Abstractα-Synuclein (α-syn) protein has been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We report here that human wild-type α-syn, PD-linked mutant α-syn(Ala30Pro) and mutant α-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. The mutant α-syn forms more β-sheet and mature filaments than the wild-type protein. These findings suggest that accumulation of α-syn as insoluble deposits of amyloid may play a major role in the pathogenesis of these neurodegenerative diseases

Curran, Martin D

El-Agnaf, Omar M.A

Jakes, Ross

Wallace, Andrew

Elsevier - Publisher Connector 

Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α-synuclein protein implicated in Parkinson's disease 

E¡ects of the mutations Ala30 to Pro and Ala53 to Thr on the physical
and morphological properties of K-synuclein protein implicated in
Parkinson’s disease
Omar M.A. El-Agnafa;*, Ross Jakesb, Martin D. Curranc, Andrew Wallacea
aCentre for Peptide and Protein Engineering, School of Biology and Biochemistry, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK
bMedical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
cNorthern Ireland Histocompatibility and Immunogenetics Laboratory, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, UK
Received 18 September 1998; received in revised form 22 October 1998
Abstract K-Synuclein (K-syn) protein has been found in
association with the pathological lesions of a number of
neurodegenerative diseases. Recently, mutations in the K-syn
gene have been reported in families susceptible to an inherited
form of Parkinson’s disease. We report here that human wild-
type K-syn, PD-linked mutant K-syn(Ala30Pro) and mutant K-
syn(Ala53Thr) proteins can self-aggregate and form amyloid-like
filaments. The mutant K-syn forms more L-sheet and mature
filaments than the wild-type protein. These findings suggest that
accumulation of K-syn as insoluble deposits of amyloid may play
a major role in the pathogenesis of these neurodegenerative
diseases.
z 1998 Federation of European Biochemical Societies.
Key words: K-Synuclein; Parkinson’s disease; Lewy body;
Self-aggregation; Amyloid; Neurodegenerative disease
1. Introduction
Parkinson’s disease (PD) and dementia with Lewy bodies
(DLB) are pathologically characterised by the presence of
Lewy bodies (LBs) and Lewy neurites (LNs) accompanied
by dead and dying neurones (reviewed in [1]). Immunohisto-
chemical studies of LBs and LNs and immunoelectron micros-
copy studies of LB ¢laments indicate that the presynaptic
protein K-synuclein (K-syn) forms the major ¢lamentous com-
ponent of LBs and LNs [2^5]. It has also been shown that
some of the LBs in PD display thio£avine-S reactivity, indi-
cating the presence of amyloid-like ¢laments [6,7]. This sug-
gests that abnormal deposition of K-syn may be involved in
the pathogenesis of PD and DLB. This proposal has been
supported by the recent discovery of mutations in the K-syn
gene in families susceptible to inherited forms of PD [8,9].
One mutation, found in certain Italian and Greek families,
results in an Ala to Thr substitution at position 53 of K-syn [8]
in a region predicted to adopt an K-helical structure sur-
rounded by L-sheets. The other mutation, Ala30 to Pro, has
been detected in a family of German origin, unrelated to the
families in which the Ala53Thr mutation was found [9]. These
substitutions have been proposed to disrupt the K-helical
structure in this region and extend the L-sheet, rendering the
protein more prone to self-aggregation [8^11]. This may lead
to the formation of abnormal deposits which could contribute
to nerve degeneration in the brain, similar to the accumula-
tion of the L-amyloid protein (AL) in Alzheimer’s disease
(AD), Prion protein in prion diseases and Huntingtin protein
in Huntington’s disease (HD) (reviewed in [12]).
Studies of neurodegenerative disorders such as AD [13^15],
prion protein diseases [16^18], and HD [19] have highlighted
the importance of the biophysical properties of mutant cellu-
lar proteins in initiating and propagating neuronal lesions
found in these neurodegenerative disorders. We therefore in-
vestigated some of the biophysical properties of recombinant
human wild-type K-syn and the PD-linked mutants K-syn-
(Ala30Pro) and K-syn(Ala53Thr). We report here that mutant
K-syn proteins developed more L-sheet structure and formed
more mature ¢laments than wild-type as revealed by SDS-
PAGE, thio£avine-S staining, electron microscopy and circu-
lar dichroism studies.
2. Materials and methods
2.1. Preparation of K-syn proteins
Recombinant K-syn proteins were expressed in Escherichia coli and
puri¢ed as previously described [20].
2.2. Preparation of aggregated ‘aged’ solutions of K-syn proteins
K-Syn proteins were dissolved in PBS (phosphate-bu¡ered saline
pH 7.4) at a concentration of 110 WM, and the resulting solutions
were aged for 7 days at 37‡C.
2.3. Electron microscopy (EM)
A 10 Wl sample was placed on a carbon-coated copper grid, and
incubated for 5 min. The droplet was then displaced with 10 Wl of
0.5% (v/v) glutaraldehyde and incubated for an additional 5 min. The
grid was then washed with ¢ve drops of water and wicked dry. Fi-
nally, the sample was stained with 10 Wl of 2% (w/v) uranyl acetate
solution for 2 min. This solution was wicked o¡, the grid was air-
dried, then viewed on a JEOL TEMCX 100 II electron microscope
and photographed.
2.4. Circular dichroism
Spectra were recorded at 21‡C over a range 190^250 nm on a
JASCO J720 spectropolarimeter. An aliquot of the protein solution
(about 130 Wl) was placed in a quartz cell (0.2 mm pathlength). All
spectra were corrected by subtracting the baseline of the appropriate
solvent. Results are expressed as molar ellipticity, [a], in units of deg
cm2 dmol31.
3. Results and discussion
Interest in the K-syn protein was spurred by reports of its
FEBS 21172 30-11-98
0014-5793/98/$19.00 ß 1998 Federation of European Biochemical Societies. All rights reserved.
PII: S 0 0 1 4 - 5 7 9 3 ( 9 8 ) 0 1 4 1 9 - 7
*Corresponding author. Fax: (44) (1232) 236505.
E-mail: o.elagnaf@qub.ac.uk
Abbreviations: AD, Alzheimer’s disease; AL, L-amyloid protein;
DLB, dementia with Lewy bodies; HD, Huntington’s disease; LB,
Lewy body; LN, Lewy neurite; NAC, non-AL component of AD
amyloid; PD, Parkinson’s disease
FEBS 21172 FEBS Letters 440 (1998) 67^70
association with the pathological lesions of a number of neu-
rodegenerative diseases [2^5,21^23]. The recent discovery that
mutations in the K-syn gene give rise to families susceptible to
an inherited form of PD [8,9] has aroused scienti¢c interest in
this protein, but the mechanism by which these mutations give
rise to PD is not known. However, one possibility is that
K-syn containing these mutations aggregates more rapidly
[8^11]. We therefore decided to examine the biophysical prop-
erties of K-syn so that we could determine whether the PD-
linked mutations had a major impact on these properties.
In order to examine the possible self-aggregation of K-syn,
we aged solutions of recombinant K-syn for 7 days and we
investigated whether any self-oligomerisation was taking
place. As expected, self-oligomerisation of wild-type and mu-
tant K-syn proteins was detected as revealed by SDS-PAGE
(data not shown). Larger amounts of dimeric species and
oligomers were formed in aged than in fresh solutions. Fur-
thermore, aggregated K-syn species were found in the stacking
gel in aged samples, suggesting the formation of larger aggre-
gates that did not enter the separating gel. Similar observa-
tions have been reported for K-syn isolated from LBs [4].
During the preparation of this article, Hashimoto et al. [7]
reported that wild-type K-syn can self-aggregate from solution
and form amyloid-like ¢laments at pH 6.9, but not at pH 7.4.
We, in contrast, were able to detect aggregation of both wild-
type and mutant K-syn at pH 7.4 when we incubated the
FEBS 21172 30-11-98
Fig. 1. Negatively stained electron micrographs (EM) of ¢laments obtained from aged K-syn proteins. A: K-syn. B,C: K-syn(Ala30Pro). D^G:
K-syn(Ala53Thr). The EM showed di¡erent morphology of ¢laments including: short irregular 6^8 nm wide ¢laments (A); short straight 8^10
nm wide ¢laments (B); long straight bundles of 10^12 nm wide ¢laments (C); long straight individual 10^12 nm wide ¢laments (D); twisted
(arrowed) ¢laments with varying width of 7^12 nm (E); two 6 nm wide ¢laments coiled around each other and joined at intervals (arrowed) to
form a ¢lament with a variable diameter (F); 25 nm wide branched ¢lament with 6^10 nm extensions (arrowed) at the end (G). Scale bar,
200 nm.
O.M.A. El-Agnaf et al./FEBS Letters 440 (1998) 67^7068
proteins at a higher concentration and for a longer period of
time than reported in [7].
The formation of K-syn ¢laments in a crossed L-pleated
sheet conformation was assessed by thio£avine-S staining
and by EM. Aggregates were formed from both aged wild-
type and mutant K-syn proteins and were thio£avine-S pos-
itive (data not shown), indicative of the presence of aggregates
with L-pleated sheet conformation characteristic of amyloid-
like ¢laments [24]. In contrast, fresh samples were thio£avine-
S negative, indicating the absence of any aggregates. We used
EM to examine uranyl acetate-stained ¢lament preparations,
made from fresh and aged samples formed under similar con-
ditions as used for the aggregation assay (Fig. 1). This re-
vealed ¢laments of variable length and of 6^25 nm in diameter
from aged samples of K-syn proteins, similar in size to those
found in LBs and LNs ([3] and references therein). Filaments
of diameter 6^8 nm wide are referred to as proto¢laments
while those of diameter 10 nm and above are referred to as
mature ¢laments. Clumps of amorphous material were also
present in some preparations. We did not detect any ¢laments
in the fresh samples. We observed di¡erences in the morphol-
ogy of the ¢laments formed by wild-type and mutant K-syn
under similar conditions. The wild-type K-syn forms mainly
short, irregular 6^8 nm wide unbranched proto¢laments (Fig.
1A) together with a very few long single straight 10 nm wide
mature ¢laments (data not shown). The ¢laments were some-
times associated with small clumps of amorphous material. In
the case of the K-syn mutants, some small amorphous clumps
were also seen, but the samples mostly consisted of single,
isolated ¢laments. Representative examples of the latter are
shown in Fig. 1B^G, with ¢laments of diameter between 6
and 25 nm and of variable length of 50^600 nm. The aggre-
gates of mutant K-syn(Ala30Pro) formed various morpholo-
gies, including proto¢laments, similar to those seen in the
wild-type (Fig. 1A), as well as short 8^10 nm wide single
¢laments (Fig. 1B), but the majority of long mature ¢laments
were seen either individually (data not shown) or as bundles
of 10^12 nm wide ¢laments (Fig. 1C). In the case of K-syn-
(Ala53Thr), similar ¢laments were observed (Fig. 1D) as well
as twisted single ¢laments with a width varying between 7 and
12 nm (Fig. 1E) and also coiling of 6 nm wide proto¢laments
to produce cable-like structures joined at intervals (Fig. 1F).
We have also seen branched mature ¢laments with slender 6^
10 nm wide extensions at the end of a 25 nm wide ¢lament
(Fig. 1G). Goedert and co-workers have proposed a model in
which the K-syn molecules in LBs and LNs assembled ¢rst to
form proto¢laments, two of which could associate to produce
a variable twisted mature ¢lament [3]. Indeed, we have ob-
served such a morphology (Fig. 1G) in a sample of aggregated
K-syn(Ala53Thr). This model is also supported by our ¢nding
that wild-type K-syn formed mainly proto¢laments (Fig. 1A)
compared to the K-syn mutants which formed mainly mature
¢laments under similar conditions (Fig. 1B^G).
Having found that K-syn proteins can self-aggregate and
form ¢laments in a crossed L-pleated sheet conformation as
revealed by SDS-PAGE, EM and thio£avine-S staining, cir-
cular dichroism (CD) spectroscopy was used to study the
conformational preferences of the K-syn proteins. CD spectra
have been used to distinguish between proteins or peptides
displaying predominantly K-helical, L-sheet or random coil
conformations. The ¢rst are characterised by a maximum at
192 nm and minima at 208 and 222 nm and the second by a
maximum at 195 nm and a single minimum at 218 nm. In
both cases, the maximum has greater intensity than the mini-
mum. Random conformations are characterised by a mini-
mum at 197 nm. CD spectra were recorded from fresh and
aged solutions of K-syn proteins. The spectrum of the fresh
K-syn(Ala53Thr), with a minimum at 196 nm, changed over
7 days in PBS to a spectrum with a maximum at 190 nm and
a minimum at 198 nm (Fig. 2A). We interpret this change in
the spectrum as indicating a transition from predominantly
random conformation to a mixture of L-sheet and random
conformations. Similar observations have been reported for
comparison studies between fragments of wild-type AL and
AL containing the Flemish mutation [13]. In contrast, when a
similar experiment was carried out on wild-type K-syn and K-
syn(Ala30Pro) under similar conditions, the spectrum of both
proteins showed little change upon ageing of the solutions for
7 days in PBS, with only a slight decrease in intensity in the
minimum at 197 nm which is characteristic of random con-
FEBS 21172 30-11-98
Fig. 2. CD spectra. A: E¡ect of ageing on the CD spectra of solu-
tions of K-syn, K-syn(Ala53Thr) and K-syn(Ala30Pro). CD spectra
of solutions of K-syn proteins (110 WM) in PBS were obtained im-
mediately upon preparation and after incubation at 37‡C for 7 days.
CD spectra were obtained after dilution to a ¢nal concentration of
10 WM in PBS: K-syn at time 0 (light solid line); K-syn after 7 days
(heavy solid line); K-syn(Ala53Thr) at time 0 (light dashed line);
K-syn(Ala53Thr) after 7 days (heavy dashed line); K-syn(Ala30Pro)
at time 0 (light dotted line); K-syn(Ala30Pro) after 7 days (heavy
dotted line). B: CD spectra of solutions of K-syn proteins in aceto-
nitrile/PBS (1:1). CD spectra were obtained after dilution of a fresh
stock solutions of K-syn proteins (110 WM) in PBS with appropriate
concentration of acetonitrile/PBS to give a ¢nal concentration of
acetonitrile 50% and 10 WM of the proteins: K-syn (solid line);
K-syn(Ala53Thr) (dashed line); K-syn(Ala30Pro) (dotted line).
O.M.A. El-Agnaf et al./FEBS Letters 440 (1998) 67^70 69
formations (Fig. 2A). As discussed above, examination by
SDS-PAGE and EM showed signs of self-aggregation and
¢lament formation in all the K-syn proteins examined. These
aggregates had a structure in crossed L-pleated sheet confor-
mation as revealed by thio£avine-S staining (data not shown).
Thus the fact that we did not directly observe a transition to
L-sheet during ageing of wild-type K-syn and the mutant K-
syn(Ala30Pro) using CD may be due to the particular kinetics
of aggregation for these proteins (reviewed in [25]). Similar
¢ndings during aggregation of AL fragments [13] as well as
a bacterial protein OsmB [26] were interpreted by the authors
as suggesting that the aggregate does not contribute to the CD
signal because the concentration of soluble aggregates is low.
The use of the membrane-mimicking solvent aqueous acetoni-
trile, which is known to stabilise L-sheet [13,27^30], empha-
sises the structural di¡erences between wild-type and mutant
K-syn proteins. Freshly prepared solutions in acetonitrile/PBS
mixtures (1:1) of K-syn mutants exhibit L-sheet conformation
featuring a minimum at around 218 nm and a maximum
about 195 nm (Fig. 2B). The L-sheet content of mutant K-
syn(Ala53Thr) is even higher than that of the mutant K-syn-
(Ala30Pro) as suggested by the intensity of the CD curves. In
contrast, wild-type K-syn under similar conditions gave a
spectrum with minima around 225 nm and 202 nm and with
a maximum at 194 nm (Fig. 2B). We interpreted this spectrum
as a mixture of random and L-sheet conformations.
Here we have presented data showing that PD-linked mu-
tant K-syn proteins developed more L-sheet structure and
formed more mature ¢laments than wild-type K-syn. These
results suggest that mutant K-syn could form ¢laments and
deposit in LBs and LNs faster than the wild-type protein. Our
results may therefore explain how patients carrying these mu-
tations in the K-syn gene could develop PD early in life. Dep-
osition of aggregates of certain neuronal proteins to form
neurotoxic ¢laments is a popular concept in neurodegenera-
tive diseases such as AD [31], prion diseases [32] and HD [19].
Our results are in keeping with the emerging theme of con-
formation-dependent toxicity and extend this theme to include
PD and DLB.
Acknowledgements: We would like to thank Mr R. Murphy, School
of Chemistry, QUB for assistance with the CD work and Ms O.
O’Shea and Mr P. Larkin, School of Biomedical Science, QUB for
their assistance with the EM work. We would also like to thank Dr
Janet Johnston of the School of Biology and Biochemistry, QUB for
advice and comments on the manuscript. This work was supported by
funding from the UK Medical Research Council (Grant G9626372N).
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Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α-synuclein protein implicated in Parkinson's disease

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