糖鎖構造解析を支援するためのGALAXYデータベース○矢木宏和1、高橋禮子1、加藤晃一1,2
1名古屋市立大学院薬学研究科、2自然科学研究機構岡崎統合バイオサイエンスセンター

Introduction

http://www.glycoanalysis.info/

糖鎖修飾は、主要な翻訳後修飾の一つである。糖鎖は分岐性、構造異性、構成残基の類似性から糖鎖の構造解析においては，DNAやタンパク質のような簡便なシーケンシング技術は存在しない。こうした状況下で，私たちは高速液体クロマトグラフィー（HPLC）を利用した糖タンパク質糖鎖の構造解析に着手し，これまでに600種類にのぼる糖鎖構造のHPLCの溶出時間データを蓄積してきた。また、得られた糖鎖の溶出データをGALAXYデータベース（http://www.glycoanalysis.info/galaxy2/）として公開することで、HPLC溶出時間に基づいて糖鎖の構造解析が行うことため基盤を整えることができている。本発表では、HPLCの溶出データに基づいた糖鎖構造の解析法を示すとともに，本糖鎖データベースの応用例について紹介したい。

Multi-HPLC mapping methods

Licensed under a Creative Commons表示4.0国際ライセンス(c)2017 矢木宏和（名古屋市立大学大学院薬学研究科）

GALAXYdatabase

The make up of the 2-D/3-D map

Extended HPLC map ofanionic oligosaccharides

The N-glycans are first released from theprotein portion by digestion withglycoamidase A, and the reducing ends ofthe oligosaccharides are fluorescent labeledwith 2-aminopyridine. These pyridylamino(PA)-glycans are then separated by HPLC
using three different columns sequentially.
Based on the elution positions on the threekinds of HPLC columns, the structures of theseparated PA-glycans are estimated bycomparing their elution positions with thepositions of the known reference N-glycans
plotted on the HPLC map. Then, the sample
PA-glycan and one or more of the candidatereference PA-glycans is co-injected into two
HPLC columns to confirm its identity (total
coincidence of elution position). In addition,
the sample PA-glycan is digested withseveral glycosidases, then the changes in theelution positions are again compared withthose of the reference N-glycans.

In order to familiarize the reader with the actual application of these methods to N-glycan analysis, the authors describe several examples from our publishedresults. During the process, it will be demonstrated that the oligosaccharides of different structure, even very closely related ones can be separated by this 3-D map
approach. The method is useful not only as an analytical procedure for N-glycan structures, but also as a means for large-scale isolation of samples for NMR
spectroscopy or MS spectrometry.

GALAXY database
The basic view of the system (2)Graph selection from the three types ofcombination of the axesODS-MWMWAmideODS-Amide

ODS

ODS

Selection of the sialic acid numbers

Amide-MWMW

The basic view of the system (1)Amide

Three types of drawing of glycan structures

Unite contribution
The GlycoTree diagram of N-glycans
57

Galα1,4-0.39

NeuAcα2,631-0.5152

53

Galβ1,4-0.05

Galβ1,40.39

57
16Manα1,2-1.00

8932

NeuAcα2,30.38

17

Galβ1,40.24

33

GlcNβ1,31.52

18

GalNβ1,40.33

58

Galα1,41.25

56

Galβ1,4-1.07

34

NeuGcα2,6-0.90

35

NeuAcα2,61.63

1920Manα1,20.86

α- Galactosidase

ODSManα1,6-0.19β- Galactosidase

GlcNβ1,6-2.21β- HexNAcase
α- Sialidase

1011GlcNβ1,41.214

NeuAcα2,30.92

21Galβ1,40.1337

NeuGcα2,31.1022

GalNβ1,41.04

38

Galα1,32.74

39

GlcNβ1,30.8859

Galα1,4-0.29

Manα1,3-0.67

GlcNβ1,2121.75

53

NeuAcα2,62.46

NeuAcα2,3410.31

54

Galβ1,41.24

523

Galβ1,40.90

Manα1,60.72

554052Fucα1,6-0.2036

NeuAcα2,60.49
6GlcNβ1,44.44

1Galβ1,40.61Manβ1,4Xylβ1,20.09

Galα1,40.85

NeuAcα2,6310.362Manα1,21.06

8Manα1,61.04

9GlcNβ1,60.7032

NeuAcα2,3-0.61

17Galβ1,40.81

33

GlcNβ1,30.45

18GalNβ1,40.4458

Galα1,40.37

56

Galβ1,40.98

1019

Manα1,20.58

Manα1,3110.89

34

NeuGcα2,60.3735

NeuAcα2,60.1420

Fucα1,61.06

NeuAcα2,336-0.4021Galβ1,40.89

37

NeuGcα2,30.03

22GalNβ1,40.5638

Galα1,30.76

39GlcNβ1,30.3259

Galα1,40.90

GlcN-PA40

Fucα1,3-1.96

23422442Galα1,30.89

24Galβ1,40.91

60Galα1,4-0.58

25Fucα1,3-0.15

60Galα1,40.75

25

Fucα1,30.79

43

NeuAcα2,31.89

26

Galβ1,30.67

43

NeuAcα2,3-0.31

26Galβ1,30.77

44Galα1,32.79

44Galα1,30.83

45

NeuGcα2,6-3.0645

NeuGcα2,60.51

46

NeuAcα2,6-1.88

27

Fucα1,6-0.75

46

NeuAcα2,60.1727

Fucα1,61.05

47

NeuAcα2,30.31

28

Galβ1,41.3347

NeuAcα2,3-0.4628Galβ1,40.9348

Galα1,3-2.6229

GalNβ1,40.9848

Galα1,30.9729

GalNβ1,40.5249

GlcNβ1,3-0.2950Fucα1,20.30

61

Galα1,40.7151

Glcα1,30.694950

GlcNβ1,4132.03

7Manα1,3-0.53

β- HexNAcase
α- Sialidase
αFucsidase
α- Fucsidase

Fucα1,28.87

61

Galα1,4-2.0051

Glcα1,30.80

α- Galactosidase
β- Galactosidase

GlcNβ1,31.30

54

Galβ1,41.36

β- Xylosidase

β- Xylosidase

30Manα1,2-0.40

1512GlcNβ1,40.58

4Manα1,61.08

GlcNβ1,20.35Manα1,2-0.48

5GlcNβ1,40.21

6Xylβ1,20.37

7Manα1,31.18

NeuAcα2,6-0.33

Galβ1,40.54

GlcNβ1,20.06

β- HexNAcase
α- Sialidase

NeuAcα2,60.26

NeuAcα2,341-0.38

1Galβ1,41.01
Manβ1,42

Fucα1,60.45

GlcNβ1,42.10
3GlcN-PAFucα1,30.96

GlcNβ1,4130.38

14

GlcNβ1,20.33

α- Fucsidase
β- Xylosidase

30Manα1,20.90

15We introduced a concept, parameterization ofunit contribution (UC). In this case, unit means
each monosaccharide component at a specificposition in a whole oligosaccharide. N-glycanstructures can be arranged in a tree-like
diagram, which spreads its branches on atrunk of a tri-mannosyl core structure. The
basic assumption used in the parameterizationis that the glucose unit of a given PAoligosaccharide on an ODS or amide column
can be represented by the total of thecontributions of all its monosaccharide units.
This approach is developed into a useful way
to utilize unit contribution values.

Manα1,20.81

Application for characterization of glycosyltransferaseGlucuronylation schemes of
tri-antennary N-glycans

Prediction of digestion precursors
of a selected N-glycan

β- Galactosidase

55

Galα1,32.01

14

α- Galactosidase

Amide-silica

Fucα1,63.43

GlcNβ1,47.20
3Galβ1,41.09

16

Display of products resulting fromglycosidase treatments

Glycosylation profiles ofreaction mixture of GlcAT-P

Time courses of
glucuronylation reactions ofa triantennary glycan
catalyzed by GlcAT-P

Application for glycosylation profiles
Neural stem cell

Elution profiles of PA-glycans derived from Neural stem
cells before and after differentiation on ODS column

Possible reaction products ofglucuronyltransferases

H. Yagi et al. 2008 Open Glycosci., 1, 8-18.

Neural stem cells are undifferentiated neural cells
characterized by their high proliferative potential and thecapacity for self-renewal and retention of multipotency todifferentiated into neurons and glial cells.
H. Yagi et al. 2012 J. Biol Chem., 287, 24356-64.