糖鎖のシークエンス解析および立体構造解析

TIAナノバイオサマースクール（糖鎖・レクチン）

2019年9月３日お茶の水女子大 国際交流留学生プラザ多目的ホール

名古屋市立大学大学院薬学研究科

矢木 宏和

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

Contents

Monosaccharide structure

Fischer Haworth

b-D-Glucose

Common monosaccharides found in vertebrates

D-Glucose (Glc) N-acetyl D-Glucosamine(GlcNAc)

D-Galactose (Gal)

N-acetyl D-Galactosamine(GalNAc)

D-Mannonse(Man)

D-Xylose (Xyl)

L-Fucose(Fuc)

D-Glucronic acid(GlcA)

N-acetylnuraminic acid(NeuAc)

グリコシド結合の形成

Glycoside formation

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

a1-4 linkageb1-6 linkage

Gentibiose Maltose

Glca1-a1Glc Glcb1- b1Glc Glca1- b1GlcGlca1-2Glc Glcb1-2GlcGlca1-3Glc Glcb1-3GlcGlca1-4Glc Glcb1-4GlcGlca1-6Glc Glcb1-6Glc

Dimers composed of two glucose resides

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

8Isomers

Glycans in MammalsAsparagine (N)-linked glycans

Mucin-typeO-glycans

Chondroitin sulfate

GPI-anchor

Glycosphingolipids

Hyaluronic acid

Cell surface

Cytosol

Glucose (Glc)Galactose (Gal)Mannose (Man)

N-Acetylglucosamine (GlcNAc)Glucosamine (GlcN)N-Acetylgalactosamine (GalNAc)

Glucuronic acid (GlcA)

Sialic acid (Sia)Xylose (Xyl)

Iduronic acid (IdoA)

Sulfate

Heparin

Basic components of glycans

EtNP

Symbolic representations

http://www.functionalglycomics.org/static/consortium/CFGnomenclature.pdf

“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

Glycan function of therapeutic antibody and biologics

Heterogeneity Mobility

Glycoprotein glycans

・N-linked glycans（Asn）

・O-linked glycans（Ser/Thr）

15

Examples of typical N- and O-linked glycans

16

N-linked glycan O-linked glycan

Classification of N-linked glycans

High-mannose型

Complex型

Hybrid型

Manb4GlcNAcb4GlcNAc

Mana2Mana2Mana3

Mana6

Mana2Mana6

Mana2Mana3

Manb4GlcNAcb4GlcNAc

Galb4GlcNAcb2Mana3

Mana6

Mana2Mana6

Mana2Mana3

Manb4GlcNAcb4GlcNAc

Galb4GlcNAcb2Mana3

Galb4GlcNAcb2Mana6

17

Type

Core 1

Core 2

Core 3

Galb1-3GAlNac

Galb1-3GalNAc

GalNAcb1 -

6

GalNAcb1-3GalNAc

Core 4

Core 5

Core 6

GalNAcb1-3GalNAc

GalNAcb1 -

6

GalNAca1-3GalNAc

GalNAc

GlcNAcb1 -

6

Structure Type Structure

18

Classification of O-linked glycans

・Highly branched structures

・Microheterogeneity

・Conformational fluctuations

Sugar chains

Protein

Glycan

・Protein solubility and stability

・ Structural integrity of protein functional sites

・Cell-cell communication

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

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

Contents

Glycoprotein Glycopeptides

Free N-glycans

Labeled N-glycans

HPLC Capillary Electrophoresis(CE)

・PNGase F treatment・Glycoamidease A treatment・Hydrazinolysis

Reductive amination

Permethylation

Permethylated glycans

MS MS/MS

Protease treatmentLC-MS/MS

Site specific glycosylation

Glycan structure

Glycan structure

Scheme of N-glycan structural analyses

・Composition analysis・Linkage analysis・NMR stricture determination

HPLC CE MSDetection Fluorescence MS Fluorescence MS MS MSn

Analysis time long rapid rapid middle

Sensitivity ◎ 〇 ◎ 〇 〇 △

Discrimination of isomeric product

◎ ◎ 〇 〇 × △

Identification of isomeric product

◎ △ △ △ × 〇

Index of determination of glycan structures

Elution position

Molecular mass

Elution position

Molecular mass

Molecular mass

Fragmentation

Database or analytical web application

・GALAXY・Glycobase

Glycostore ・GlycoMod・jCGGDB

・Glycan Mass Spectral DataBase

Comparison of analytical methods for N-glycans

N-glycan-releasing methodsHydrozynolysis 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 and tissues)

・Direct glycan-releasing from glycoproteins

・Possible for releasing to core a1,3 fucosylation

Demerit ・Since N-acetyl and N-glycoryl gropus are removed by hydrazinolysis, reacetylation is nessesary for sialylatedglycans (Undistinguishable for molecular species of sialic acid )・Production of Byproducts

・Uncleavable to core a1,3 fucosylatedoligosacchairdes

・Uncleavable to whole glycoproteins (cleavable to glycopepetides)

O-glycan-releasing method

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

ABA:2-Aminobenzoic acid2-ABAD:2-Aminobenzamide3-ABAD:3-AminobenzamideABEE:Ethyl p-aminobenzoateABN:p-AminobenzonitrileACP:2-Amino-6-cyanoethylpyridineAMAC:2-AminoacridoneAMC:7-Amino-4-methylcoumarinANTS:8-Aminonaphthalene-1,3,6-trisulfonic acidANDS:7-Aminonaphthalene-1,3-disulfonic acidAP:2-AminopyridineAPTS:8-Aminopyrene-1,3,6-trisulfonic acid

Florescence labeling of oligosaccharides

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

Reductive amination

Separation of oligosaccharides by HPLCSeparation modes Anion exchange

columnNormal phase column

Reverse phase column

Species ・DEAE・mono Q

・amide・amino・cellurose

・ODS・C30

Principal According to negative charge degree such as number of sialic acid residues and sulfate groups

Separation is carried out using hydrogen bonds between the resin and sugar chains.

Separation is carried out using hydrophobic interaction between the resin and sugar chains.

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

0.0 5.0 10.0 15.0 20.0 25.00.0 5.0 10.0 15.0 20.0Elution time (min)

Fluo

resc

ence

inte

nsity

Elution time (min)

Elution time (min)

Fluo

resc

ence

inte

nsity

Fluo

resc

ence

inte

nsity

Amide column

ODS column

DEAE column

Examination of glycosylation profiles

Neutral

G4

G5G6 G7

G8 G9 G10 G11

Glucose oligomer

Glucose oligomerG4

G5G6

G7

G8G9

G10

G11G12G13 G14

G16G18

G20

S1

S2

S3S4

S1:mono-sialylS2:di-sialylS3:tri-sialyS4:tetra-sialyl

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

Inconsistence between standard and sample

Comparison with elution accumulated data

GALAXY(http://www.glycoanalysis.info/)

Glycobase(http://glycobase.nibrt.ie/glycobase/show_nibrt.action)

Over 500 data of PA-N-oligosaccharides

Over 675 data of AB-oligosaccharides(containing O-glycans)

Consistence between standard and sample

5.0 10.0 15.0 20.0 25.0 30.0 35.0

Incase of unknow oligosaccharide which is not registered in database↓

Estimation/identification by the enzyme treatment

S

S

Two Neu5Ac residues were released by neuraminidasetreatment

Before neuraminidase

After neuraminidase

Composition and linkage analyses

The CCRC Spectral Database for Partially Methylated Alditol Acetate

https://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 analysisThe following figure illustrates the general nomenclature scheme for glycan fragments.

2,5X2

2,5A1

Y2

B1

Z2

C1

Y1

B2

Z1

C2

1,5X0

1,5A3

a) 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 from the cetuximab. The cartoons of possible structures of glycans were adapted from Glycoworkbench and structure is depicted following the CFG notation.

MALDI-TOF MS spectrum of N-glycans enzymatically released from the biosimilar of cetuximab and cetuximab

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

Complex type glycans

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

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

Native mass analysisMS can be used to measure the stoichiometry and composition of protein complexes, the presence of small molecules

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

(a) Schematic of the rhEPObackbone sequence and its reported PTM sites. (b) The zero-charge deconvoluted native MS spectrum of rhEPO.

Detail information of N-glycans structural analysis by using HPLC

mapping method

2-aminopyridine

ODS column

Amide column

Glycoamidase A (E.C. 3.5.1.52)

DEAE column

The multi-dimensional HPLC mapping technique

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

DEAE

col

umn

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

Elution time (min)

Fluo

resc

ence

inte

nsity

4

56

7

8

9

10

11 12 13 1415 16

17 18 19

2120

12.8

The elution position of each peak is expressed in glucose units (gu).

N-glycosylation profiles on ODS cplumn

a1-6Glucose oligomer Sample

The elution positions of peaks in an unknown glycan pool are assigned an overall gu value by comparison with the standard a1-6glucose oligomers.

y = 86055x - 39159

R² = 0.9994

0 50 100 150

y = 81857x + 31562

R² = 0.9963

0 50 100 150

arbi

trary

are

a un

it

arbi

trary

are

a un

it

PA-oligosaccharide (pmol) PA-oligosaccharide (pmol)

GNGNGNGN

M

MM-GN-GN-PA

G-GN-MM-GN-GN-PA

G-GN-M

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

HPLC-based discrimination of glycol-isomers

410.15410.14410.13

ODS : 14.1

Amide : 9.5

ODS : 13.8

Amide : 9.3

ODS : 13.7

Amide : 9.2

ODS : 12.5

Amide : 8.9

GNG-GNG-GNG-GN

M

MM-GN-GN-

F

410.12

G-GNG-GN

GNG-GN

M

MM-GN-GN-

FG-GN

GNG-GNG-GN

M

MM-GN-GN-

FG-GNG-GNG-GN

GN

M

MM-GN-GN-

F

Distinguish a2-6 from a2-3!

1A1-100.4

ODS :7.8

Amide : 6.0

1A2-100.4

ODS : 9.1

Amide : 5.4

NeuAcα2-6G-GN

M

MM-GN-GN-

NeuAcα2-3G-GN

M

MM-GN-GN-

Elution time (min) Elution time (min)

Mono-sialyl Di-sialyla2-3a2-6

quail quail

chicken chicken

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

Glycoamidase A (E.C. 3.5.1.52)

Anion-exchange column

ODS column

Amide column

2-aminopyridine

Sugar Library

A principal of HPLC mapping method

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

Multiple structuresLectin＝Glycan binding protein

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

Imm

obili

zed

lect

inLigand

solution Library of PA-glycans

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/glycotechnology/GT-C07J.html

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.3FAC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/glycotechnology/GT-C07J.html

http://www.glycoanalysis.info/

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

GALAXY database

Information page for the individual N-glycans

Galβ1,40.61

55

GlcNβ1,44.44

5

Xylβ1,20.09

6

Manα1,3-0.53

7

Manα1,60.72

4GlcNβ1,41.21

10

GlcNβ1,6-2.21

9

Manα1,3-0.67

11

GlcNβ1,21.75

12

Manα1,6-0.19

8

GalNβ1,40.33

18

Galβ1,40.24

17

Galβ1,4-1.07

56

Manα1,20.86

19

Manα1,2-1.00

16

GalNβ1,41.04

22

Galβ1,40.13

21

Fucα1,6-0.20

20

Galα1,41.25

58

GlcNβ1,31.52

33

Galα1,32.74

38

GlcNβ1,30.88

39

Galα1,4-0.29

59

Galα1,4-0.39

57

Galβ1,40.39

53

Galβ1,4-0.05

52

GlcNβ1,20.06

14

GlcNβ1,42.03

13

Manα1,2-0.48

15

Galβ1,40.54

24

Fucα1,3-0.15

25

Galβ1,30.67

26

Fucα1,6-0.75

27

Galβ1,41.33

28

GalNβ1,40.98

29

Manα1,2-0.40

30Glcα1,30.80

51

Galα1,32.01

42

Galα1,4-0.58

60

Galα1,32.79

44

Galα1,3-2.62

48

GlcNβ1,31.30

49

Fucα1,28.87

50

Galα1,4-2.00

61

Galβ1,41.24

54

NeuAcα2,6-0.51

31

NeuAcα2,30.38

32

NeuGcα2,6-0.90

34

NeuAcα2,61.63

35

NeuAcα2,30.92

36

NeuGcα2,31.10

37

NeuAcα2,60.49

23

NeuAcα2,62.46

40

NeuAcα2,30.31

41

NeuAcα2,31.89

43

NeuGcα2,6-3.06

45

NeuAcα2,6-1.88

46

NeuAcα2,30.31

47

Fucα1,63.43

2

Manβ1,4 GlcNβ1,47.20

GlcN-PA1

Fucα1,3-1.96

3

The GlycoTree diagram

Galβ1,40.61

55

GlcNβ1,44.44

5

Xylβ1,20.09

6

Manα1,3-0.53

7

Manα1,60.72

4GlcNβ1,4

1.2110

GlcNβ1,6-2.21

9

Manα1,3-0.67

11

GlcNβ1,21.75

12

Manα1,6-0.19

8

GalNβ1,40.33

18

Galβ1,40.24

17

Galβ1,4-1.07

56

Manα1,20.86

19

Manα1,2-1.00

16

GalNβ1,41.04

22

Galβ1,40.13

21

Fucα1,6-0.20

20

Galα1,41.25

58

GlcNβ1,31.52

33

Galα1,32.74

38

GlcNβ1,30.88

39

Galα1,4-0.29

59

Galα1,4-0.39

57

Galβ1,40.39

53

Galβ1,4-0.05

52

GlcNβ1,20.06

14

GlcNβ1,42.03

13

Manα1,2-0.48

15

Galβ1,40.54

24

Fucα1,3-0.15

25

Galβ1,30.67

26

Fucα1,6-0.75

27

Galβ1,41.33

28

GalNβ1,40.98

29

Manα1,2-0.40

30Glcα1,3

0.8051

Galα1,32.01

42

Galα1,4-0.58

60

Galα1,32.79

44

Galα1,3-2.62

48

GlcNβ1,31.30

49

Fucα1,28.87

50

Galα1,4-2.00

61

Galβ1,41.24

54

NeuAcα2,6-0.51

31

NeuAcα2,30.38

32

NeuGcα2,6-0.90

34

NeuAcα2,61.63

35

NeuAcα2,30.92

36

NeuGcα2,31.10

37

NeuAcα2,60.49

23

NeuAcα2,62.46

40

NeuAcα2,30.31

41

NeuAcα2,31.89

43

NeuGcα2,6-3.06

45

NeuAcα2,6-1.88

46

NeuAcα2,30.31

47

Fucα1,63.43

2

Manβ1,4 GlcNβ1,47.20

GlcN-PA1

Fucα1,3-1.96

3

α- Galactosidase

β- Galactosidase

β- HexNAcase

α- Sialidase

α- Fucsidase

β- Xylosidase

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

Graph selection from the three types of combination of the axes

ODS-Amide ODS-MW Amide-MW

MW

MW

Amide

ODS ODS Amide

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

MW 2302 ?

5 10 15 20 25

3

5

7

9

11

13

ODS(G.U.)

Am

ide

(G.U

.)Expanded HPLC map including sulfated oligosaccharides

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

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

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

Fluo

resc

ence

inte

nsity

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

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

Contents

Conformation analysis

ψΦC1

C2

C1’

C6’

C3’

Conformations of saccharide linkages- information available X-ray crystallography –Most oligosaccharides and glycoproteins either do not crystallize or give no resolvable electron density for the glycan. Glycans that can be seen are incomplete. → average properties of linkages

Nuclear Magnetic Resonance Spectroscopy –Experimental structural parameters (inter-nuclear distances and torsion 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 that the theory is correct.

CH2

D1

D2

CH3

CH2

CH3

Glycosylated FcgRIIINon-fucosylated Fc

Crystal structures of IgG1-Fc/FcgRIII complex

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

FcgRIII

Fc

Asn162

Complex type

FcgRIII

Fc

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

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

Glycoprotein (X-ray)3,290 (3.49%)

Glycoprotein (NMR)19 (0.02%)

Non-glycosylated protein (NMR)80,791 (10.85%)

010002000300040005000600070008000900010000

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

N-g

lyca

n st

ruct

ures

8680

3190

1416512 445259 88 63177 38 2 8 0 0 2

0

5

10

15

20

25

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

20

21

3

0 0 1 20 0 0 0 0 0 0

N-g

lyca

n st

ruct

ures

NMR

X-ray

Nuclear Magnetic Resonance Spectroscopy

Nuclear Overhauser effect (NOE) < 5 Å

Pseudocontact Shift (PCS) < 40 Å

rθ

φ

H

χz

χyχx

J coupling :Dihedral angles

MD simulationMultiscale modeling of glycosaminoglycans from disaccharide to polysaccharide is necessitated by their size and heterogeneity

! = #$%&'(

)$(+ − +-)/+ #1&2+3(

)1(4 − 4-)/+ #5%6(7%&(

8&/ 9 + :%((&∅ − ∅-)

#<=9

>?9

#7=<@9

>

A7, <C-7<C7<

9/− /

C-7<C7<

D

#<=9

>?9

#7=<@9

>E7E<

FGA%C7<

#<=9

>?9

#7=<@9

>H7<C7<9/

−I7<

C7<9-

+

+

+

van der Waals

electrostatic

hydrogen bonding

Harmonic oscillator-like bonding, angular, torsional terms

3D Structural Information 3D Structural Model

Compare

Validated conformational ensemble

Computational CalculationExperiment

Disagree

Information averaged over dynamic

conformational ensemble

Results depending on force field,

initial state and simulation time

Molecular Dynamics Simulation (MD)Nuclear Magnetic Resonance (NMR)

Agree

Paramagnetic NMR-Validated Molecular Dynamics Simulation

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

Conformational dynamics of GM9 dodecamerGM9 conformational ensemble based on NMR-validated MD simulation Conformational dynamics of trisaccharide on GM9

Glc

ManD1

ManC

Man4

Φ : O5-C1-O1-CʹXΨ : C1-O1-CʹX-CʹX-1

ΦΨ

Glc-ManD1-ManC-Man4

Density maps of glycosidic linkage torsion angles

0

-90

180

90

-180

Ψ/ d

egre

e

900-90Φ / degree

-180 900-90Φ / degree

900-90Φ / degree

Glc-ManD1 ManD1-ManC ManC-Man4

Low density

High density

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

Glc-ManD1-ManC-Man4

0

-90

180

90

-180

Ψ/ d

egre

e

900-90Φ / degree

-180 900-90Φ / degree

900-90Φ / degree

Glc-ManD1 ManD1-ManC ManC-Man4

Low density

High density

180

Density maps of glycosidic linkage torsion anglesKozlov, G.; et al, J. Biol. Chem. 2010, 285, 38612-38620

Calreticulinbinding state

Conformational dynamics of trisaccharide on GM9

Glc

ManD1

ManC

Man4

Φ : O5-C1-O1-CʹXΨ : C1-O1-CʹX-CʹX-1

ΦΨ

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

Kyowa Hakko Kirin K. ShitaraM. SatohS. IIda Taiyo Nippon Sanso

Shizuoka Univ. E.Y. ParkT. Kato

NIHSN. HashiiS. Nakazawa

NIASM. Nakamura

AISTN. FukuzawaT. MatsumuraH. Tateno

Yokoyama City Univ.N. KawasakiGeorgia Regent Univ.

R.K. Yu

Kato’s lab members

Acknowledgement

Kyoto Univ.S. OkaN. Nakagawa

Academia SinicaK.H. KhooC.W. Kuo