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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