From the Department of Biochemistry, University of
Illinois, Urbana, Illinois 61801 and the § Department of
Cell and Structural Biology, University of Illinois,
Urbana, Illinois 61801
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ABSTRACT |
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-Synuclein is a highly conserved presynaptic
protein of unknown function. A mutation in the protein has been
causally linked to Parkinson's disease in humans, and the normal
protein is an abundant component of the intraneuronal inclusions (Lewy
bodies) characteristic of the disease.
-Synuclein is also the
precursor to an intrinsic component of extracellular plaques in
Alzheimer's disease. The
-synuclein sequence is largely composed of
degenerate 11-residue repeats reminiscent of the amphipathic
-helical domains of the exchangeable apolipoproteins. We
hypothesized that
-synuclein should associate with phospholipid
bilayers and that this lipid association should stabilize an
-helical secondary structure in the protein. We report that
-synuclein binds to small unilamellar phospholipid vesicles
containing acidic phospholipids, but not to vesicles with a net neutral
charge. We further show that the protein associates preferentially with
vesicles of smaller diameter (20-25 nm) as opposed to larger (~125
nm) vesicles. Lipid binding is accompanied by an increase in
-helicity from 3% to approximately 80%. These observations are
consistent with a role in vesicle function at the presynaptic
terminal.
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INTRODUCTION |
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Our studies in songbird brain resulted in the identification of a presynaptic protein, which we called synelfin, whose expression is regulated in the avian song control circuit during the critical period for song learning (1). We hypothesized that this protein might play an important role in the modulation of synaptic plasticity, and have focused on determining its normal cellular and biochemical function.
Independent evidence has emerged that implicates this protein in human
neurodegenerative disease. Ueda et al. (2) reported that a
fragment of the homologous human protein is an intrinsic component of
the amyloid plaques which deposit extracellularly in the brains of
individuals with Alzheimer's disease; they called the protein
NACP,1 for
non-amyloid component
precursor. More recently, Polymeropoulos et al.
(3) described a mutation in the identical protein -synuclein, which
apparently causes a hereditary form of Parkinson's disease. This
neurodegenerative disease is also characterized by the abnormal deposition of cellular material, in this case the intracellular inclusions known as Lewy bodies (4), and these structures are also
immunopositive for
-synuclein (5).
Synelfin/-synuclein/NACP (which we will henceforth refer to as
-synuclein) is a small, soluble protein of 140-143 amino acids
which is highly enriched in presynaptic nerve terminals (1, 6, 7). The
mechanism of its localization is unclear, however, because unlike most
presynaptic proteins,
-synuclein is not tightly associated with
either the synaptic vesicle or the synaptic plasma membrane (1, 8). The
-synuclein sequence has been extremely well conserved in evolution
(1, 8, 9), implying functional constraints on its three-dimensional
structure. Paradoxically, its conformation in solution appears to be
largely random, as estimated from circular dichroism measurements
(10).
The most notable feature of the sequence is a recurring 11-residue
periodicity, which is consistent with the capacity to fold into an
amphipathic -helix (1). When projected onto a helical wheel (11),
several of the
-synuclein 11-mers display a distinctive distribution
of polar and nonpolar residues to opposite faces of the helix, which
conforms very well to the consensus class A2 amphipathic
-helix, found in the lipid-binding domains of the exchangeable
apolipoproteins A-II, C-I, C-II, and C-III (12, 13). Observation of
this structural motif led us to hypothesize that: 1)
-synuclein
would have the capacity to associate with phospholipid membranes, and
2) membrane association would be dependent upon an
-helical
secondary structure. Here we show data in support of both of these
hypotheses. Furthermore, we demonstrate that, in contrast to the
exchangeable apolipoproteins,
-synuclein binds exclusively to acidic
phospholipids and preferentially to vesicles with smaller diameters.
These observations suggest that the secondary structure of
-synuclein might be sufficient to target it to a particular subset
of vesicles in the presynaptic terminal, perhaps facilitating selective
regulation of some aspect of vesicle function.
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EXPERIMENTAL PROCEDURES |
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Materials
Bovine brain phospholipid extract (Folch extract 1) and egg ovalbumin were purchased from Sigma (+99% grade). 1-Palmitoyl 2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl 2-oleoyl phosphatidic acid (POPA), 1-palmitoyl 2-oleoyl phosphatidylserine (POPS), 1-palmitoyl 2-oleoyl phosphatidylethanolamine (POPE), and liver phosphatidylinositol (PI) were obtained from Avanti Polar Lipids (Birmingham, AL). All other reagents were analytical grade.
Methods
Purification of Recombinant -Synuclein--
The canary
-synuclein cDNA was subcloned into the plasmid pET28a (Novagen),
which directs expression in Escherichia coli via an
isopropyl-1-thio-
-D-galactopyranoside-inducible T7
promoter. Purification of
-synuclein was based on a modification of
Weinreb et al. (10), who documented the heat stability of
the protein; briefly, cell lysates are boiled, and the soluble fraction
is precipitated with 60% ammonium sulfate. The pellet is resuspended in 25 mM Tris, pH 9.0, loaded onto a Poros HQ column
(Perseptive Biosystems), and eluted with 0-1 M NaCl.
-Synuclein containing fractions are pooled and exchanged into sodium
acetate buffer, pH 4.0, loaded onto a Poros HS column (Perseptive
Biosystems), and eluted with 0-1 M NaCl.
-Synuclein
fractions are finally loaded onto a gel exclusion column (Superose 6, Pharmacia) to confirm purity and achieve final buffer exchange.
The Binding of -Synuclein to Small (SUV) and Large (LUV)
Unilamellar Vesicles--
SUV of various lipid compositions were
prepared by the sonication technique of Barenholz et al.
(14). The SUV were reisolated by ultracentrifugation at 55,000 rpm in a
TLA 100.3 rotor for 2 h at 25 °C and exhibited hydrodynamic
diameters of 25 ± 5 nm as estimated by gel filtration
chromatography (15). LUV were prepared as described by Hope et
al. (16). Briefly, phospholipid multilamellar liposomes in buffer
were placed into an extrusion device (Lipex Biomembranes Inc.,
Vancouver, British Columbia, Canada) which passed the mixture through
polycarbonate filters of 100-nm pore size under pressures of 100-500
p.s.i. This procedure routinely produced unilamellar vesicles with
hydrodynamic diameters of 125 ± 30 nm. SUV or LUV preparations in
Tris buffer were mixed with
-synuclein to determine the affinity of
-synuclein for lipid surfaces of varying curvature. The vesicles and
-synuclein at a 20:1 mass ratio of phospholipid to
-synuclein
were incubated in Tris buffer for 2-24 h at 25 °C. The resulting
complexes were separated from unreacted lipid and protein on a
calibrated Superose 6 (10 mm × 30 cm) gel filtration column
(Pharmacia) eluted at 0.5 ml/min with Tris buffer. The protein content
of the fractions was determined using the Markwell modification of the
Lowry protein assay (17) while phospholipids were determined as
inorganic phosphorus by the method of Sokoloff and Rothblat (18). In
some experiments, the fractions corresponding to lipid-free protein, SUV and any multilamellar vesicles were pooled and analyzed by immunoblotting with
-synuclein monoclonal H3C, as described in Ref.
1.
Secondary Structure Analysis--
The structural predictions
based on the primary sequence of canary -synuclein (1) were
determined using programs ANTHEPROT (19) and the Wisconsin Sequence
Analysis program (Genetics Computer Group) running on an IBM PC. Three
algorithms, the Levin homologue method (20), the GOR 1 prediction
method (21), and the Chou-Fasman prediction method (22) were used for
the sequence analysis because of their distinct prediction strategies.
Consensus predictions were obtained from the results of all three
methods by manual comparison of the predictions for each amino acid in
the sequence (see the legend to Fig. 5 for more detail). For more
information on the application of these established prediction methods
to lipid-binding proteins, see Ref. 23.
Circular Dichroism Measurements and Analysis--
The average
secondary structure contents of lipid-free and lipid-bound
-synuclein were determined by circular dichroism (CD) spectroscopy
using a Jasco J-720 spectropolarimeter. Spectra were taken at 25 °C
in a 0.1-cm path length quartz cuvette containing the sample at
concentrations of 0.1-0.2 mg/ml of protein in 20 mM
phosphate buffer, pH 8.0, without NaCl. The spectra were corrected for
buffer and vesicle contributions as appropriate. The percent
-helix
was determined from the molar ellipticities at 222 nm (42) as well as
by computer fitting to a library of CD spectra from proteins of known
structure using the learning neural network program K2D which is based
on the algorithm published by Andrade et al. (24).
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RESULTS |
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Characterization of -Synuclein with Respect to
Apolipoproteins--
The identification of several predicted class
A2 amphipathic
-helices in the
-synuclein sequence
suggested that
-synuclein might exhibit traits that are
characteristic of the plasma apolipoproteins. Experiments showed that
recombinant
-synuclein could not clear multilamellar
dimyristoylphosphatidylcholine liposomes, whereas apoA-I readily
cleared them into small disc-shaped particles (data not shown).
Furthermore,
-synuclein did not yield protein-lipid complexes using
the sodium cholate method of reconstituted high density lipoprotein
preparation commonly used with apoA-I (data not shown). Finally,
studies using the chemical cross-linking agent BS3 failed
to demonstrate self-association of
-synuclein when free in solution
(not shown), a common observation with apolipoproteins. Taken together,
these results indicate that despite similarities in predicted
structure,
-synuclein does not possess the critical properties of
apolipoproteins.
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Structural Analysis of -Synuclein in the Lipid-free and
Lipid-bound States--
Circular dichroism spectroscopy was used
to determine the effects of lipid binding on the secondary structure of
-synuclein. The spectrum of lipid-free
-synuclein is shown in
Fig. 4A. The prominent minimum
ellipticity at 200 nm was characteristic of a high percentage of random
coil. Table II lists the predicted secondary structure compositions based on the CD spectrum from 200 to
241 nm. The data indicate that lipid-free
-synuclein is highly
unstructured in solution. This is in strong agreement with the results
of Weinreb et al. (10) for human
-synuclein. Upon lipid
binding,
-synuclein undergoes a striking change in conformation (Fig. 4A, Table II), shifting to a highly
-helical
conformation as evidenced by the characteristic minima at 208 and 222 nm. The helical character of the protein increased from less than 3%
to greater than 71%
-helix when bound to lipid. A similar change in
conformation was observed when
-synuclein bound to PC/PS vesicles (Fig. 4B), although the helical content was not as high as
in the case of the PC/PA vesicles. This difference is due to the spectral contribution of lipid-free
-synuclein, which was present only in the PC/PS incubations (see Fig. 2). The incubation of
-synuclein with PC-only vesicles resulted in a CD spectrum that resembled lipid-free
-synuclein, confirming that
-synuclein did
not bind to these vesicles. The secondary structure content of
-synuclein bound to PC/PA vesicles was also measured under conditions of increasing ionic strength. Table
III shows that as the NaCl concentration
was increased, the degree of
-helix content decreased with a
corresponding increase in the estimated amount of random coil content.
These changes in structure generally correlated with the decreases in
-synuclein binding shown in Fig. 3. In a single experiment with a
small amount of recombinant human NACP/
-synuclein (a kind gift of
Peter Lansbury), we observed a qualitatively similar increase in
-helical content when the protein associated with PC/PA vesicles
(data not shown).
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DISCUSSION |
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-Synuclein binds best to vesicles containing at least 30%
acidic phospholipid. Because the requirement for acidic phospholipid was determined at a lipid/protein mass ratio of 20:1 (molar ratio ~400:1), there must be an excess of acidic sites available even at
20% acidic phospholipid, suggesting that binding requires multiple interactions.
A preference for acidic phospholipids could be a mechanism for
targeting the protein to a particular membrane or vesicle population. Synuclein was first isolated from the electric fish Torpedo because of
its association with the abundant cholinergic vesicles of that animal's electric organ, essentially a massive synapse (25), and EM
immunohistochemistry suggests that the protein is concentrated in the
cytoplasmic matrix surrounding synaptic vesicles in the presynaptic
terminal (25, 27). Our studies of primary hippocampal neurons show a
tight colocalization of -synuclein with the synaptive vesicle
protein synapsin by immunofluorescence, further indication that the
protein associates with this vesicle population (6). However, in
subcellular fractionation experiments,
-synuclein does not tightly
copurify with synaptic vesicles, and the majority of
-synuclein in
cytosolic and synaptosomal subfractions is freely soluble (1, 8, 26).
This paradoxical feature of
-synuclein could result from rapid
exchange of the protein between membrane surface and cytosol, modulated
by factors that have yet to be identified.
The average composition of synaptic vesicles is estimated to be 0-2%
PA, 40-48% PC, 24-36% PE, 4-12% sphingomyelin, 7-12% PS, 3-4%
PI, with a cholesterol:phospholipid ratio of ~0.5-0.6 (28-30); the
proportion of acidic phospholipids (10-18%) is somewhat less than the
30% acidic phospholipid required for maximal binding of -synuclein
to our synthetic membranes. However, these estimates of composition do
not take into account the asymmetric distribution of phospholipid
species between the two leaflets of the membrane, a critical property
of many biological membranes, including synaptic vesicles.
Determination of phospholipid asymmetry in synaptic vesicles has been
somewhat confounded by methodological concerns, but it is estimated
that 100% of the PI in synaptic vesicles is localized to the
cytoplasmic face (30). Also, it is increasingly clear that
phospholipids can become locally concentrated within a single membrane
leaflet, with important consequences for protein localization. For
example, microdomains of PS form in response to increased
[Ca2+], encouraging translocation and activation of
protein kinase C (31). Bilayer asymmetry and/or phospholipid
microdomains might be critical to
-synuclein localization in
vivo, yet difficult to reproduce in vitro.
The observation that -synuclein binds small (20-25 nm) in
preference to large (~125 nm) unilamellar vesicles of the same composition most likely results from differences in phospholipid packing on the vesicle surface. The phospholipid bilayer of an LUV is
an essentially planar surface in which the fatty acyl side chains are
motionally restricted (32), compared with the curved surface of an SUV
(33). It has been demonstrated that the initial insertion of the
amphipathic
-helical regions of plasma lipoproteins into a
phospholipid bilayer is facilitated by the existence of lipid packing
defects caused by extreme membrane curvature or by the differential
packing properties of different phospholipids within the membrane (34).
It is likely that these factors also affect the partitioning of
-synuclein between the lipid-bound and lipid-free states. This
observation may be important for in vivo targeting of
-synuclein to vesicles or membranes with irregular or curved
surfaces.
The binding of -synuclein to acidic phospholipid vesicles (PC/PA or
PC/PS) is accompanied by a large increase in its
-helical content;
no such increase is observed in the presence of PC vesicles, to which
-synuclein does not bind (Table II).
-Helicity declines in
parallel with lipid binding as salt concentration is increased (Table
III). These observations suggest that lipid binding is mediated by
-helical structures which are themselves stable only in the presence
of lipid, and are consistent with our initial identification of
consensus domains for lipid-binding amphipathic
-helices in the
-synuclein sequence (1). We have now extended our analysis of the
-synuclein sequence, and have identified 5 potential amphipathic
-helical domains representing 2 different classes, in accordance with the criteria established by Segrest et al. (13) for
analysis of the exchangeable apolipoproteins.
A key feature of the -synuclein peptide sequence is a set of 7 degenerate 11-residue repeats. 11-mer repeats are also a hallmark of
the amphipathic
-helices of the exchangeable apolipoproteins, which
mediate a variety of lipid and protein interactions. These
-helices
are divided into classes depending upon the distribution of residues to
the polar and nonpolar faces of the helices (13). Class A helices are
typically lipid-binding, and are characterized by a clustering of basic
residues at the polar/nonpolar interface and acidic residues at the
center of the polar face. Class G* helices are implicated
in protein interactions; this class is less well defined and is
typically characterized by a random radial distribution of charged
residues to the polar face of the helix. Class Y helices (which are
rare) also mediate lipid interactions (35, 36), and are characterized
by 2 clusters of negative residues separating 3 clusters of positive
residues on the polar face of the helix.
Class A helices are further subdivided as class A1, A2, or A4. Class A4 is least common and is distinguished by clusters of basic residues ±120° from the center of the nonpolar face. Class A1 helices typically have arginines positioned ±90° from the center of the nonpolar face, with leucines at the center of the nonpolar face. Class A2, the motif which is best defined and correlated with the highest lipid affinity, is characterized by lysines positioned ±100° from the center of the nonpolar face, and glutamates clustered in the center of the polar face. Segrest et al. (12) proposed a model for the interaction of class A helices with phospholipid membranes, wherein positively charged lysine or arginine residues positioned at the polar/nonpolar interface interact with negatively charged phosphate groups in the phospholipid backbone. The amphipathicity of these amino acid residues, particularly lysine with its longer aliphatic side chain, allows the hydrophobic face of the helix to penetrate more deeply into the bilayer interior, stabilizing the lipid-protein interaction (12).
Helices 1-4 of -synuclein are clearly class A, and most resemble
the class A2 consensus in the positioning of the
interfacial basic residues at ±100° from the center of the nonpolar
face, in the preponderence of lysines over arginines, and in the
presence of glutamate residues on the polar face. These domains would
be predicted to mediate the binding of
-synuclein to phospholipid bilayers. Helix 5 resembles the class G* helix, and thus is
a candidate for protein-protein interactions. Interestingly, this
putative helix (residues 61-93) corresponds almost exactly to the
non-amyloid component peptide (residues 61-95) purified from the
Alzheimer's senile plaque.
A few notable features distinguish the putative amphipathic
-synuclein helices from those in the exchangeable apolipoproteins. The threonine residues at the center of the nonpolar faces of helices
2-4 are unique to
-synuclein and are perfectly conserved among
sequences from canary, human, and rat (1), which might represent a site
for regulation by phosphorylation. Threonine, although polar, can
reside on the nonpolar face of the helix due to its relatively long
aliphatic side chain (13). Phosphorylation of these residues would
introduce negative charges which would be expected to disrupt lipid
binding. Evidence exists that a closely related isoform of this
protein,
-synuclein, is phosphorylated in vivo and
in vitro (37), although the precise sites remain to be
mapped.
Another unique aspect of the -synuclein sequence is the absence of
prolines among the 11-mer repeating domains. The amphipathic
-helical motifs of the exchangeable apolipoproteins are typically divided into anti-parallel 22-residue segments by proline residues, which introduce helix-breaking hairpin turns. The resulting short helical segments function cooperatively, and this cooperativity enhances many of the functions of the apolipoproteins, including their
dynamic ability to bind and solubilize lipid particles (for a review,
see Ref. 13) In contrast, the
-synuclein helices 1-4 seem to be
punctuated at various points by nonpolar residues that are predicted to
disrupt the amphipathicity of a helix (Fig. 6). It is not known whether
these residues mediate breaks to form distinct helices or merely
introduce kinks that do not totally disrupt a single, long helix.
Borhani et al. (38) have recently solved the x-ray crystal structure of a fragment of apoA-I. These authors suggest that the helices in apoA-I are not arranged in an antiparallel fashion when associated with spherical lipid particles, in contrast with the evidence for an antiparallel orientation in discoidal particles (39). The intrahelical regions in this model form kinks that are significantly less than 180° which allows the protein to wrap around the sphere of lipid in a ring or horseshoe topology. Thus, apoA-I may have the ability to adjust the degree of turn between adjacent helical segments, with turns approximating 180° in discs but of lesser degree in spherical particles.
The absence of proline-induced hairpin turns may explain why
-synuclein can bind spherical and planar surfaces, yet fail to form
discoidal particles. It should be noted that among the many
protein/lipid incubations performed in the course of the work described
here, we never detected structures in the size range of discs (7-12
nm). In addition,
-synuclein failed to clear multilamellar
dimyristoylphosphatidylcholine liposomes, which are readily cleared
into discoidal particles by apoA-I (data not shown), and did not form
discs using the sodium cholate dialysis method, which is well
established for the preparation of reconstituted high density
lipoprotein with apoA-I (data not shown).
Clearly, identification of the physiological substrates for
-synuclein binding will be critical to discerning its function in vivo. It is interesting to note that the class A helices
are typically stabilizing to membranes, inhibiting membrane
fusion and lysis by relieving negative monolayer curvature
strain (40, 41). Whether
-synuclein exerts similar stabilizing
effects on its target membrane, e.g. to modulate some aspect
of vesicle trafficking, remains a question for future research. The
very dynamic structure of
-synuclein in response to its environment will likely be important in understanding its propensity to aggregate and accumulate in both Alzheimer's and Parkinson's
diseases.
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ACKNOWLEDGEMENTS |
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We thank Dr. Wendi Rodrigueza for providing LUV and Wendy Woods and Michael Urban for invaluable technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 1R01 AG13762.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Cell and Structural Biology, B107 Chemical and Life Science Laboratory, 601 S. Goodwin, Urbana, IL 61801. Tel.: 217-244-4525; Fax: 217-244-1648; E-mail: j-george{at}uiuc.edu.
1 The abbreviations used are: NACP, non-amyloid component precursor; POPC, 1-palmitoyl 2-oleoyl phosphatidylcholine; POPA, 1-palmitoyl 2-oleoyl phosphatidic acid; POPS, 1-palmitoyl 2-oleoyl phosphatidylserine; POPE, 1-palmitoyl 2-oleoyl phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; SUV, small unilamellar vesicle; LUV, large unilamellar vesicle; MLV, multilamellar vesicle.
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REFERENCES |
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