2019年9月３日
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TIAナノバイオサマースクール（糖鎖・レクチン）

糖鎖のシークエンス解析および立体構造解析

名古屋市立大学大学院薬学研究科

矢木 宏和

Contents
I. Introduction
・Chemical character

II. Sequence analysis
・Released glycan analysis
・Mass spectrometric analysis
・HPLC mapping method

III. Conformational analysis
・Digest for conformational analysis
・Our recent topics

Monosaccharide structureb-D-Glucose
FischerHaworthCommon monosaccharides found in vertebrates

D-Glucose (Glc)

N-acetyl D-Glucosamine(GlcNAc)

N-acetyl D-Galactosamine(GalNAc)

D-Mannonse(Man)

L-Fucose(Fuc)

D-Glucronic acid
(GlcA)

D-Galactose (Gal)D-Xylose (Xyl)N-acetylnuraminic acid
(NeuAc)Glycoside formation

グリコシド結合の形成

a-D-Glucose

Metyl a-D-Glucose

Reducing and nonreducing ends of a disaccharide

糖鎖の末端

Nonreducing
end
Reducing end

Two isomeric disaccharides

異性体b1-6 linkage

Gentibiose

a1-4 linkage

MaltoseIsomers

8

Essentials of Carbohydrate Chemistry and Biochemistry (2003) より引用

Dimers composed of two glucose resides
Glca1-a1Glc Glcb1- b1Glc Glca1- b1Glc
Glca1-2Glc Glcb1-2Glc
Glca1-3Glc Glcb1-3Glc
Glca1-4Glc Glcb1-4Glc
Glca1-6Glc Glcb1-6Glc

Glycans in Mammals
Asparagine (N)-linked glycans

Hyaluronic acid

Chondroitin sulfate

Heparin

EtNP

Mucin-typeO-glycans

Glycosphingolipids

GPI-anchor

Cell surface
Cytosol

Basic components of glycans
Glucose (Glc)

N-Acetylglucosamine (GlcNAc)

Glucuronic acid (GlcA)

Galactose (Gal)Glucosamine (GlcN)

Iduronic acid (IdoA)

Mannose (Man)N-Acetylgalactosamine (GalNAc)

Sialic acid (Sia)

Xylose (Xyl)SulfateSymbolic representations

http://www.functionalglycomics.org/static/c
onsortium/CFGnomenclature.pdf

Glycan function of therapeutic antibody and biologics“Naked” proteinGlycan function of therapeutic antibody and biologics
Cell-Cell Communication
Protein Quality Control
Protein Solubility & Stability

Glycan function of therapeutic antibody and biologics
Effecter functions
Serum half-life
Antigenicity

Heterogeneity
MobilityGlycoprotein glycans
・N-linked glycans（Asn）・O-linked glycans（Ser/Thr）

15

Examples of typical N- and O-linked glycans

N-linked glycan

O-linked glycan

16Classification of N-linked glycans
Mana2Mana6
Mana6
Mana2Mana3
Manb4GlcNAcb4GlcNAc

High-mannose型

Mana2Mana2Mana3

Galb4GlcNAcb2Mana6
Manb4GlcNAcb4GlcNAc

Complex型

Mana6
Mana2Mana3
Manb4GlcNAcb4GlcNAc

Hybrid型

Galb4GlcNAcb2Mana3
Mana2Mana6

Galb4GlcNAcb2Mana317

Classification of O-linked glycans
Structure

Core 1

Galb1-3GAlNac-

Core 2

GalNAcb1

6
Galb1-3GalNAc

Structure
GalNAcb1

Core 4

6
GalNAcb1-3GalNAc

Core 5

GalNAca1-3GalNAc

Core 6

GlcNAcb1-

Core 3 GalNAcb1-3GalNAc

Type

-

Type

6
GalNAc18

Sugar chains・Protein solubility and stability

Protein・ Structural integrity of proteinfunctional sites
Glycan・Cell-cell communication

・Highly branched structures
・Microheterogeneity
・Conformational fluctuations

Such structural complexity, diversity, andfluctuation hamper the structural biology-based
approaches for understanding the function of
glycoprotein as well as oligosaccharides.

Contents
I. Introduction
・Chemical character

II. Sequence analysis
・Released glycan analysis
・Mass spectrometric analysis
・HPLC mapping method

III. Conformational analysis
・Digest for conformational analysis
・Our recent topics

Scheme of N-glycan structural analyses
Protease treatment

Glycoprotein

Glycopeptides

・PNGase F treatment
・Glycoamidease A treatment
・Hydrazinolysis

LC-MS/MS
Site specific glycosylation

Free N-glycans

Permethylation

Reductive amination

Permethylated glycans

Labeled N-glycans
MS

MS/MS
Glycan structure

HPLC

Capillary Electrophoresis(CE)
Glycan structure・Composition analysis
・Linkage analysis
・NMR stricture determination

Comparison of analytical methods for N-glycans
HPLC
Detection

Fluorescence

Analysis time

CE
Fluorescence

MS

long

MS

MS

MSn

MS

rapid

rapid

middleSensitivity

◎

〇

◎

〇

〇

△

Discrimination ofisomeric product

◎

◎

〇

〇

×

△

Identification ofisomeric product

◎

△

△

△

×

〇

Index ofdetermination ofglycan structures

Elutionposition

Database or analytical
web application

・GALAXY
・Glycobase

Molecular Elution
mass
position
Glycostore

Molecular Molecular Fragment
mass
mass
ation
・GlycoMod
・jCGGDB・Glycan
Mass
Spectral
DataBaseN-glycan-releasing methods
Hydrozynolysis

peptide:N-glycanase
F (PNGase F)

glycoamidase A

Chemical reaction（hydrazine）

Enzyme reaction（recombinant protein）
optimal pH 7-8

Enzyme reaction（Extract of alamondseeds） optimal pH 4

Merit・Application for crude
sample (Cells andtissues)・Direct glycan-releasingfrom glycoproteins・Possible for releasing
to core a1,3
fucosylation

Demerit・Since N-acetyl and N・Uncleavable to core
glycoryl gropus are
a1,3 fucosylated
removed byoligosacchairdes
hydrazinolysis,
reacetylation isnessesary for sialylated
glycans(Undistinguishable formolecular species ofsialic acid )・Production of Byproducts

・Uncleavable to whole
glycoproteins(cleavable toglycopepetides)

O-glycan-releasing methodb-Elimination in common O-glycoside linkages with Ser or
Thr residues in alkaline conditions and a plausible
mechanism of subsequent peeling reaction.

Florescence labeling of oligosaccharides
ABA:2-Aminobenzoic acid
2-ABAD:2-Aminobenzamide
3-ABAD:3-Aminobenzamide
ABEE:Ethyl p-aminobenzoate
ABN:p-Aminobenzonitrile
ACP:2-Amino-6-cyanoethylpyridine
AMAC:2-Aminoacridone
AMC:7-Amino-4-methylcoumarin
ANTS:8-Aminonaphthalene-1,3,6-trisulfonic acid
ANDS:7-Aminonaphthalene-1,3-disulfonic acid
AP:2-Aminopyridine
APTS:8-Aminopyrene-1,3,6-trisulfonic acid

Reductive amination

吉年正宏, 鈴木茂生: 糖の高感度分析法 -Update-. Glycoword. GT-C03.
https://www.glycoforum.gr.jp/glycoword/glycotechnology/GT-C03upJ.html

Separation of oligosaccharides by HPLC
Separation modes

Anion exchange
columnNormal phasecolumn

Reverse phasecolumnSpecies・DEAE・mono Q・amide・amino・cellurose

・ODS
・C30

Principal

According tonegative charge
degree such as
number of sialic acidresidues and sulfate
groupsSeparation is carriedout using hydrogenbonds between theresin and sugar
chains.

Separation is carriedout usinghydrophobic
interaction between
the resin and sugar
chains.

DEAE column

Fluorescence intensity

Neutral

S1:mono-sialyl
S2:di-sialyl
S3:tri-sialy
S4:tetra-sialyl

S2

5.0

10.0

15.0

Elution time (min)

S4

20.0

Glucose oligomer

G4
G5
G6

G10
G8
G9

0.0

5.0

10.0

G20
G12
G16
G11 G13 G14
G18

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

Elution time (min)Amide column

0.0

Glucose oligomer

G4

Fluorescence intensity

S1

S3
0.0

G7
Fluorescence intensity

Examination of
glycosylation profiles

ODS column

G8

G5
G6

5.0

10.0

G9

G10

G7

15.0

Elution time (min)

20.0

25.0

G11

Identification of glycan structures by HPLC
・Coinjection with standard glycans
・Evaluation by mass spectrometric data

Comparison with elution accumulated data

Consistence between standard and sample

GALAXY(http://www.glycoanalysis.info/)
Over 500 data of PA-N-oligosaccharides

Glycobase(http://glycobase.nibrt.ie/glycobas Inconsistence between standard and sample
e/show_nibrt.action)
Over 675 data of AB-oligosaccharides(containing O-glycans)Incase of unknow oligosaccharide which is not registered in database↓
Estimation/identification by the enzyme treatment
Before neuraminidase

S

Two Neu5Ac residues werereleased by neuraminidase
treatment

After neuraminidase

S

5.0

10.0

15.0

20.0

25.0

30.0

35.0

Composition and linkage
analyses

The CCRC Spectral Database forPartially Methylated Alditol Acetatehttps://www.ccrc.uga.edu/specdb/ms/pmaa/pframe.html

Ferdosi S, Ho TH, Castle EP, Stanton ML, Borges CR (2018)
Behavior of blood plasma glycan features in bladder cancer.
PLoS ONE 13(7): e0201208.
https://doi.org/10.1371/journal.pone.0201208

Structural identification by NMR
H-13C HSQC spectrum of the VPS-PS with 1H NMR trace.

Yildiz F, Fong J, Sadovskaya I, Grard T, Vinogradov E (2014) Structural Characterization of the Extracellular Polysaccharide from Vibrio
cholerae O1 El-Tor. PLoS ONE 9(1): e86751. https://doi.org/10.1371/journal.pone.0086751

Mass spectrometric analysis
The following figure illustrates the general nomenclaturescheme for glycan fragments.

2,5X

Z2

2

Z1

Y2

Y1

C1
2,5A

1

B1

1,5X

C2
B2

1,5A

3

0

MALDI-TOF MS spectrum of N-glycans enzymaticallyreleased from the biosimilar of cetuximab and cetuximaba) native N-glycans
before mild alkali
treatment (pH 10
ammonium hydroxide);b) native N-glycans of
the biosimilar after mild
alkali treatment; c)native N-glycans fromthe cetuximab. The
cartoons of possible
structures of glycans
were adapted from
Glycoworkbench andstructure is depicted
following the CFG
notation.

Liu S, Gao W, Wang Y, He Z, Feng X, Liu B-F, et al. (2017) Comprehensive N-Glycan Profiling of Cetuximab Biosimilar
Candidate by NP-HPLC and MALDI-MS. PLoS ONE 12(1): e0170013. https://doi.org/10.1371/journal.pone.0170013

NanoLC-ESI-MS/MS spectrum of native glycans

MS/MS spectra of m/z 2060 with chemical composition of GlcNAc4Man3Gal2NeuAcLac1; b) MS/MS
spectra of m/z 2078 with chemical composition of GlcNAc4Man3Gal2NeuAc1.

Liu S, Gao W, Wang Y, He Z, Feng X, Liu B-F, et al. (2017) Comprehensive N-Glycan Profiling of Cetuximab Biosimilar
Candidate by NP-HPLC and MALDI-MS. PLoS ONE 12(1): e0170013. https://doi.org/10.1371/journal.pone.0170013

MS profiling of site-specific glycoforms of the serum sFcγRIIIb,

H. Yagi et al. Sci. rep. ,9: 2719, 2018

Molecular model of sFcγRIIIb with N-glycans
on the basis of our LC-MS/MS data.

Complex typeglycans

H. Yagi et al. Sci. rep. ,9: 2719, 2018

Native mass analysis

MS can be used to measure the stoichiometry and compositionof protein complexes, the presence of small molecules(a) Schematic of the rhEPO
backbone sequence and its
reported PTM sites. (b) The zerocharge deconvoluted native MS
spectrum of rhEPO.

Yang, Y., Liu, F., Franc, V. et al. Hybrid mass spectrometry approaches in glycoprotein analysis and theirusage in scoring biosimilarity. Nat Commun 7, 13397 (2016). https://doi.org/10.1038/ncomms13397

Detail information of N-glycans
structural analysis by using HPLC
mapping method

The multi-dimensional HPLC mapping technique
Glycoamidase A (E.C. 3.5.1.52)

2-aminopyridine

DEAE column
ODS column

Amide column
Dr. Noriko Takahashi
Prog. Nucl. Magn. Reson. Spectrosc. 56, 346-359 (2010)

DEAE column

The elution position of each peak is expressed in glucose units (gu).
The elution positions of peaks in an unknown glycan pool are assigned an
overall gu value by comparison with the standard a1-6glucose oligomers.

N-glycosylation profiles on ODS cplumn
7

Fluorescence intensity

45

a1-6Glucose oligomer
Sample
12.8

6

10
8
20
15 16

9
11

0.0

5.0

10.0

15.0

20.0

25.0

12

13

30.0

Elution time (min)

14

35.0

17

40.0

45.0

18

19

50.0

55.0

21

HPLC peak areas of PA-glycans can show a linearity plot from
0.1 to 100 pmol (in a quantitative manner)GN
M
GN
M-GN-GN-PA
GNM
GN

G-GN-M

arbitrary area unit

arbitrary area unit

G-GN-M

M-GN-GN-PA

y = 81857x + 31562
R² = 0.9963

0

50

100

PA-oligosaccharide (pmol)

150

y = 86055x - 39159
R² = 0.9994

0

50

100

PA-oligosaccharide (pmol)

150

HPLC-based discrimination of glycol-isomers
GN
G-GN
G-GN
G-GN
M
F
M
F
M
F
M
F
G-GN
G-GN
GN
G-GN
M-GN-GNM-GN-GNM-GN-GNM-GN-GNG-GN
GN
G-GN
G-GN
M
M
M
M
G-GN
G-GN
G-GN
GN
410.12410.13410.14410.15

ODS : 14.1

ODS : 13.8

ODS : 13.7

ODS : 12.5

Amide : 9.5

Amide : 9.3

Amide : 9.2

Amide : 8.9

Distinguish a2-6 from a2-3!
M

M
M-GN-GN-

NeuAcα2-6G-GN

M

M-GN-GNNeuAcα2-3G-GN

1A1-100.4

M
1A2-100.4

ODS :7.8

ODS : 9.1

Amide : 6.0

Amide : 5.4

Expression of a2-6 sialylated N-glycans in avian intestines
Mono-sialyl

chickenDi-sialylchicken a2-3
a2-6

quail

quail

Elution time (min)Elution time (min)A principal of HPLC mapping method
Glycoamidase A (E.C. 3.5.1.52)

Sugar Library

2-aminopyridine

Anion-exchange column

ODS column
Amide column

The HPLC mapping method
enable us to collect thestandard oligosaccharides
according to HPLC separation.

Lectin＝Glycan binding protein

Multiple structures

Systematic analysis of sugar chain-protein interactions
by frontal affinity chromatography (FAC) method

Immobilized
lectinLigandsolution

平林淳: フロンタル分析を利用する糖-タンパク
質相互作用解析. Glycoword. GT-C07.
https://www.glycoforum.gr.jp/glycoword/gl
ycotechnology/GT-C07J.html

Library of PA-glycans

レクチンアフィニティカラムからのPA化糖鎖の溶出プロファイル

図.3
FACの解析例：C. elegansガレクチン
LEC-6を固定化したカラム（7.44
mg/mlゲル）に糖脂質由来のオリゴ糖溶液（ピリジルアミノ化体、10 nM）
6種を2 mlのサンプルループを介して0.25 ml/minの流速で注入する。V0はラムノースの溶出位置として求めている。各オリゴ糖に対するKd値はp-ア
ミノフェニルラクトシドの濃度変化解析からBtを求め、FACの基本式（1）から算出している。

平林淳: フロンタル分析を利用する糖-タンパク
質相互作用解析. Glycoword. GT-C07.
https://www.glycoforum.gr.jp/glycoword/gl
ycotechnology/GT-C07J.html

GALAXY
database

http://www.glycoanalysis.info/

Trends Glycosci. Glycotech. 15, 235-251 (2003)

Information page for the individual N-glycans

The GlycoTree diagram

52

53

Galβ1,4-0.05

Galβ1,40.39

57

Galα1,4-0.39

31

NeuAcα2,6
-0.51

32

16

Manα1,2-1.00

8Manα1,6-0.19

NeuAcα2,30.38

17Galβ1,40.24

9GlcNβ1,6-2.21

33

GlcNβ1,3
1.52

18

GalNβ1,4
0.33

58

Galα1,41.25

56

Galβ1,4-1.07

10

GlcNβ1,4
1.21

NeuGcα2,6
-0.90

19

Manα1,2
0.86

11

34

Manα1,3-0.67

35

NeuAcα2,61.63

20

Fucα1,6-0.20

36

NeuAcα2,30.92

21

Galβ1,40.13

12GlcNβ1,2
1.75

37

NeuGcα2,31.1022GalNβ1,4
1.04

38

Galα1,32.74

39

GlcNβ1,3
0.88

59Galα1,4-0.29

4Manα1,6
0.72

55
40
41

54

Galβ1,4
1.24

NeuAcα2,6
2.46
NeuAcα2,30.31

5GlcNβ1,44.44

6Xylβ1,2
0.09

7Manα1,3-0.53

NeuAcα2,6
23
0.49

42

Galα1,32.01

24

Galβ1,40.5460

Galα1,4-0.58

25Fucα1,3-0.15

43

NeuAcα2,31.89

26

Galβ1,30.67

44Galα1,3
2.79

45

NeuGcα2,6
-3.06

46

NeuAcα2,6
-1.88

27

Fucα1,6-0.75

47

NeuAcα2,30.31

28

Galβ1,4
1.33

48

Galα1,3-2.62

29

GalNβ1,40.98

49

GlcNβ1,31.30

50Fucα1,2
8.87

61

Galα1,4-2.00

51

Glcα1,30.80

30Manα1,2-0.40

13GlcNβ1,42.03

14GlcNβ1,2
0.06

15Manα1,2-0.48

1

Galβ1,4
0.61
Manβ1,42

Fucα1,6
3.43

GlcNβ1,4
7.20
3

GlcN-PAFucα1,3
-1.96

52

53

Galβ1,4-0.05

Galβ1,40.39

57

Galα1,4-0.39

31

NeuAcα2,6
-0.51

32

16

Manα1,2-1.00

8Manα1,6-0.19

NeuAcα2,30.38

17Galβ1,40.24

9GlcNβ1,6-2.21

33

GlcNβ1,3
1.52

GalNβ1,4
18
0.33

58

Galα1,41.25

56

Galβ1,4-1.07

19

Manα1,20.86

NeuAcα2,61.63

20

Fucα1,6-0.20

36

NeuAcα2,30.92

21

Galβ1,40.13

37

NeuGcα2,31.1022GalNβ1,4
1.04

38

Galα1,32.74

39

GlcNβ1,3
0.88

59Galα1,4-0.29

34

NeuGcα2,6
-0.90

35

α- Galactosidase
β- Galactosidase
β- HexNAcase
α- Sialidase

10

GlcNβ1,4
1.21

11Manα1,3-0.67

12GlcNβ1,2
1.75

4Manα1,6
0.72

55
40
41

54

Galβ1,4
1.24

NeuAcα2,6
2.46
NeuAcα2,30.31

5GlcNβ1,44.44

6Xylβ1,2
0.09

7Manα1,3-0.53

NeuAcα2,6
23
0.49

42

Galα1,32.01

24

Galβ1,40.5460

Galα1,4-0.58

25Fucα1,3-0.15

43

NeuAcα2,31.89

26

Galβ1,30.67

44Galα1,3
2.79

45

NeuGcα2,6
-3.06

46

NeuAcα2,6
-1.88

27

Fucα1,6-0.75

47

NeuAcα2,30.31

28

Galβ1,4
1.33

48

Galα1,3-2.62

29

GalNβ1,40.98

49

GlcNβ1,31.30

50Fucα1,2
8.87

61

Galα1,4-2.00

51

Glcα1,30.80

13GlcNβ1,42.03

14GlcNβ1,2
0.06

1Galβ1,4
0.61
Manβ1,4α- Fucsidase
β- Xylosidase

30

Manα1,2-0.40

15Manα1,2-0.48

2Fucα1,6
3.43

GlcNβ1,4
7.20
3

GlcN-PAFucα1,3-1.96

Display of products resulting from glycosidase treatments

Prediction of digestion precursors of a selected N-glycan

Prediction of digestion precursors of a selected N-glycan

MW

MW

Amide

Graph selection from the three types of combination of the axes

ODS

ODS

ODS-Amide

ODS-MW

Amide

Amide-MW

Trends Glycosci. Glycotech. 15, 235-251 (2003)

MW 2302 ?

Expanded HPLC map including sulfated oligosaccharides
13

Amide (G.U.)

11

9

7

5
Sulfated Asialo ---------40
Sulfated Sialo -----------23
Glucuronylated --------49
HNK-1--------- 5

3
5

10

15
ODS(G.U.)

20

25

Trends in Glycoscience and Glycotechnology, 21, pp95-104, 2009

Fluorescence intensity

N-glycosylation profiles derived from two different influenza A virusesgrown in MDCK cells and embryonated eggs

Elution time (min)Open Glycoscience, 5, pp2-12, 2012

Contents
I. Introduction
・Chemical character

II. Sequence analysis
・Released glycan analysis
・Mass spectrometric analysis
・HPLC mapping method

III. Conformational analysis
・Digest for conformational analysis
・Our recent topics

Conformation analysis
C6’
C1’
C3’

Φ
ψ C2
C1

Conformations of saccharide linkages- information available
X-ray crystallography –Most oligosaccharides and glycoproteins either do not crystallize orgive no resolvable electron density for the glycan. Glycans that canbe seen are incomplete.
→ average properties of linkages
Nuclear Magnetic Resonance Spectroscopy –Experimental structural parameters (inter-nuclear distances andtorsion angles) averaged on a msec timescale.
→ a single well-defined conformation as an average structure.
Molecular Dynamics Simulations –Theoretical dynamic structures on a nsec timescale.
→ a conformational amassable of the structure if it is assumed thatthe theory is correct.

Crystal structures of IgG1-Fc/FcgRIII complex
Glycosylated FcgRIII
Non-fucosylated Fc
D1

FcgRIII

D2

Complex type

FcgRIII

Asn162

CH 2
CH 2

Fc

CH 3

CH 3

Fc

Mizushima et al. Genes Cells. 2011 Nov;16(11):1071-80. doi: 10.1111/j.1365-2443.2011.01552.x.

Statistics of N-linked glycoproteins from PDB(94,336 structures, 2013.10.02)

Non-glycosylated protein (NMR)
80,791 (10.85%)
Glycoprotein (X-ray)
3,290 (3.49%)

N-glycan structures

Glycoprotein (NMR)
19 (0.02%)
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0

8680

X-ray
3190
1416
512 445259 177 88 63 38 2

8

0

0

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Size (14880 sites)

Non-glycosylated protein (X-ray)
80,791 (85.64%)

N-glycan structures

25
20

20

NMR

15
10
5
0

2

3
1

0

0

0

1

2
0

0

0

0

0

0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Size (29 sites)

Nuclear Magnetic Resonance Spectroscopy
J coupling :Dihedral angles

Nuclear Overhauser effect (NOE) < 5 Å

Pseudocontact Shift (PCS) < 40 Å

χz
χx

H θ
r
φ

χy

MD simulation
Multiscale modeling of glycosaminoglycans from
disaccharide to polysaccharide is necessitated bytheir size and heterogeneity! = # )$(+ − +-)/ + # )1(4 − 4-)/ +$%&'(1&2+3(#
5%6(7%&(

8&
9 + :%((&∅ − ∅-)/Harmonic oscillator-like bonding, angular, torsional terms>?9>+ # # A7, <<=9 7=<@9>?9+C-7< 9/C-7< D−/C7<
C7<

van der Waals

>

E7E<
# #
FGA%C7<

electrostatic<=9 7=<@9

>?9

+

>

H7<
I7<
# #
−
hydrogen bonding
C7<9/ C7<9<=9 7=<@9Paramagnetic NMR-Validated Molecular Dynamics Simulation
Experiment

Computational Calculation

Nuclear Magnetic Resonance (NMR)

Molecular Dynamics Simulation (MD)Results depending on
force field,
initial state andsimulation time

Information averaged
over dynamic
conformational ensemble

3D Structural Information

3D Structural Model
CompareDisagree

Agree

Validated conformational ensemble
The combination between NMR and MD data enable us to obtain validated conformational
ensemble.

Conformational dynamics of GM9 dodecamer
GM9 conformational ensemble basedon NMR-validated MD simulation

Conformational dynamics of trisaccharide on GM9

ManC

Glc
Ψ

Man4

Φ
Glc-ManD1-ManC-Man4

ManD1
Φ : O5-C1-O1-CʹX
Ψ : C1-O1-CʹX-CʹX-1

Density maps of glycosidic linkage torsion angles
180

Glc-ManD1

ManD1-ManC

ManC-Man4
High density

Ψ / degree

90
0

-90
Low density
-180
-180

-90

0
90
Φ / degree

-90

0
90
Φ / degree

-90

0
90
Φ / degree

180

Suzuki et. al. Chembiochem . 2017 Feb 16;18(4):396-401. doi: 10.1002/cbic.201600595

The carbohydrate recognition by the ER chaperone
calreticulin involves an induced-fit mechanism

3D-structural models of the sugar-binding
mode of calreticulin

Conformational dynamics of trisaccharide on GM9

ManC

Glc
Ψ

Man4

Φ
Glc-ManD1-ManC-Man4

ManD1
Φ : O5-C1-O1-CʹX
Ψ : C1-O1-CʹX-CʹX-1

Kozlov, G.; et al, J. Biol. Chem. 2010, 285, 38612-38620

Density maps of glycosidic linkage torsion angles
180

Glc-ManD1

ManD1-ManC

Calreticulinbinding state90
Ψ / degree

ManC-Man4
High density0-90
Low density
-180
-180

-90

0
90
Φ / degree

-90

0
90
Φ / degree

-90

0
90
Φ / degree

180

Suzuki et. al. Chembiochem . 2017 Feb 16;18(4):396-401. doi: 10.1002/cbic.201600595

Take home message!
糖鎖の構造解析をした際に、どこまでの詳細構造をキャラクラライズしているかのかを理解しておくことが重要！
Hex5NexNAc4Sia2
Gal2Man3GlcNAc4Neu5Ac2

Acknowledgement

Kato’s lab members

NIAS
M. Nakamura

AIST
Georgia Regent Univ.
R.K. Yu

Kyowa Hakko Kirin
K. Shitara
M. Satoh
S. IIdaN. Fukuzawa
T. Matsumura
H. Tateno

Kyoto Univ.
S. OkaN. Nakagawa

Academia Sinica
K.H. Khoo
C.W. Kuo

NIHS
N. Hashii
S. Nakazawa

Yokoyama City Univ.
N. Kawasaki

Shizuoka Univ.
E.Y. Park
T. KatoTaiyo Nippon Sanso