From the Department of Medical Biochemistry, University of Aarhus,
DK-8000 Aarhus C, Denmark
Received for publication, February 7, 2001, and in revised form, April 17, 2001
-Synuclein is a protein normally involved in
presynaptic vesicle homeostasis. It participates in the development of
Parkinson's disease, in which the nerve cell lesions, Lewy bodies,
accumulate
-synuclein filaments. The synaptic neurotransmitter
release is primarily dependent on Ca2+-regulated
processes. A microdialysis technique was applied showing that
-synuclein binds Ca2+ with an IC50 of about
2-300 µM and in a reaction uninhibited by a 50-fold
excess of Mg2+. The Ca2+-binding site consists
of a novel C-terminally localized acidic 32-amino acid domain also
present in the homologue
-synuclein, as shown by Ca2+
binding to truncated recombinant and synthetic
-synuclein peptides. Ca2+ binding affects the functional properties of
-synuclein. First, the ligand binding of 125I-labeled
bovine microtubule-associated protein 1A is stimulated by
Ca2+ ions in the 1-500 µM range and is
dependent on an intact Ca2+ binding site in
-synuclein.
Second, the Ca2+ binding stimulates the proportion of
125I-
-synuclein-containing oligomers. This suggests that
Ca2+ ions may both participate in normal
-synuclein
functions in the nerve terminal and exercise pathological
effects involved in the formation of Lewy bodies.
 |
INTRODUCTION |
Parkinson's disease
(PD)1 and other common
neurodegenerative disorders, e.g. dementia with Lewy bodies
and the Lewy body variant of Alzheimer's disease, are characterized by
the development of the proteinaceous inclusions called Lewy bodies in
the degenerating nerve cells (1). Lewy bodies comprise
-synuclein
(AS)-containing filaments, and purified AS readily forms amyloid-like
filaments in vitro (2-5). Moreover, missense mutations in
the AS gene cause heritable autosomal dominant PD (6, 7). Transgenic
animal models support the direct link between AS and neurodegeneration because overexpression of AS leads to neuronal loss, nerve terminal pathology, and formation of Lewy body-like inclusions (8-11). It has
been proposed that the pathogenic mechanisms triggered by AS rely on
structural changes occurring during the transition from the monomeric
to the
-folded filamentous state (12). AS is a member of the
synuclein family, which, in man, is dominated by
-,
-, and
-synuclein (13). The synucleins are acidic proteins of about 140 amino acids that display a "natively unfolded" structure (14). The
N-terminal part of the proteins is highly conserved and contains
several KTKEGV consensus repeats, whereas the C-terminal portion is
less well conserved and possesses no known structural elements
(15). The differences in its primary structure are reflected in
segregated functional domains, e.g. brain vesicles bind to
the N-terminal part, whereas the microtubule-associated proteins tau
and microtubule-associated protein 1B bind to the C-terminal part
(16-18). The AS gene is dispensable for normal development and
breeding as demonstrated in AS knockout mice (19). However, these mice
do exhibit subtle changes in the contents of certain neurotransmitters
and in synaptic transmission (19), and antisense suppression in primary
nerve cell cultures causes a reduced distal pool of synaptic vesicles
(20). This indicates that AS plays a role in the cellular signaling
events, an observation that is in agreement with biochemical studies
demonstrating that AS can affect phospholipase D2 and protein kinases
and modulate phosphorylation of nerve cell proteins (17, 21, 22).
Ca2+ ions regulate a plethora of cellular processes. This
functionality has been refined in neurons, where the propagation of action potentials over long distances and the fine-tuned
neurotransmitter release from nerve terminals represent such
Ca2+-regulated processes (23-25).
The actions of Ca2+ ions are mediated by several
mechanisms. The Ca2+-calmodulin complex and its diverse
downstream signaling pathways represent common cellular mechanisms
(26). More neuron-specific Ca2+-regulated proteins are
represented by synaptic vesicle-associated proteins and abundant
Ca2+-binding neuronal proteins like parvalbumin and
calbindin (27). The latter group may function as a slow buffer that
modulates synaptic plasticity (28). The importance of cellular
Ca2+ homeostasis is highlighted by the central role of
Ca2+ ions in apoptotic processes and neuronal
excitotoxicity (29, 30).
The purpose of the present study was to investigate the binding of
Ca2+ to AS to ascertain whether Ca2+ can
regulate normal and pathological AS functions.
 |
MATERIALS AND METHODS |
Miscellaneous--
45Ca and 125I were
obtained from Amersham Pharmacia Biotech. All reagents were of analytic
grade, unless stated otherwise. The synthetic peptide
AS-(109-140), corresponding to amino acid residues 109-140 in
human AS, was from Shaefer-N (Copenhagen, Denmark).
Proteins--
The novel deletion mutants AS-(1-110) and
AS-(1-125) were produced by PCR-based mutagenesis as described
previously for AS-(1-95) and AS-(55-140) (18). The constructs were
verified by DNA sequencing. The mutant proteins were expressed in
Escherichia coli and purified essentially as described for
wild type AS (31). The peptides were more than 95% pure as assessed by
Coomassie Blue staining (Fig. 2, middle panel, A, inset),
and their identities were verified by mass spectrometry (data not
shown). Purified bovine microtubule-associated protein (MAP)-1A was
kindly provided by Dr. Khalid Islam (32). It consisted essentially of
the pure ~360-kDa heavy chain (Fig. 3, top panel, inset, lane
1). The MAP-1A was iodinated to a specific activity of about 250 mCi/mg using chloramin T as the oxidizing agent, as described
previously for MAP-1B (18). The electrophoretic migration of the
iodinated MAP-1A consisted of a single slow-migrating band
corresponding to the nonlabeled protein (Fig. 3, top panel, inset, lane 2). All protein concentrations were determined using the Bio-Rad protein assay using bovine serum albumin as standard.
45Ca2+ Equilibrium Dialysis
Assay--
First buffers and protein stock solutions were passed
through a Chelex 100 column (Bio-Rad) to remove Ca2+ ions
to negligible levels (33). Next, solutions containing 1 mM
45Ca2+ and different concentrations of
unlabeled Ca2+ were prepared with and without a constant
concentration of AS-(1-140), AS-(1-125), AS-(1-110), AS-(1-95),
-synuclein, and
-synuclein. All experiments were performed at
4 °C in a solution containing 150 mM KCl and 20 mM HEPES, pH 7.4. The concentration of the synucleins varied from 20 to 300 µM. Binding was measured by
equilibrium dialysis. For equilibrium dialysis, 30-µl plexiglass
chambers were used (34). Each chamber was divided into two equal
compartments by a cellulose membrane cut from dialysis tubing
(Spectrum, Houston, Texas; cutoff, 3,500 Da). The left-side
compartments contained 25 µl of calcium-containing samples, with or
without the synucleins, and the right-side compartments contained 25 µl of buffer. Control experiments showed that equilibrium was
established within 2 h (data not shown). Accordingly, the chambers were
emptied after 9 h, before the samples were assayed for radioactivity
and protein. The Ca2+ concentration was determined by
liquid scintillation counting with a LKB Wallac 1209 Rackbeta counter
(Turku, Finland). No quenching of the radioactivity of
45Ca2+ by the synucleins was observed. The
recovery of 45Ca2+ was 97%, demonstrating that
no significant adsorption of calcium to the cellulose membrane or
dialysis chamber had occurred. The radioactivity of
Ca2+-containing solutions that had not been dialyzed was
taken to represent the known concentration of total Ca2+.
In the binding experiments, the concentrations of bound and free
Ca2+ were calculated by using the radioactivity samples
taken from the synuclein-containing chambers (representing bound plus
free Ca2+) and the corresponding synuclein-free chambers
(representing free Ca2+). The concentrations of the
synucleins were measured by spectroscopy at 280 nm using the extinction
coefficient calculated for each of them. The protein content in the
isolated samples was determined by SDS-polyacrylamide gel
electrophoresis and silver staining to assure the absence of
degradation and leakage through the membrane.
MAP-1A Binding Assay--
The 125I-MAP-1A binding to
AS peptides immobilized in Polysorb microtiter plates (Nunc,
Copenhagen, Denmark) was performed essentially as described previously
for tau (17). The binding buffer consisted of 150 mM KCl,
20 mM HEPES, pH 7.4, 0.01% extensively dialyzed bovine
serum albumin, 0.1 mM EDTA, and 0.1 mM EGTA
supplemented with various concentrations of CaCl2 and
MgCl2. The even immobilization of the C-terminally
truncated AS peptides was verified by their similar specific binding of
a 125I-labeled affinity-purified antibody (ASY-3) raised
against a synthetic peptide corresponding to the N-terminal 31 residues of AS (data not shown).
Chemical Cross-linking of AS Oligomers--
AS and
C-terminal-truncated peptides (1 µM) supplemented with
500 pM of the corresponding 125I-labeled AS
were incubated for 2 h at 20 °C in 150 mM KCl, 20 mM 4-morpholinepropanesulfonic acid, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, and 0.5 mM
dithioerythreitol in the absence and presence of Ca2+. The
distribution of monomers, oligomers, and higher aggregates was
subsequently stabilized by the addition of a short-length hydrophilic
chemical cross-linker, bis(sulfosuccinimidyl)suberate (BS3) (1 mM), for 15 min, and then the cross-linker was quenched by
the addition of an equal volume of Tris-containing SDS,
dithioerythreitol loading buffer. The samples were subsequently
resolved by reducing gradient SDS-polyacrylamide gel electrophoresis
followed by visualization by autoradiography.
 |
RESULTS |
AS Contains a Novel Ca2+-binding
Motif--
45Ca2+ equilibrium dialysis was
performed to determine whether AS is a Ca2+-binding
protein. Fig. 1 demonstrates that human
recombinant AS binds Ca2+ with a half-saturation of about
300 µM. The saturation of the binding approaches 0.5 mol
Ca2+/mol AS at 1 mM Ca2+, which
indicates the presence of a single binding site. Mg2+ (8 mM) fails to inhibit the 45Ca2+
tracer binding (1 µM) significantly as compared with the
~85% inhibition obtained by 1.5 mM unlabeled
Ca2+. Hence, the binding site displays a Ca2+
selectivity among the dominating intracellular divalent cations (Fig.
2, middle panel, A).

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Fig. 1.
Ca2+ binding isotherm for
AS-(1-140). Recombinant human AS (~100 µM) was
incubated with 1 µM 45Ca2+and
increasing concentrations of unlabeled Ca2+ in a
microdialysis apparatus for 9 h at 4 °C, and then the
45Ca2+ concentration was measured on each side
of the dialysis membrane. The abscissa shows the free
Ca2+ concentration, and the ordinate shows mol
Ca2+ bound/mol -synuclein. The points
represent the mean ± 1 S.D. of five experiments. The
square in the bottom left corner of the graph
represents the data demonstrated in the bottom panel of Fig.
2.
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Fig. 2.
Both - and
-synuclein contain a C-terminal Ca2+
binding site. Top part of the top panel,
alignment of the C-terminal 32 amino acid residues in human -, -,
and -synuclein. Acidic residues are shown in bold, and
those positions where acidic residues are identical to AS are marked by
gray boxes. Bottom part of the top
panel, the acidic amino acids in the C terminus of - and
-synuclein represent a 16-residue acidic tandem repeat. Residues
109-124 are aligned with residues 125-140 in AS, and residues
103-118 are aligned with residues 119-134 in -synuclein. Repeated
acidic residues are indicated by gray boxes. Middle
panel, binding of 45Ca2+ to recombinant
synucleins and truncated recombinant synuclein peptides. The
45Ca2+ binding experiments were performed as
described in the Fig. 1 legend. The ordinate demonstrates
the percentage of tracer binding as the mean ± 1 S.D. of three
independent experiments. A, Ca2+ binding to
human recombinant wild type AS, AS containing the Parkinson's
disease-causing point mutations A30P and A53T, -synuclein, and
-synuclein. Supplementing the buffer with 8 mM unlabeled
Mg2+ did not significantly inhibit the tracer binding as
compared with 1.5 mM Ca2+. B,
Ca2+ binding to AS, truncated AS proteins, and a synthetic
peptide corresponding to C-terminal residues 109-140. The
columns represent the mean ± 1 S.D. of three
independent experiments. The numbers below the columns
correspond to the amino acid residues in the peptides, e.g.
1-140 for full-length AS. The inset demonstrates the purity
of the recombinant proteins by a Coomassie Blue-stained
SDS-polyacrylamide gel with the molecular size markers in kDa (60, 36, 22, and 6) indicated to the left. Bottom panel,
comparison of Ca2+ binding to recombinant AS and the
synthetic peptide AS-(109-140). Both peptides (100 µM)
were incubated with 1 µM 45Ca2+
and increasing concentrations of Ca2+ as described in the
Fig. 1 legend. The abscissa shows the free Ca2+
concentration, and the ordinate shows mol Ca2+
bound/mol peptide. The points represent the mean ± 1 S.D. of three experiments. Open circles represent AS,
closed circles represent AS-(109-140).
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Truncated recombinant AS peptides with deletions of the N-terminal 29- and 54-amino acid residues and the C-terminal 45-amino acid residues
were used for initial localization of the Ca2+-binding
site. Fig. 2, middle panel, B demonstrates that only the
C-terminal truncation inhibited the binding, whereas the N-terminal truncation has no effect. No inhibition of the Ca2+ binding
is observed when testing the mutations causing PD (A30P and A53T) (Fig.
2, middle panel, A). Acidic amino acid residues often
participate in the binding of Ca2+ ions as noted in the
EF-hand, C2-domain and the low-affinity Ca2+-binding sites in S100 proteins (35-37), and such
residues account for 33% of the C-terminal 45 residues. Fig. 2,
top panel, demonstrates a striking identity in the spacing
of 10 of the 12 acidic residues in the C-terminal 32 residues of
-
and
-synuclein.
-Synuclein, however, shows no such similarity.
The similarity in the spacing of acidic residues is reflected at the
functional level, where
- and
-synuclein, but not
-synuclein,
bind Ca2+ (Fig. 2, middle panel, A). The acidic
residues in the C terminus of
- and
-synuclein are organized as a
tandem repeat of 16 amino acids (Fig. 2, top panel), and the
integrity of this structure may be required for the binding of
Ca2+ ions. This hypothesis was explored by examining the
expression and purification of recombinant truncated AS peptides
lacking (i) the C-terminal repeat AS-(1-125) and (ii) both
repeats AS-(1-110) and AS-(1-95) (Fig. 2, middle panel, B,
inset). Fig. 2, middle panel, B shows that removal of
the single C-terminal repeat in AS-(1-125) inhibits the
Ca2+ binding to the level of the peptides lacking both
repeats or the entire 45 C-terminal residues. Moreover, a synthetic
peptide corresponding to the tandem repeat structure, AS-(109-140),
binds 45Ca2+ to the same extent as wild type AS
(Fig. 2, middle panel, B), and its binding isotherms reveal
indistinguishable affinities for Ca2+ (Fig. 2, bottom
panel). Accordingly, the C-terminal repeat structure in
- and
-synuclein is necessary and sufficient to bind Ca2+ and
represents a bona fide Ca2+-binding domain.
Ca2+ Binding to
-Synuclein Modulates Ligand
Interactions--
The propensity of Ca2+ ions to modulate
ligand binding to AS was analyzed in terms of the effect of such
binding of (i) the amyloidogenic A
(1-40) peptide and (ii) the
microtubule-associated proteins tau and MAP-1B. Ca2+ ions
have no significant effect on these interactions (data not shown).
MAP-1A is a novel AS ligand, as demonstrated by the binding of
125I-labeled bovine MAP-1A to immobilized AS (Fig.
3, top panel). The association
of 50 pM 125I-MAP-1A reaches a plateau within
9 h at 4 °C (data not shown), and all incubations are therefore
performed for 16 h. The interaction is specific, as demonstrated
by the inhibition of 125I-MAP-1A binding by both unlabeled
MAP-1A and AS, and it exhibits a high affinity (IC50 ~ 30 nM; Fig. 3, top panel).

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Fig. 3.
Ca2+ binding to AS
regulates the interaction with MAP-1A. Top panel,
recombinant AS, immobilized in microtiter plates, was incubated
with 50 pM 125I-MAP-1A and increasing
concentrations of unlabeled MAP-1A ( ) or AS ( ). The
ordinate represents the percentage of bound/free
(B/F) ligand, and the abcissa represents the
concentration of free ligand. The points represent the
mean ± 1 S.D. of four replicates in one of four similar
experiments. Inset, lane 1, purified bovine MAP-1A (4 µg)
was mixed with 10,000 cpm 125I-MAP-1A, resolved by 8-16%
reducing SDS-polyacrylamide gel, and stained with Coomassie Blue.
Lane 2, autoradiogram of the same gel. The molecular size
markers (in kDa) are shown on the left. Middle
panel, the Ca2+ dependence of 125I-MAP-1A
binding to AS. The binding (as described in the top panel)
was determined in the presence of increasing concentrations of
Ca2+ ions. The ordinate represents the ratio
between bound MAP-1A and the control binding of MAP-1A in the absence
of Ca2+ ions, and the abscissa represents the
Ca2+ concentration. The points represent the
mean ± 1 S.D. of four replicates in one of three similar
experiments. Bottom panel, A, effect of Ca2+ and
Mg2+ on the binding of 125I-MAP-1A to AS. The
binding was determined in the absence (Control) and presence
of the indicated concentrations of divalent cations. Bottom
panel, B, the binding of 125I-MAP-1A to immobilized
full-length AS-(1-140) and the C-terminal-truncated AS peptides
1-125, 1-110, and 1-95 was determined in the presence of 1.5 mM Ca2+. The columns in A
and B represent the mean ± 1 S.D. of four replicates
in one of three similar experiments. The equal immobilization of
the different AS peptides was verified by their similar binding of the
125I-labeled ASY-3 antibody that recognizes the N terminus
of AS.
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The binding of MAP-1A to AS is enhanced by Ca2+ ions (Fig.
3, middle panel), and a maximal stimulatory effect of about
90% is obtained at concentrations greater than 0.5 mM
(Fig. 3, middle panel), with a half-maximal stimulation at
about 0.3 mM Ca2+ (Fig. 3, middle
panel). Both Mg2+ and Ca2+ (1.5 mM) stimulate MAP-1A binding to AS, but the effect of
Ca2+ alone is about 60% greater than that for
Mg2+ ions alone; when combined, their effect is synergistic
(Fig. 3, bottom panel, A). Disruption of the
Ca2+-binding domain in AS obtained by removal of the
C-terminal 15, 30, and 45 amino acid residues completely abrogates the
Ca2+-stimulatory effect on MAP-1A binding (Fig. 3,
bottom panel, B) and demonstrates that the Ca2+
effect was indeed based on the AS moiety. Removal of the
Ca2+-binding site in AS increases the binding of MAP-1A to
the truncated AS peptide (data not shown), but it abrogates the
stimulatory Ca2+ effect (Fig. 3, bottom panel,
B). This indicates a negative regulatory effect of the
C-terminal segment of AS on the MAP-1B interaction that is alleviated
by binding of Ca2+ ions. The stimulatory effect of
Mg2+ ions on MAP-1A binding may thus be mediated via the
MAP-1A moiety. Many Ca2+ effects are mediated through the
binding of Ca2+ to calmodulin, but AS does not bind to
calmodulin-Sepharose in either the absence or presence of
Ca2 (data not shown).
Ca2+ Ions Regulate the Oligomeric Distribution of AS
Molecules--
Abnormal filamentous AS is a characteristic of diseased
brain tissue, and AS aggregation represents a
nucleation-dependent process, where the nucleation by
oligomeric AS species may represent a rate-limiting step. We used the
short-length hydrophilic chemical cross-linker BS3 to covalently
stabilize AS oligomers in the absence and presence of Ca2.
This analysis is likely to underestimate the oligomeric content because
the cross-linking efficiency is <100%. However, the method has the
advantage of visualizing molecules associated through low-affinity
interactions. Gel filtration methods and other time-consuming procedures for separating oligomerized and monomeric species may not be
able to reveal such interactions due to dissociation during the
procedures. No significant AS-(1-140) oligomers are present without
cross-linking (Fig. 4). The same applies
to AS-(1-125) and AS-(1-110) (data not shown). Supplementing the AS
solution with 1 mM BS3 for 15 min before reducing
SDS-polyacrylamide gel electrophoresis causes the formation of
125I-labeled bands compatible with AS dimers, trimers, and
higher oligomers with a higher oligomeric content among the
C-terminal-truncated peptides (Fig. 4). Saturation of the
Ca2+ binding site (1.5 mM) increases the
oligomeric content of AS-(1-140) 2-fold for dimers and 2.5-fold for
trimers, and higher aggregates, whereas no Ca2
effect is observed for the truncated peptides. The oligomers are not an
artifact of the iodination of AS because the distinct oligomeric
pattern is absent without the presence of 1 µM unlabeled AS. Accordingly, Ca2+ binding to the C-terminal tandem
repeat domain favors the formation of AS oligomers.

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Fig. 4.
Ca2+ regulates AS
oligomerization. AS and the AS peptides (1 µM) with
truncations in the Ca2+-binding domain were supplemented
with 500 pM of the corresponding 125I-labeled
AS peptide and incubated in the absence and presence of 1.5 mM Ca2+ for 2 h at 20 °C. The incubates
were subsequently cross-linked with BS3 (1 mM), resolved by
SDS-polyacrylamide gel electrophoresis, and processed for
autoradiography. The panels represent the autoradiographic image of
AS-(1-140) and the C-terminal-truncated peptides AS-(1-125) and
AS-(1-110). The presence of Ca2+ and BS3 is indicated
below the panels. Brackets to the left
indicate the localization of dimers, trimers, and larger
oligomers. The monomer is indicated by an arrow.
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DISCUSSION |
The present study identifies a novel Ca2+-binding
motif in the C terminus of AS. The binding of Ca2+ alters
the interactions between AS molecules in the process of oligomerization
and between AS and certain nerve cell proteins as exemplified by
MAP-1A. AS binds Ca2+ ions selectively as compared with the
predominant cytosolic divalent cation Mg2+, which suggests
that AS functions can be regulated by Ca2+ ions in a
cellular context.
Several synuclein genes are expressed in man; the most predominant of
these are AS,
-synuclein, and
-synuclein (15). The localization
of
- and
-synuclein in normal nervous tissue is restricted to the
nerve terminals (38, 39), in contrast to
-synuclein, which is
localized in the somatodendritic compartment (40). The nerve terminal
localization parallels the Ca2+ binding properties of the
proteins because
- and
-synuclein, but not
-synuclein, bind
Ca2+. We therefore wish to suggest a functional
significance of Ca2+ binding to AS in the nerve terminals
where high local Ca2+ concentrations are reached (41) and
AS regulates complex nerve terminal processes related to
neurotransmitter homeostasis and maintenance of the distal pool of
synaptic vesicles (19, 20).
The Ca2+-binding motif is localized to the C-terminal 32 residues of AS and comprises an acidic tandem repeat rich in proline residues. This structure is sufficient and necessary to confer Ca2+ binding activity and requires the presence of both
repeats as demonstrated both by the full binding activity of the
synthetic peptide AS-(109-140) and the absence of binding to
AS-(1-125). The Ca2+-binding domain does not resemble any
of the hitherto recognized Ca2+-binding structures such as
the EF-hand, the C2-domain, or the less defined
low-affinity binding sites in the SH-100 class of proteins, with the
exception of the clustering of negatively charged residues (35-37). AS
is natively unfolded, and circular dichroism spectroscopy does not
reveal any structural changes in the absence or presence of
Ca2+ (14). However, it is not always necessary for
Ca2+ ions to cause gross structural changes for functional
effects to arise, as shown for the C2A domain in
synaptotagmin I, where Ca2+ works as an electrostatic
switch that facilitates binding to syntaxin I and acidic phospholipids
(42, 43). The IC50 for the Ca2+ binding to AS
is about 300 µM, and Ca2+ concentrations
close to this magnitude are only encountered in normal nerve cells
close to Ca2+ channels at the plasma membrane during
propagation of action potentials and at neurotransmitter release (41).
However, cofactors may increase the Ca2+ affinity and thus
increase the potential significance of the Ca2+ binding in
analogy with the approximate 1000-fold increase in the apparent
Ca2+ affinity of the synaptotagmin C2A domain
upon phospholipid binding (43). Candidate cofactors are the
kinases casein kinase-1, casein kinase-2, src, and fyn that have
been implicated in the phosphorylation of Ser129 and
Tyr125 (44-46). Tyr125 is conserved from fish
and birds to man. Such phosphorylation will increase the negative
charge of the Ca2+-binding domain and thereby potentially
increase the Ca2+ affinity.
-Synuclein and
-synuclein are soluble proteins with
vesicle-binding properties that are localized to nerve terminals. Their local concentration is very high because they constitute about 0.1% of
the total protein in rat brain extracts (47), and this may make
them suited to be presynaptic Ca2+ buffers.
The Ca2+ binding to the C-terminal domain in AS stimulates
binding of the novel ligand MAP-1A, and this domain probably plays a
negative regulatory role because its removal increases MAP-1A binding
(data not shown) but abrogates the stimulatory Ca2+ effect.
MAP-1A belongs to the same group of microtubule-associated proteins as
the AS ligands tau and microtubule-associated protein-1B (17, 18), and
several characteristics favor a physiological interaction between
MAP-1A and AS. First, their developmental expression profiles are
parallel with a low to absent expression in the fetal period, followed
by increased expression during postnatal development (48-51). Second,
both proteins are predominantly carried as part of the slow component b
of axonal transport, indicating subcellular contacts to the same
transporting structures (52, 53). Third, a significant part of the
transported proteins is incorporated into stationary axonal
structures (52, 53). The functional significance of such a putative
interaction remains unsolved, but AS is known to change the functional
properties of its ligands (17, 21).
AS-containing filaments accumulate in Lewy bodies during the year-long
process of neurodegeneration in PD. In vitro, filament formation is a nucleation-dependent process, as
demonstrated, where preformed oligomers/filaments can seed the growth
of filaments (54). Accordingly, if oligomer formation represents
a rate-limiting step, then even a small increase in their rate of
formation, regulated by pathogenic factors, may enhance filament growth
significantly (12). Known factors with this property are: (i) AS
mutations linked to familial Parkinson's disease (5), and (ii)
proteolytic activities directed against the AS C-terminal because
C-terminally truncated AS preparations more readily form fibrils (4),
contain a higher proportion of oligomers, as revealed by chemical
cross-linkers (Fig. 4), and such peptides are recovered from
pathological brain tissue and isolated Lewy bodies (3, 18). Increased
Ca2+ concentrations represent a novel fibrillogenic factor, as
demonstrated by the increased oligomeric content upon binding of
Ca2+ to the tandem repeat domain in AS. This makes AS resemble
synaptotagmin VII whose oligomerization is stimulated by Ca2+
(55). High levels of AS filaments have been reported in preparations of
recombinant protein (56). However, this study was performed with
prolonged incubation, elevated temperature, and acidic pH as compared
with our 2-h incubation at pH 7.4. The low oligomeric content in
Ca2+-stimulated wild type AS is, by contrast, in accordance
with gel filtration experiments demonstrating oligomers with low
solubility (<10%) even after 66 days of incubation at pH 7.4 (5). The inhibitory role of the C-terminal part of AS on fibril formation may
rely on an electrostatic repulsion from these negatively charged segments. The molecular mechanism exploited by proteolysis and Ca2+ binding would then be similar because both remove
negative charges from the C terminus.
Conclusively, our study extends our knowledge of AS functions in
relation to both normal and pathological nerve cell paradigms by
linking AS functions to the important cellular messenger
Ca2+. This may facilitate future studies on the still
poorly understood mechanisms underlying the gain in toxic function by
AS in neurodegenerative disorders.
We thank Dr. Khalil Islam for generously
providing purified bovine MAP-1A. We also thank Lis Hygom for excellent
technical assistance.
The abbreviations used are:
, PD, Parkinson's
disease;
BS3, bis(sulfosuccinimidyl)suberate;
AS,
-synuclein;
MAP, microtubule-associated protein.
1.
|
McKeith, I. G.,
Perry, E. K.,
and Perry, R. H.
(1999)
Neurology
53,
902-905[Abstract/Free Full Text]
|
2.
|
Spillantini, M. G.,
Schmidt, M. L.,
Lee, W. M.,
Trojanowski, J. Q.,
Jakes, R.,
and Goedert, M.
(1997)
Nature
388,
839-840[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Baba, M.,
Nakajo, S.,
Tu, P. H.,
Tomita, T.,
Nakaya, K.,
Lee, V. M.,
Trojanowski, J. Q.,
and Iwatsubo, T.
(1998)
Am. J. Pathol.
152,
879-884[Abstract]
|
4.
|
Crowther, R. A.,
Jakes, R.,
Spillantini, M. G.,
and Goedert, M.
(1998)
FEBS Lett.
436,
309-312[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Conway, K. A.,
Lee, S. J.,
Rochet, J. C.,
Ding, T. T.,
Williamson, R. E.,
and Lansbury, P. T., Jr.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
571-576[Abstract/Free Full Text]
|
6.
|
Polymeropoulos, M. H.,
Lavedan, C.,
Leroy, E.,
Ide, S. E.,
Dehejia, A.,
Dutra, A.,
Pike, B.,
Root, H.,
Rubenstein, J.,
Boyer, R.,
Stenroos, E. S.,
Chandrasekharappa, S.,
Athanassiadou, A.,
Papapetropoulos, T.,
Johnson, W. G.,
Lazzarini, A. M.,
Duvoisin, R. C.,
Di Iorio, G.,
Golbe, L. I.,
and Nussbaum, R. L.
(1997)
Science
276,
2045-2047[Abstract/Free Full Text]
|
7.
|
Kruger, R.,
Kuhn, W.,
Muller, T.,
Woitalla, D.,
Graeber, M.,
Kosel, S.,
Przuntek, H.,
Epplen, J. T.,
Schols, L.,
and Riess, O.
(1998)
Nat. Genet.
18,
106-108[Medline]
[Order article via Infotrieve]
|
8.
|
Masliah, E.,
Rockenstein, E.,
Veinbergs, I.,
Mallory, M.,
Hashimoto, M.,
Takeda, A.,
Sagara, Y.,
Sisk, A.,
and Mucke, L.
(2000)
Science
287,
1265-1269[Abstract/Free Full Text]
|
9.
|
Kahle, P. J.,
Neumann, M.,
Ozmen, L.,
Muller, V.,
Jacobsen, H.,
Schindzielorz, A.,
Okochi, M.,
Leimer, U.,
van Der Putten, H.,
Probst, A.,
Kremmer, E.,
Kretzschmar, H. A.,
and Haass, C.
(2000)
J. Neurosci.
20,
6365-6373[Abstract/Free Full Text]
|
10.
|
van der Putten, H.,
Wiederhold, K. H.,
Probst, A.,
Barbieri, S.,
Mistl, C.,
Danner, S.,
Kauffmann, S.,
Hofele, K.,
Spooren, W. P.,
Ruegg, M. A.,
Lin, S.,
Caroni, P.,
Sommer, B.,
Tolnay, M.,
and Bilbe, G.
(2000)
J. Neurosci.
20,
6021-6029[Abstract/Free Full Text]
|
11.
|
Feany, M. B.,
and Bender, W. W.
(2000)
Nature
404,
394-398[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Goldberg, M. S.,
and Lansbury, P. T., Jr.
(2000)
Nat. Cell Biol.
2,
E115-E119[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Duda, J. E.,
Lee, V. M.,
and Trojanowski, J. Q.
(2000)
J. Neurosci. Res.
61,
121-127[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Weinreb, P. H.,
Zhen, W.,
Poon, A. W.,
Conway, K. A.,
and Lansbury, P. T.
(1996)
Biochemistry
35,
13709-13715[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Clayton, D. F.,
and George, J. M.
(1999)
J. Neurosci. Res.
58,
120-129[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Jensen, P. H.,
Nielsen, M. S.,
Jakes, R.,
Dotti, C.,
and Goedert, M.
(1998)
J. Biol. Chem.
273,
26292-26294[Abstract/Free Full Text]
|
17.
|
Jensen, P. H.,
Hager, H.,
Nielsen, M. S.,
Højrup, P.,
Gliemann, J.,
and Jakes, R.
(1999)
J. Biol. Chem.
274,
25481-25489[Abstract/Free Full Text]
|
18.
|
Jensen, P. H.,
Islam, K.,
Kenney, J. M.,
Nielsen, M. S.,
Power, J.,
and Gai, W. P.
(2000)
J. Biol. Chem.
275,
21500-21507[Abstract/Free Full Text]
|
19.
|
Abeliovich, A.,
Schmitz, Y.,
Farinas, I.,
Choi-Lundberg, D.,
Ho, W. H.,
Castillo, P. E.,
Shinsky, N.,
Verdugo, J. M.,
Armanini, M.,
Ryan, A.,
Hynes, M.,
Phillips, H.,
Sulzer, D.,
and Rosenthal, A.
(2000)
Neuron
25,
239-252[Medline]
[Order article via Infotrieve]
|
20.
|
Murphy, D. D.,
Rueter, S. M.,
Trojanowski, J. Q.,
and Lee, V. M.
(2000)
J. Neurosci.
20,
3214-3220[Abstract/Free Full Text]
|
21.
|
Jenco, J. M.,
Rawlingson, A.,
Daniels, B.,
and Morris, A. J.
(1998)
Biochemistry
37,
4901-4909[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Ostrerova, N.,
Petrucelli, L.,
Farrer, M.,
Mehta, N.,
Choi, P.,
Hardy, J.,
and Wolozin, B.
(1999)
J. Neurosci.
19,
5782-5791[Abstract/Free Full Text]
|
23.
|
Ghosh, A.,
and Greenberg, M. E.
(1995)
Science
268,
239-247[Medline]
[Order article via Infotrieve]
|
24.
|
Goda, Y.,
and Südhof, T. C.
(1997)
Curr. Opin. Cell Biol.
9,
513-518[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Berridge, M. J.
(1998a)
Neuron
21,
13-26[Medline]
[Order article via Infotrieve]
|
26.
|
Chin, D.,
and Means, A. R.
(2000)
Trends Cell Biol.
10,
322-328[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Baimbridge, K. G.,
Celio, M. R.,
and Rogers, J. H.
(1992)
Trends Neurosci.
15,
303-308[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Caillard, O.,
Moreno, H.,
Schwaller, B.,
Llano, I.,
Celio, M. R.,
and Marty, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13372-13377[Abstract/Free Full Text]
|
29.
|
Choi, D. W.
(1992)
J. Neurobiol.
23,
1261-1276[Medline]
[Order article via Infotrieve]
|
30.
|
Berridge, M. J.,
Bootman, M. D.,
and Lipp, P.
(1998)
Nature
395,
645-648[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Jensen, P. H.,
Hojrup, P.,
Hager, H.,
Nielsen, M. S.,
Jacobsen, L.,
Olesen, O. F.,
Gliemann, J.,
and Jakes, R.
(1997)
Biochem. J.
323,
539-546[Medline]
[Order article via Infotrieve]
|
32.
|
Pedrotti, B.,
and Islam, K.
(1994)
Biochemistry
33,
12463-12470[Medline]
[Order article via Infotrieve]
|
33.
|
Vorum, H.,
Madsen, P.,
Rasmussen, H. H.,
Etzerodt, M.,
Svendsen, I.,
Celis, J. E.,
and Honoré, B.
(1996)
Electrophoresis
17,
1787-1796[Medline]
[Order article via Infotrieve]
|
34.
|
Kragh-Hansen, U.,
and Vorum, H.
(1993)
Clin. Chem.
39,
202-208[Abstract/Free Full Text]
|
35.
|
Branden, C.,
and Tooze, J.
(1991)
Introduction to Protein Structure
, pp. 22-23, Garland Publishing, New York
|
36.
|
Rizo, J.,
and Sudhof, T. C.
(1998)
J. Biol. Chem.
273,
15879-15882[Free Full Text]
|
37.
|
Tjoelker, L. W.,
Seyfried, C. E.,
Eddy, R. L.,
Byers, M. G.,
Shows, T. B.,
Calderon, J.,
Schreiber, R. B.,
and Gray, P. W.
(1994)
Biochemistry
33,
3229-3236[Medline]
[Order article via Infotrieve]
|
38.
|
Jakes, R.,
Spillantini, M. G.,
and Goedert, M.
(1994)
FEBS Lett.
345,
27-32[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Iwai, A.,
Masliah, E.,
Yoshimoto, M.,
Ge, N.,
Flanagan, L.,
de Silva, H. A.,
Kittel, A.,
and Saitoh, T.
(1995)
Neuron
14,
467-475[Medline]
[Order article via Infotrieve]
|
40.
|
Buchman, V. L.,
Adu, J.,
Pinõn, L. G. P.,
Ninkina, N. N.,
and Davies, A. M.
(1998)
Nat. Neurosci.
1,
101-103[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Llinas, R.,
Sugimori, M.,
and Silver, R. B.
(1992)
Science
256,
677-679[Medline]
[Order article via Infotrieve]
|
42.
|
Shao, X.,
Davletov, B. A.,
Sutton, R. B.,
Südhof, T. C.,
and Rizo, J.
(1996)
Science
273,
248-251[Abstract]
|
43.
|
Davletov, B. A.,
and Südhof, T. C.
(1993)
J. Biol. Chem.
268,
26386-26390[Abstract/Free Full Text]
|
44.
|
Okochi, M.,
Walter, J.,
Koyama, A.,
Nakajo, S.,
Baba, M.,
Iwatsubo, T.,
Meijer, L.,
Kahle, P. J.,
and Hass, C.
(2000)
J. Biol. Chem.
275,
390-397[Abstract/Free Full Text]
|
45.
|
Nakamura, T.,
Yamashita, H.,
Takahashi, T.,
and Nakamura, S.
(2001)
Biochem. Biophys. Res. Commun.
280,
1085-1092[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Ellis, C. E.,
Schwartzberg, P. L.,
Grider, T. L.,
Fink, D. W.,
and Nussbaum, R. L.
(2001)
J. Biol. Chem.
276,
3879-3884[Abstract/Free Full Text]
|
47.
|
Shibayama-Imazu, T.,
Okahash, I.,
Omata, K.,
Nakajo, S.,
Ochiai, H.,
Nakai, Y.,
Hama, T.,
Nakamura, Y.,
and Nakaya, K.
(1993)
Brain Res.
622,
17-25[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Hsu, L. J.,
Mallory, M.,
Xia, Y.,
Veinbergs, I.,
Hashimoto, M.,
Yoshimoto, M.,
Thal, L. J.,
Saitoh, T.,
and Masliah, E.
(1998)
J. Neurochem.
71,
338-344[Medline]
[Order article via Infotrieve]
|
49.
|
Petersen, K.,
Olesen, O. F.,
and Mikkelsen, J. D.
(1999)
Neuroscience
91,
651-659[CrossRef][Medline]
[Order article via Infotrieve]
|
50.
|
Bayer, T. A.,
Jakala, P.,
Hartmann, T.,
Egensperger, R.,
Buslei, R.,
Falkai, P.,
and Beyreuther, K.
(1999)
Neuroreport
10,
2799-2803[Medline]
[Order article via Infotrieve]
|
51.
|
Fink, J. K.,
Jones, S. M.,
Esposito, C.,
and Wilkowski, J.
(1996)
Genomics
35,
577-585[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Jensen, P. H.,
Li, J.-Y.,
Dahlström, A.,
and Dotti, C.
(1999b)
Eur. J. Neurosci.
11,
3369-3376[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Nixon, R. A.,
Fischer, I.,
and Lewis, S. E.
(1990)
J. Cell Biol.
110,
437-448[Abstract]
|
54.
|
Wood, S. J.,
Wypych, J.,
Steavenson, S.,
Louis, J. C.,
Citron, M.,
and Biere, A. L.
(1999)
J. Biol. Chem.
274,
19509-19512[Abstract/Free Full Text]
|
55.
|
Fukuda, M.,
and Mikoshiba, K.
(2000)
J. Biol. Chem.
275,
28180-28185[Abstract/Free Full Text]
|
56.
|
Hashimoto, M.,
Hsu, L. J.,
Sisk, A.,
Xia, Y.,
Takeda, A.,
Sundsmo, M.,
and Masliah, E.
(1998)
Brain Res.
799,
301-306[CrossRef][Medline]
[Order article via Infotrieve]
|