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Volume 582, Issue 27 p. 3788-3792
Short communication
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Membrane binding of oligomeric α-synuclein depends on bilayer charge and packing

Bart D. van Rooijen

Bart D. van Rooijen

Biophysical Engineering Group, MESA+ Institute for Nanotechnology and Institute for Biomedical Technology, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands

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Mireille M.A.E. Claessens

Mireille M.A.E. Claessens

Biophysical Engineering Group, MESA+ Institute for Nanotechnology and Institute for Biomedical Technology, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands

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Vinod Subramaniam

Corresponding Author

Vinod Subramaniam

Biophysical Engineering Group, MESA+ Institute for Nanotechnology and Institute for Biomedical Technology, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands

Corresponding author. Fax: +31 53 4891105.Search for more papers by this author
First published: 16 October 2008
Citations: 61

Abstract

Membrane disruption by oligomeric α-synuclein (αS) is considered a likely mechanism of cytotoxicity in Parkinson's disease (PD). However, the mechanism of oligomer binding and the relation between binding and membrane disruption is not known. We have visualized αS oligomer-lipid binding by fluorescence microscopy and have measured membrane disruption using a dye release assay. The data reveal that oligomeric αS selectively binds to membranes containing anionic lipids and preferentially accumulates into liquid disordered (Ld) domains. Furthermore, we show that binding of oligomers to the membrane and disruption of the membrane require different lipid properties. Thus membrane-bound oligomeric αS does not always cause bilayer disruption.

1 Introduction

α-Synuclein (αS) is a 140 amino acid protein abundantly expressed throughout the central nervous system [1] and is likely to be involved in the pathogenesis of Parkinson's disease (PD). It is the major constituent of Lewy bodies found in PD [2] and three mutant forms of the protein, that lead to a rare familial form of PD, have been identified [3-5]. In vitro, αS is an intrinsically disordered protein with little secondary structure [6]. Although the role of αS in the cell is not known, lipid binding is thought to be involved in its function [7]. Under specific conditions, αS aggregates into densely packed β-sheet rich fibrils, similar to those found in vivo [8]. Monomeric αS can bind to negatively charged phospholipid vesicles in the liquid disordered (Ld) phase [9-11]. Binding of αS monomers to membranes is mediated by the N-terminus which folds into an α-helical conformation [12]. It is often suggested that a gain of toxic function of αS relates the protein to PD [7]. However, the mechanism of toxicity and the relation between aggregation, lipid binding and toxicity is not unequivocally established [13-16]. In recent years, oligomeric intermediates in the aggregation of αS are considered as a likely toxic species [17, 18]. Membrane disruption and subsequent calcium leakage have been suggested as a possible mechanism of oligomer toxicity [14]. In vitro, oligomeric αS can permeabilize lipid vesicles, possibly through a pore-like mechanism [19-21]. However, surprisingly little is known about the lipid binding properties of oligomeric αS. Using confocal fluorescence microscopy and a dye release assay we investigated the relation between lipid binding and membrane disruption by oligomeric αS. Confocal microscopy images of giant unilamellar vesicles (GUVs) and fluorescently labeled oligomeric αS indicate that oligomers preferentially bind to GUVs composed of anionic phospholipids in the Ld phase. Furthermore, we observe that the binding of oligomers to the negatively charged vesicle bilayer does not result in membrane permeabilization for all lipid compositions.

2 Materials and methods

2.1 Purification of αS

Since wild-type (wt) αS does not contain any cysteine residues necessary for fluorescent labeling, an alanine to cysteine mutation was introduced at residue 140. αS-wt and the αS-A140C mutant were expressed in Escherichia coli strain BL21(DE3) using the pT7-7 expression plasmid and purified in the presence of 1 mM DTT as previously reported [22].

2.2 αS labeling

Prior to labeling, αS-A140C was reduced with a five-fold molar excess of DTT for 30 min at room temperature. The samples were desalted with Pierce Zeba desalting columns, followed by the addition of a two-fold molar excess of Alexa 488 (AL488) C5 maleimide dye (Invitrogen) and incubated for one hour in the dark at room temperature. Free label was removed using two desalting steps. The protein labeling efficiency was estimated to be ∼80% from the absorption spectrum.

2.3 Oligomer preparation

αS-wt was mixed with αS-A140C-AL488 at a 7:1 ratio in 10 mM Tris–HCl, pH 7.4 and lyophilized. The protein was redissolved at a concentration of 1 mM using MilliQ water and incubated for 16 h in the dark in an Eppendorf thermomixer at 500 rpm at room temperature. The oligomer containing sample was filtered through a 0.2 μm Spin-X centrifugal filter (Corning) to remove possible larger aggregates. Oligomeric species were then purified on a Superdex 200 (GE) gel filtration column using 10 mM HEPES pH 7.4, 150 mM NaCl as eluant. Fractions containing αS-wt/A140C-AL488 oligomers were pooled and concentrated using a Vivaspin (Sartorius) concentrator with 10 kDa molecular weight cutoff. The oligomer concentration and final labeling ratio were determined from the absorption spectra. Generally, stock solutions with a concentration of ∼20 μM (equivalent monomer concentration) and 10–15% labeling were obtained.

2.4 Vesicle preparation

1-Palmitoyl, 2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl, 2-oleoyl phosphatidylglycerol (POPG), 1,2-dipalmitoyl phosphatidylglycerol (DPPG), 1,2-dilinoleoyl phosphatidylglycerol (18:2-PG), 1-palmitoyl, 2-oleoyl phosphatidylserine (POPS), 1,2-dioleoyl phosphatidic acid (DOPA), 1,2-dioleoyl phosphatidylethanolamine-(lissamine rhodamine B) (DOPE-Rhod) and cholesterol (Chol) were obtained from Avanti Polar lipids. Vesicles of various compositions were prepared by mixing the lipids from stock solutions in chloroform. 0.05% DOPE-Rhod was added to all compositions. GUVs containing anionic lipids were created by gentle hydration. Approximately 0.5 mg of lipid was deposited in a glass vial and dried using nitrogen gas. Residual chloroform was removed by drying under vacuum for 4 h. The lipid film was hydrated for 6 h at room temperature in 500 μl of sucrose solution of equal osmolarity to 10 mM HEPES pH 7.4, 150 mM NaCl. GUVs containing saturated lipids were hydrated at 50 °C. Electroswelling [23] was used to prepare POPC GUVs. A dried lipid film was prepared on the conducting side of an ITO coated slide and joined with a second ITO slide separated by a spacer and hydrated overnight at room temperature in 400 μl of sucrose solution in the presence of an AC electric field (1 V/mm, 10 Hz).

2.5 Confocal microscopy

Confocal microscopy was performed on a Zeiss LSM 510 confocal microscope. DOPE-Rhod was excited at 543 nm using a green He–Ne laser and αS-AL488 was excited using the 488 nm Argon laser line. Fluorescence was detected using multitrack imaging to minimize crosstalk. GUVs were diluted to approximately 60 μM in 10 mM HEPES, pH 7.4, 150 mM NaCl and mixed with fluorescently labeled oligomeric αS at a 1 μM concentration (equivalent monomer concentration). In the quantitive binding experiments all images were acquired at exactly the same instrument settings. The amount of binding was quantified from the GUV images as the maximum value of the radial profile. For each data point images of at least 15 GUVs were acquired. The experiment was performed twice.

2.6 Dye release assay

To prepare large unilamellar vesicles (LUVs), a lipid film was formed by drying ∼0.5 mg of lipid. The lipid film was hydrated for 1 h, with regular vortexing in a solution of 50 mM calcein (Sigma), 10 mM HEPES, pH 7.4. To prevent osmotic pressure differences between the vesicle interior and the solution outside, NaCl was added to maintain an osmotic strength equal to 10 mM HEPES, pH 7.4, 150 mM NaCl. The sample went through 5 freeze-thaw cycles and was then extruded 11 times through a 100 nm polycarbonate membrane filter. The extrusion procedure was repeated twice. Unencapsulated dye was removed by gel filtration through a column packed with Sephadex G-100 (GE). Finally, the phospholipid concentration was determined [24]. For the efflux assay the vesicle solution was mixed with an equal volume of protein solution. After 30 min incubation, the maximum intensity of the fluorescence emission spectrum for excitation at 497 nm was recorded on a Varian Cary Eclipse fluorimeter. After background subtraction, leakage was expressed as a percentage of the maximum effect induced by the addition of 0.5% (w/v) Triton X-100.

3 Results

3.1 Lipid binding headgroup specificity

To obtain fluorescently labeled oligomers, αS-A140C-AL488 was aggregated with αS-wt in a 1:7 ratio. Labeled αS efficiently incorporated into the oligomeric species and the labeling ratio was spectroscopically determined to be 10–15%. After purification no monomeric αS could be detected in the oligomer solution by native gradient PAGE (Supplementary information, Fig. S1). The labeled oligomers migrated at a similar molecular weight as αS-wt oligomers and showed a comparable tendency to disrupt POPG vesicles as the unlabeled species (Supplementary information, Fig. S2). It can therefore be concluded that the introduction of αS-A140C-AL488 does not affect the oligomer properties.

The binding of oligomers to GUVs was studied using confocal fluorescence microscopy. First GUVs from different anionic lipids with 20% cholesterol were prepared. Labeled αS clearly showed binding to these negatively charged GUVs (Fig. 1 A–C). GUV morphology was not affected by binding of oligomeric αS and the GUVs remained stable during the time of the experiment. Binding of αS oligomers to neutrally charged POPC GUVs could not be detected (Fig. 1D). Increasing the protein to lipid ratio 12 times to ∼1:5 did not change this observation.

figure image
Confocal Microscopy images of DOPE-Rhod labeled GUVs (left panels) and αS-A140C-AL488 oligomers (right panels). The scale bars indicate 5 μm. The lipid compositions of the GUVs were: (A) POPG:Chol 5:1, (B) DOPA:Chol 5:1, (C) POPS:Chol 5:1, and (D) POPC.

To further investigate the specificity for negatively charged headgroups, mixtures of POPC and anionic phospholipids in a 1:1 ratio were used (Fig. 2 ). The αS oligomers showed binding to POPC:DOPA and POPC:POPG GUVs but much lower binding was detected to POPC:POPS.

figure image
Confocal microscopy images of DOPE-Rhod labeled GUVs from 1:1 mixture of anionic lipids with POPC (left panels) and αS-A140C-AL488 oligomers (right panels). The scale bars indicate 5 μm. The lipid compositions of the GUVs were, (A) POPC:POPG, (B) POPC: POPS, and (C) POPC:DOPA.

3.2 Oligomer membrane permeabilization

The lipid disruption properties of oligomeric αS critically depend on the lipid headgroup composition [20]. To investigate if this originates from a different binding affinity or, for instance, a different mode of binding, a dye release assay together with a quantitive binding experiment was performed. The results shown in Fig. 3 indicate that POPC:POPG LUVs are not permeabilized whereas POPC:DOPA LUVs showed a concentration dependent calcein release upon addition of oligomeric αS. Next labeled αS oligomers were added to POPC:POPG and POPC:DOPA GUVs at different concentrations and binding was quantified from the confocal images. These results indicate that αS oligomers bind POPC:DOPA with higher affinity than POPC:POPG but the difference in binding affinity is much smaller than the difference in permeabilization properties.

figure image
Calcein release from LUVs (open symbols, dotted lines) and binding to GUVs (closed symbols, solid lines) from POPC:DOPA (grey circles) and POPC:POPG vesicles (black triangles) at a lipid concentration of 40 μM and at different αS-A140C-AL488 concentrations (oligomer concentration is expressed in equivalent monomer concentration). The error bars denote the standard deviation (n = 2).

3.3 Lipid binding phase specificity

Lipid packing can be a critical parameter in determining protein lipid interactions. We have previously observed (van Rooijen et al., submitted) that vesicle disruption by oligomeric αS is sensitive to lipid packing. Denser packed membranes were found to be less sensitive to disruption. Lipids in the liquid ordered (Lo) phase are much more densely packed than lipids in the Ld phase. Thus the effect of lipid phase on αS oligomer-lipid binding was examined. A binding specificity to Ld or Lo phases can be observed by examining binding to GUVs containing coexisting domains [25]. This can be visualized since DOPE-Rhod preferentially accumulates in Ld domains [26]. 18:2-PG:DPPG:Chol GUVs were prepared and formation of segregated Ld and Lo domains was observed (Fig. 4 A). Labeled αS oligomers preferentially accumulated in the DOPE-Rhod rich domains. This specificity for Ld domains was so strong that it was also observed using widefield fluorescence microscopy (Fig. 4B). In addition, for GUVs of other compositions containing only lipids in an ordered phase, no oligomer binding could be detected (Supplementary information, Fig. S3). Oligomeric αS thus preferentially binds to lipids in the Ld phase.

figure image
Confocal microscopy image (A) and wide field fluorescence microscopy image (B) of DOPE-Rhod labeled DLPG:DPPG:Chol 1:1:1GUVs (left panels) and αS-A140C-AL488 oligomers (right panels). The scale bar indicates 5 μm.

4 Discussion

4.1 αS Oligomers only bind to anionic lipids

αS oligomers preferentially bind to GUVs consisting of anionic lipids and no binding was detected to zwitterionic POPC GUVs. Oligomer binding was observed in mixtures of anionic lipids and POPC. Although oligomeric αS showed binding to POPC:POPG and POPC:DOPA GUVs, little binding to POPC:POPS was observed. Differences in lipid headgroup structure might be responsible for the lower apparent affinity of αS oligomers for POPS in mixed GUVs. The binding preference for anionic lipids agrees with an earlier report, indicating binding to PG and PC:PA but not to PC [21]. However, these authors did not check binding to other lipids and lipid mixtures. Others have reported oligomer binding [27] to the zwitterionic lipid PC. A potential explanation is that different intermediates in the aggregation of αS could possess different lipid binding properties. Detailed binding studies require stable purified samples. For earlier intermediates in the aggregation this might be technically challenging.

4.2 Binding does not always lead to permeabilization

In spite of the observation that αS oligomers bind POPC:POPG GUVs, our dye release experiments show that the POPC:POPG LUVs are not permeabilized. The quantitive imaging experiment revealed a smaller binding affinity for POPC:POPG. However this difference is too small to explain why POPC:POPG LUVs are not affected even at high oligomer concentrations. Thus, binding of αS oligomers to the membrane does not necessarily lead to membrane permeabilization. We postulate that membrane disruption is a two step process in which the initial binding and the subsequent disruption require different lipid properties. The mechanism of permeabilization is currently still debated [19, 20, 28]. When comparing the concentration dependence of leakage with the binding curves it appears that the oligomers do not need to saturate the membranes before leakage occurs, which is consistent with a pore model. However, one must be cautious in comparing the GUV and LUV systems.

4.3 αS Oligomers only bind to Ld domains

Lipid packing is an important parameter in determining protein-lipid interactions. We have previously shown that bilayer disruption is sensitive to the accessibility of the hydrophobic bilayer core (van Rooijen et al., submitted). The results presented here are consistent with these data, and show that αS oligomers preferentially bind to lipids in the Ld phase, and do not show affinity for Lo bilayers. In the Lo phase lipids are much more densely packed with the lipid tails extended and a reduced area per headgroup. Possibly, the higher surface charge density in the Lo phase makes it energetically unfavorable for the negatively charged αS to adsorb on the membrane. Alternatively steric hindrance or inaccessibility of the membrane hydrophobic core offer possible explanations for why oligomeric αS does not bind to the Lo phase. Interestingly, monomeric αS has a similar binding specificity for anionic lipids in the Ld phase [9, 10]. Lipid binding of monomeric αS occurs at residues ∼1–100 [12]. Although the protein has a net negative charge, the N-terminal residues contain many hydrophobic and positively charged amino acids, explaining the specificity for anionic lipids. The similar specificity for oligomeric αS could suggest that in the oligomers, the N-terminal is available for lipid binding. We have not found evidence for a structural reorganization of αS oligomers upon lipid interaction by circular dichroism spectroscopy, consistent with earlier work [21]. Folding of the N-terminus into a helical conformation upon lipid binding, as occurs with monomeric αS, is therefore unlikely for oligomeric αS.

Summarizing, we have systematically studied the lipid binding properties of oligomeric αS and its relation to bilayer disruption. The data show that oligomeric αS preferentially binds to anionic lipids in the Ld phase. Furthermore, binding does not lead to membrane disruption for all lipid compositions, which suggests that binding and disruption are distinct processes.

Acknowledgements

This work is part of the research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)”, which is financially supported by the “Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)”.

    Appendix A A

    Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2008.10.009.