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 Kai Simons & Elina Ikonen, NATURE |VOL 387 | 5 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). 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 Langmuir-Blodgett technique 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. Molecular structures of lipids for L-B film preparation O DPPC OO
O dipalmitoylphosphatidylcholine O O P O O Me + N Me Me O OO O DOPC O dioleoylphosphatidylcholine O O OO O DPPE
O O P O O P O dipalmitoylphosphatidylethanolamine O Me + N Me Me NH2 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
Chemical structure of ganglioside GM1 OH HO HO HO HO O O HO O AcNH O -OOC OH HO O AcHN
O OH O O HO HO OH O HO H HN O O H OH OH OH Ganglioside was composed of carbohydrate part and lipid part (Ceramide). Ganglioside GM1 has a sialyl 23 galactose residue. Membrane properties of binary and ternary systems of ganglioside GM1/DPPC/DOPC
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. Yumiko Ohta, Shoko Yokoyama, Hideki Sakai, Masahiko Abe Colloids and Surfaces B: Biointerfaces 34 (2004) 147153. 1. Research on ganglioside GM1a raft GM1 is not commercially available, We must synthesize it. GM1 in DOPC/DPPC monolayers and hybrid bilayers are prepared. Distribution of GM1 is investigated by AFM. Chemical structure of GM1 HO OH O AcHN HO HO HO -OOC
OH OH O HO HO O O O AcNH O HO OH O O HO HO OH O HO H HN O
O H OH -Series ganglioside containing a sialyl -26-N-acetylgaractosamine residue are exclusive y localized on cholinergic neurons. Chemical synthesis of GM1 (1) MeO 6 steps from glucosamine O O AcO OH OBn O BnO O SEt BnO OBn 2 1 NPhth OBn O
O OBn OSE a OH OBn R2O O OBn O O BnO 1 MeO O O R O AcO O SEt BnO OSE AcO O O OBn OBn 2 1 NPhth
OBn PhthN OBn O BnO Oa BnO OSE R2O O OBn 1 O R O 1 2 OBn AcO6H4OMe-p O (91%) OBn b 3 R1, R = CHC 2 PhthN OBn 4 R = H, R = PMB (88%) O BnO 7 steps from lactose
O OSE O OBn OBn 6H4OMe-p (91%) b 3 R1, R c= CHC OPMB 4 R = H, R2 = PMB (88%) BnO R2O 1 2 O HO d R2O O R1 HO
BnO OPMB OBn OO c OBn BnO O OOBn 1 OSE R OBn O BnOO OBn O BnO OSE O 5 R1 = NPhth, R2 = Ac (83%) OBn OBn OBn R12 = NPhth, R2 = Ac (83%) d 5R 6 R1 = NHAc, 1 = H (89%) 2
BzO OBz 6 R = NHAc, R = H (89%) e BzO BzO f e BzO OBz HO OR OBz HO ORO O O O O BzO O OBz O OAcHNOBn BnO OBz AcHN 8 R = PMB (58%) O f 9 RBnO = H (91%)
8 R = PMB (58%) 9 R = H (91%) BzO BzO O OBz O O SLau SLau 7 OBz OBz 7 BzO OBn O BnO O BnO
OBn OBn 4 steps from galactose OBn OSE OBn O OBn O OBn OSE 23 steps Chemical synthesis of GM1 (2) AcO 9 AcO AcO 3
R HN COOMe AcO COOMe O TCAHN SEt AcO OAc 10 a O O AcO OAc 2 BzO OBz R O O O O
BzO O OBz AcHN R1O 9 steps from glucosamine 1 OR OR1 O R1O O OSE O OR1 1 OR 11 R1 = Bn, R2 = H, R3 = TCA (60%) b 1 2 3 12 R = Bn, R = H, R = Ac (96%) c 13 R1 = Bz, R2 = Bz, R3 = Ac (59%) OBz
AcO AcO AcHN COOMe O HO d (CH2)14CH3 N3 O AcO OAc BzO OBz BzO O O O BzO O OBz OBz AcHN O BzO O
BzO OBz 15 R = N3 (36%) e 16 R = NHCO(CH2)16CH3 (74%) HO HO AcHN COOH 14 7 steps from galactose OBz OBz O OBz O (CH2)14CH3 R f
Total steps steps were known in publication.) O O HO OH HO HO OH O O O HO O OH AcHN HO 17 (76%) OH O OH HO O
OH OH O OH O (CH2)14CH3 NHCO(CH2)16CH3 Surface pressure vs. area per molecule isotherms for GM1a/DOPC/DPPC(1:9:9) Surface pressure [mN / m] 80 70 DOPC 60 GM1/DOPC/DPPC /DOPC/DPPCDOPC/DOPC/DPPCDPPC GM1/DOPC/DPPC 50 40
liquid-condensed phase 30 20 liquid-expanded phase 10 0 0 50 100 150 200 Aera per molecule [sq. / molecule] 250 AFM images for GM1/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 GM1-raft Subphase: 2.0mM NaCl AFM images for GM1/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 GM1/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: GM1:DOPC:DPPC=1:9:9 Subphase: 2.0 mM NaCl Self-assembly by surface pressure GM1/DOPC/DPPC ternary monolayer and hybrid bilayer obviously shows GM1-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 Guofei Suna, Hu Zhoub, Yi Li a, Kandiah Jeyaseelanc, Arunmozhiarasi Armugamc, Tai-Shung Chung, Colloids and Surfaces B: Biointerfaces 89 (2012) 283 288. The incorporation of Histidine-tagged AquaporinZ by Nickel chelating lipids is investigated with LangmuirBlodgett technology for the first time. Detergent removal by BioBeads in the LangmuirBlodgett system is studied. AquaporinZ and detergent interaction during protein insertion is elaborated and a protein incorporation mechanism is proposed.
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 AFM images for GM3/DOPC/DPPC (1:9:9) monolayers Surface pressure: 10 mN/m Surface pressure: 20 mN/m 9 images / 9 points 9 images / 9 points Surface pressure:
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