Reconstruction of a transmembrane protein tetraspanin (CD9) into lipid bilayer by interaction of ganglioside GM3 and tetraspanin

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1 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

2 Model for the organization of rafts Kai Simons & Elina Ikonen, NATURE VL JUNE 1997 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).

3 Function of ganglioside rafts n 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?

4 Preparation monolayers by Langmuir-Blodgett technique Lipids solution were spread on the subphase using microsyringe. After evaporation of the solvent, the monolayer was compressed at 50 cm 2 / min. The half side of cell membrane can be obtained.

5 Molecular structures of lipids for L-B film preparation DPPC dipalmitoylphosphatidylcholine DPC dioleoylphosphatidylcholine DPPE dipalmitoylphosphatidylethanolamine

6 About atomic force microscopy (AFM) Atomic Force between probe and sample is able to measure. Same value of atomic force means same distance. sample Resolusion: about 0.2 nm Hight of sample surface was obsurbed from position of probe. hight map

7 Chemical structure of ganglioside GM1 Ganglioside was composed of carbohydrate part and lipid part (Ceramide). Ganglioside GM1 has a sialyl a2 3 galactose residue.

8 Membrane properties of binary and ternary systems of ganglioside GM1/DPPC/DPC Effect of surface pressure on the AFM images for the GM1/DPPC/DPC (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/DPC monolayer changed as the surface pressure was varied. Yumiko hta, Shoko Yokoyama, Hideki Sakai, Masahiko Abe Colloids and Surfaces B: Biointerfaces 34 (2004)

9 1. Research on ganglioside GM1a raft GM1a is not commercially available, We must synthesize it. GM1a in DPC/DPPC monolayers and hybrid bilayers are prepared. Distribution of GM1a is investigated by AFM.

10 Chemical structure of GM1a a-series ganglioside containing a sialyl a-2 6-N-acetylgaractosamine residue are exclusively localized on cholinergic neurons.

11 Chemical synthesis of GM1α (1) 6 steps from glucosamine Me Ac b d Bz Bz f R 2 R 1 Ac H Bn Bn SEt Bn NPhth Bn 1 2 Me Ac PhthN Bn H Bn Bn SEt Bn NPhth Bn Bn 1 2 Bn Bn R 2 a R 1 Bn Ac Bn Bn PhthN Bn Bn Bn SE Bn 3 R 1, R 2 = CHC Bn b 6 H 4 Me-p (91%) c 4 R 1 = H, R 2 = PMB (88%) Bz H R Bz AcHN Bn Bn 8 R = PMB (58%) Bn Bn Bn SE R 2 PMB c R 1 Bn Bn H Bn R 1 Bn SE Bn Bn Bn Bn SE Bn Bn Bn d 5 R 1 = NPhth, R 2 = Ac (83%) 6 R 1 = NHAc, R 2 = H (89%) Bz Bz Bz Bz e e Bz Bz SLau Bz Bz Bz 7 9 R = H (91%) Bn 3 R 1, R 2 = CHC 6 H 4 Me-p (91%) 4 R 1 = H, R 2 = PMB (88%) R 2 PMB H a Bn 5 R 1 = NPhth, R 2 = Ac (83%) 6 R 1 = NHAc, R 2 = H (89%) Bz H R Bz Bz AcHN f Bn 8 R = PMB (58%) 9 R = H (91%) Bn Bn Bn Bn Bn SE 7 SE Bn SE Bn Bn SE Bn 7 steps from lactose 4 steps from galactose 23 steps

12 Chemical synthesis of GM1α (2) Ac Ac 9 CMe Ac Ac TCAHN Ac Ac a 10 CMe SEt R 3 HN Ac Ac Bz Bz R 2 Bz R 1 Bz AcHN R 1 R 1 R 1 R 1 SE 11 R 1 = Bn, R 2 = H, R 3 = TCA (60%) R 1 b 12 R 1 = Bn, R 2 = H, R 3 = Ac (96%) c 13 R 1 = Bz, R 2 = Bz, R 3 = Ac (59%) Bz Ac H (CH 2 ) 14 CH Ac CMe 3 d N 3 14 AcHN Ac Ac Bz Bz Bz Bz Bz Bz AcHN Bz Bz Bz Bz (CH Bz 2 ) 14 CH 3 15 R = N 3 (36%) e Bz R 16 R = NHC(CH 2 ) 16 CH 3 (74%) 9 steps from glucosamine 7 steps from galactose H H CH f AcHN H H H H H H H AcHN 17 (76%) H H H H H H H (CH 2 ) 14 CH 3 NHC( CH 2 ) 16 CH 3 Total steps:45 (33 steps were known in publication.)

13 Surface pressure vs. area per molecule isotherms for GM1a/DPC/DPPC(1:9:9) Surface pressure [mn/ m] DPC liquid-condensed phase liquid-expanded phase Aera per molecule [sq. A / molecule]

14 AFM images for GM1a/DPC/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

15 AFM images for GM1a/DPC/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

16 AFM images for hybrid bilayers of GM1a/DPC/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:DPC:DPPC=1:9:9 Subphase: 2.0 mm NaCl

17 Self-assembly by surface pressure GM1a/DPC/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 enzymelocalization on signal transduction.

18 2. Reconstruction of transmembrane protein

19 A novel method of AquaporinZ incorporation via binary-lipid Langmuir monolayers Guofei Suna, Hu Zhoub, Yi Li a, Kandiah Jeyaseelanc, Arunmozhiarasi Armugamc, Tai-Shung Chung, Colloids and Surfaces B: Biointerfaces 89 (2012) 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.

20 GM3/CD9/CD81 complex Ganglioside GM3 Tetraspanin CD9 CD81 The complex regulates cell growth, proliferation. The complex regulates also cancer cell proliferation.

21 Surface pressure (mn / m) Surface pressure vs. area per molecule isotherms for GM3/DPPC/DPC(1:9:9) ternary monolayer Area / molecule (sq.a / molecule)

22 AFM images for GM3/DPC/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

23 Cross-sectional study of rafts Surface 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

24 Proposed structure of ganglioside raft GM3 rich domain (about 2 nm) DPPC rich domain (about 1 nm) DPC rich domain Mica plate

25 AFM images for hybrid bilayers of GM3/DPC/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

26 Cross-sectional study of rafts Surface 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

27 Proposed structure of ganglioside raft GM3 rich domain (about 12 nm) DPPC rich domain (about 7 nm) DPC rich domain Mica plate

28 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.

29 Cross-sectional study of rafts reconstructed by tetraspanin Surface 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

30 Proposed structure of ganglioside raft with tetraspanin GM3 rich domain (about 12 nm) +tetraspanin(about 17 nm) DPPC rich domain (about 7 nm) DPC rich domain Mica plate tetraspanin