Reconstruction of a transmembrane

protein tetraspanin (CD9) into lipid

bilayer by interaction of ganglioside

GM3 and tetraspanin

Glycobiology World Congress

August 12, 2015

Tokyo University of Science, Japan

Shigeomi Horito

Model for the organization of rafts

The rafts (red) segregate from the

other regions (blue) of the bilayer.

Rafts contain proteins attached to

the exoplasmic leaflet of the bilayer

by their GPI anchors, proteins

binding to the cytoplasmic leaflet

by acyl tails (the Src-family kinase

Yes is shown), or proteins

associating through their

transmembrane domains, like the

influenza virus proteins

neuraminidase and haemagglutinin

(HA).

Kai Simons & Elina Ikonen, NATURE |VOL 387 | 5 JUNE 1997

Function of ganglioside rafts

On outer membrane of raft, it is

very clear that many ligands link to

the corresponding receptors.

How is the signal transduced on

inner membrane of raft?

For example, autophosphorylation

enzyme or cutting enzyme of signal

peptides.

How is the enzyme recluited?

Preparation monolayers by

Lipids solution were spread on the subphase using microsyringe.

After evaporation of the solvent, the monolayer was compressed at

50 cm2 / min.

The half side of cell membrane can be obtained.

Langmuir-Blodgett technique

Molecular structures of lipids for

L-B film preparation

DPPC dipalmitoylphosphatidylcholine

DOPC

DPPE

dioleoylphosphatidylcholine

dipalmitoylphosphatidylethanolamine

About atomic force microscopy (AFM)

Atomic Force between probe and sample is able to measure.

Same value of atomic force means same distance.

sample

Hight of sample surface

was obsurbed from

position of probe.

hight map

Resolusion: about 0.2 nm

Chemical structure of ganglioside GM1

Ganglioside was composed of carbohydrate part and lipid part (Ceramide).

Ganglioside GM1 has a sialyl a2→3 galactose residue.

Membrane properties of binary and ternary

systems of ganglioside GM1/DPPC/DOPC

Yumiko Ohta, Shoko Yokoyama, Hideki Sakai, Masahiko Abe

Colloids and Surfaces B: Biointerfaces 34 (2004) 147–153.

Effect of surface pressure on the AFM images for the GM1/DPPC/DOPC (2:9:9)

monolayer. Surface pressure: (a) 30 mN/m; (b) 35 mN/m; (c) 40 mN/m.

Conclusion: The percolation pattern in the GM1/DPPC/DOPC monolayer

changed as the surface pressure was varied.

1. Research on ganglioside GM1a raft

• GM1a is not commercially available, We

must synthesize it.

• GM1a in DOPC/DPPC monolayers and

hybrid bilayers are prepared.

• Distribution of GM1a is investigated by

AFM.

Chemical structure of GM1a

a-Series ganglioside containing a sialyl a-2→6-N-acetylgaractosamine

residue are exclusiveｌy localized on cholinergic neurons.

Chemical synthesis of GM1α (1)

OOO

NPhth

SEtAcOO

O

OH OBn

OBnBnO

BnO

OBn

OBn

OSEMeO

O

1 2

OR1O

R2O

PhthNAcO

O

O

O OBn

OBnBnO

BnO

OBn

OBn

OSEO

3 R1, R

2 = CHC6H4OMe-p (91%)

4 R1

= H, R2

= PMB (88%)

a

b

O

R2O OPMB

R1

HO

O

O

O OBn

OBnBnO

BnO

OBn

OBn

OSEO

5 R1

= NPhth, R2 = Ac (83%)

6 R1

= NHAc, R2

= H (89%)d

c

O

HO OR

AcHNO

O

O

O OBn

OBnBnO

BnO

OBn

OBn

OSEO

O

BzO OBz

OBzBzO

O

BzO OBz

OBzBzO SLau

e

7

8 R = PMB (58%)

9 R = H (91%)

f

OOO

NPhth

SEtAcOO

O

OH OBn

OBnBnO

BnO

OBn

OBn

OSEMeO

O

1 2

OR1O

R2O

PhthNAcO

O

O

O OBn

OBnBnO

BnO

OBn

OBn

OSEO

3 R1, R

2 = CHC6H4OMe-p (91%)

4 R1

= H, R2

= PMB (88%)

a

b

O

R2O OPMB

R1

HO

O

O

O OBn

OBnBnO

BnO

OBn

OBn

OSEO

5 R1

= NPhth, R2 = Ac (83%)

6 R1

= NHAc, R2

= H (89%)d

c

O

HO OR

AcHNO

O

O

O OBn

OBnBnO

BnO

OBn

OBn

OSEO

O

BzO OBz

OBzBzO

O

BzO OBz

OBzBzO SLau

e

7

8 R = PMB (58%)

9 R = H (91%)

f

6 steps from

glucosamine 7 steps from lactose

4 steps from galactose

23 steps

Chemical synthesis of GM1α (2)

O SEt

COOMe

OAc

AcO

AcO

TCAHN

AcO

O

R2O

O

AcHNO

O

O

O OR1

OR1

R1O

R1O

OR1

OR1

OSEO

O

BzO OBz

OBzBzO

O

COOMe

OAc

AcO

AcO

R3HN

AcO

9

11 R1 = Bn, R

2 = H, R

3 = TCA (60%)

12 R1 = Bn, R

2 = H, R

3 = Ac (96%)

13 R1 = Bz, R

2 = Bz, R

3 = Ac (59%)

HO (CH2)14CH3N3

OBz

14

O

BzO

O

AcHNO

O

O

O OBz

OBzBzO

BzO

OBz

OBz

O

O

BzO OBz

OBzBzO

O

COOMe

OAc

AcO

AcO

AcHN

AcO

O (CH2)14CH3R

OBz

15 R = N3 (36%)

16 R = NHCO(CH2)16CH3 (74%)

O

HO

O

AcHNO

O

O

O OH

OHHO

HO

OH

OH

O

O

HO OH

OHHO

O

COOH

OH

HO

HO

AcHN

HO

O (CH2)14CH3NHCO( CH2)16CH3

OH

17 (76%)

a 10

d

b

c

e

f

9 steps from glucosamine

7 steps from galactose

Total steps：４５（３３ steps were known in publication.)

0

10

20

30

40

50

60

70

80

0 50 100 150 200 250

Aera per molecule [sq. Å/ molecule]

Sur

face

pre

ssur

e [m

N/ m

] DOPC

Surface pressure vs. area per molecule

isotherms for GM1a/DOPC/DPPC(1:9:9)

liquid-expanded phase

liquid-condensed phase

AFM images for GM1a/DOPC/DPPC

(1:9:9) monolayers

a) 15 mN/m

b) 25 mN/m

c) 30 mN/m

d) 40 mN/m

A, A’: DPPC-rich domains

→： GM1α-raft

Subphase: 2.0mM NaCl

AFM images for GM1a/DOPC/DPPC

(1:9:9) monolayers on physiologycal saline

a) 15 mN/m

b) 25 mN/m

c) 30 mN/m

d) 40 mN/m

Subphase: 157 mM NaCl

AFM images for hybrid bilayers of

GM1a/DOPC/DPPC (1:9:9)

a) 15 mN/m

b) 20 mN/m

c) 25 mN/m

d) 30 mN/m

First layer: DPPE

Subphase: 157 mM NaCl

Second layer:

GM1α:DOPC:DPPC=1:9:9

Subphase: 2.0 mM NaCl

Self-assembly by surface pressure

• GM1a/DOPC/DPPC ternary monolayer and

hybrid bilayer obviously shows GM1α-rafts.

• The raft was associated, separated and associated

again, according to increase of surface pressure.

• The association of rafts causes enzyme-

localization on signal transduction.

2. Reconstruction of

transmembrane protein

A novel method of AquaporinZ incorporation via

binary-lipid Langmuir monolayers

The incorporation of Histidine-tagged AquaporinZ by Nickel chelating

lipids is investigated with Langmuir–Blodgett technology for the first time.

Detergent removal by BioBeads in the Langmuir–Blodgett system is

studied. AquaporinZ and detergent interaction during protein insertion is

elaborated and a protein incorporation mechanism is proposed.

Guofei Suna, Hu Zhoub, Yi Li a, Kandiah Jeyaseelanc, Arunmozhiarasi Armugamc,

Tai-Shung Chung, Colloids and Surfaces B: Biointerfaces 89 (2012) 283– 288.

GM3/CD9/CD81 complex

Ganglioside GM3

Tetraspanin CD9

CD81

The complex regulates cell

growth, proliferation.

The complex regulates also

cancer cell proliferation.

Surface pressure vs. area per molecule isotherms

for GM3/DPPC/DOPC(1:9:9) ternary monolayer

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120

Su

rfa

ce p

ress

ure

(m

N/

m)

Area / molecule (sq.Å / molecule)

AFM images for GM3/DOPC/DPPC

(1:9:9) monolayers

Surface pressure:

10 mN/m

9 images / 9 points

Surface pressure:

20 mN/m

9 images / 9 points

Surface pressure:

30 mN/m

8 images / 9 points

Surface pressure:

40 mN/m

9 images / 9 points

Cross-sectional study of raftsSurface pressure: 10 mN/m

Hight: 0.9±0.1 nm

Surface pressure: 20 mN/m

Hight: 0.8±0.1 nm, 2.1±0.2 nm

Surface pressure: 30 mN/m

Hight: 0.5±0.2 nm, 1.7±0.5 nm

Surface pressure: 40 mN/m

Hight: 0.5±0.2 nm, 1.6±0.3 nm

Proposed structure of

ganglioside raft

Mica plate

DOPC rich domain

DPPC rich domain (about 1 nm)

GM3 rich domain (about 2 nm)

AFM images for hybrid bilayers

of GM3/DOPC/DPPC (1:9:9)

Surface pressure:

10 mN/m

6 images / 9 points

Surface pressure:

20 mN/m

7 images / 9 points

Surface pressure:

30 mN/m

6 images / 9 points

Surface pressure:

40 mN/m

5 images / 9 points

Cross-sectional study of raftsSurface pressure: 10 mN/m

Hight: 6.3±0.7 nm, 11.5±0.4 nm

Surface pressure: 20 mN/m

Hight: 6.7±0.6 nm, 12.5±0.2 nm

Surface pressure: 30 mN/m

Hight: 6.2±0.4 nm, 11.7±0.5 nm

Surface pressure: 40 mN/m

Hight: 6.5±0.5 nm, 11.2±0.3 nm

Proposed structure of

ganglioside raft

DPPC rich domain (about 7 nm)

Mica plate

DOPC rich domain

GM3 rich domain (about 12 nm)

Reconstruction of tetraspanin

(CD9) into lipid bilayer

The hybrid bilayer was kept in CD9 solution (2 mg/ml) for 5 min.

Surface pressure:

30 mN/m

5 images / 9 points

were changed.

Surface pressure:

40 mN/m

2 images / 9 points

were no change.

7 images / 9 points

were changed.

Cross-sectional study of rafts

reconstructed by tetraspaninSurface pressure: 30 mN/m

Hight: 6.1±0.5 nm, 11.7±1.1 nm

Surface pressure: 30 mN/m

Hight: 6.1±0.5 nm, 16.4±0.9 nm

Surface pressure: 40 mN/m, 2 / 9

Hight: 6.4±0.5 nm, 11.3±0.8 nm

Surface pressure: 40 mN/m, 7 / 9

Hight: 6.4±0.5 nm, 15.6±1.7 nm

Proposed structure of

ganglioside raft with tetraspanin

Mica plate

DOPC rich domain

GM3 rich domain (about 12 nm) +tetraspanin(about 17 nm)

tetraspanin

DPPC rich domain (about 7 nm)