Photon Factory Activity Report 2014 #32 (2015) B

BL-10C/2013G038

Interaction of amyloid-β protein with raft-model liposome under mimic environment of cell
Mitsuhiro Hirai1,* Shoki Sato1, Nobutaka Shimizu2, and Noriyuki Igarashi2
1
Graduate School of Engineering, Gunma University, Maebashi, Gunma 371-8510, Japan.
2
Photon Factory, Tsukuba 305-0801, Japan
It is now widely accepted that the amyloidβ-peptide(Aβ), which exists in fibrillar forms as a major
component of senile plaques, is central to thedevelopment of Alzheimer's disease (AD). Aβ–membraneinteractions have been extensively investigated toelucidate the molecular mechanisms of the Aβ-induced
cellular dysfunctions underlying the pathogenesis of AD.
Particularly, according to accumulating evidence using
spectroscopic methods, the importance of gangliosideclusters in the fibrillization of Aβ is emphasized.
Gangliosides, major components of glycosphingolipids(GSLs), are acidic lipids and rich in central nervoussystem. GSL-cholesterol formed microdomains in cell
membranes, so-called lipid rafts, are suggested tofunction as platforms of various membrane-associatedevents such as signal transduction, cell adhesion,
lipid/protein sorting and so on. The interaction between
mono-sialoganglioside (GM1) and Aβ is reported topromote the conformational change of Aβ to its crossbeta structure and the GM1-bound form seeds in theprocess of Aβ polymerization to amyloid fibril [1].
Recently, we reported that that the interaction between
the Aβ proteins and the model membrane at the liquidcrystal phase significantly suppresses a bending-diffusion
motion, suggesting the possibility of non-receptormediated disorder in signaling through a modulation of amembrane dynamics induced by the association ofamyloidogenic peptides on a plasma membrane [2].

2. Experimental
In the present study, we have investigated the Aβmembrane interactions under a mimic intracellularenvironment by using the time-resolved small- and wideangle X-ray scattering (SWAXS) by using thesynchrotron radiation small-angle X-ray scattering
spectrometer installed at BL10C beam port of PF at KEK,
Tsukuba, Japan. The X-ray wavelengths were 1.0 Å and1.5 Å. The sample-to-detector distances were 50 cm and300 cm. X-ray scattering intensity was recorded by ahybrid pixel detector, PILATUS3 2M (Dectris). The
exposure time was 60 seconds. Sample cells composed ofa pair of thin-quartz windows with 1 mm path length
were used. The membrane used was the small unilamellar
vesicle (SUV) composed of GM1, cholesterol, andphospholipid. The molar ratio [GM1]/[cholesterol]/[DPPCor DOPC] of the mixtures was 0.1/0.1/1, where thephospholipid concentration was fixed at 1 % w/v.
Amyloid β protein (1-40) purchased from Wako Chem.
Co. was used without further purification. The solvent

condition was 10mM Hepes, pH7.4, 50mM NaCl, 5mM
KCl, 2mM MgCl2, 2mM CaCl2. The mimic intracellularenvironment, so-called crowding environment, wasrealized by the addition of the high-molecular weightneutral polymer (polyvinyl-pyrrolidone, PVP).

3. Results and Discussion
Fig. 1 shows the time evolution of SWAXS curve ofthe SUV (GM1-cholesterol-DPPC) after addting Aβprotein. The present results are summarized as follows.
Under non-crowding environment, the interaction
between SUV and Aβ protein proceeds mostly without
affecting the SUV membrane structure. While, undercrowding environment, the presence of crowders induces
the deformation of the SUV shape and the interaction
between SUV, and Aβ protein causes further aggregation
to form the lamellar stacking. The time-course of thelamellar distance and the lamellar-peak height suggest thepreferential cohesion or intercalation of Aβ proteins in theinter-bilayer region.

logI(q) (arb. units)

1 Introduction

evolution time (min)
021
39
58
168
227
289
347

10.01

q (Å-1)0.1

1Figure 1. Time-evolution of SAWXS curve of SUVafter Aβ addition

References[1] K. Matsuzaki et al, Biochem. Boiphys. Acta.
1801 (2010) 868.
[2] M. Hirai et al, Eur. Phys. J. E. 36 (2013) 74, DOI
10.1140/epje/i2013-13074-3* mhirai@gunma-u.ac.jp