, 2008.
3) 	Kato K, Yamaguchi Y, Arata Y. Stable-isotope-assisted NMR approaches toglycoproteins using immunoglobulin G as a model system. Prog. Nucl. Magn.
Reson. Spectrosc. (56): 346-359, 2010.
4) 	Kato K, Kamiya Y. Structural views of glycoprotein-fate determination incells. Glycobiology (17): 1031-1044, 2007.
5) 	Kamiya Y, Kamiya D, Yamamoto K, Nyfeler B, Hauri H-P, Kato K. Molecularbasis of sugar recognition by the human L-type lectins ERGIC-53, VIPL andVIP36. J. Biol. Chem. (283): 1857-1861, 2008.
6) 	Quan EM, Kamiya Y, Kamiya D, Denic V, Weibezahn J, Kato K, Weissman JS.
Defining the glycan destruction signal for endoplasmic reticulum-associateddegradation. Mol. Cell (32): 870-877, 2008.

7) 	Nishio M, Kamiya Y, Mizushima T, Wakatsuki S, Sasakawa H, Yamamoto K,Uchiyama S, Noda M, McKay AR, Fukui K, Hauri H-P, Kato K. Structural
basis for the cooperative interplay between the two causative gene productsof combined factor V and factor VIII deficiency. Proc. Natl. Acad. Sci. USA(107): 4034-4039, 2010.
8) 	Mizushima T, Yagi H, Takemoto E, Shibata-Koyama M, Isoda Y, Iida S, Masuda K, Satoh M, Kato K. Structural basis for improved efficacy of therapeutic
antibodies on defucosylation of their Fc glycans. Genes Cells (16): 1071-1080,
2011.
9) 	Utsumi M, Yamaguchi Y, Sasakawa H, Yamamoto N, Yanagisawa K, Kato K.
Up-and-down topological mode of amyloid β-peptide lying on hydrophilic/hydrophobic interface of ganglioside clusters. Glycoconjugate J. (26): 9991006, 200910) 	Yagi-Utsumi M, Matsuo K, Yanagisawa K, Gekko K, Kato K. Spectroscopic
characterization of intermolecular interaction of Aβ molecules promoted on
GM1 micelles. Int. J. Alzheimer’s Dis. (2011): e925073, 2011.

Commemorative Symposium

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