THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 49, Issue of December 6, pp. 47551–47556, 2002
Printed in U.S.A.
Oxidation-induced Misfolding and Aggregation of Superoxide
Dismutase and Its Implications for Amyotrophic Lateral Sclerosis*
Received for publication, July 22, 2002, and in revised form, September 25, 2002
Published, JBC Papers in Press, September 27, 2002, DOI 10.1074/jbc.M207356200
Rishi Rakhit‡, Patricia Cunningham§, Alexandra Furtos-Matei§, Sophie Dahan§, Xiao-Fei Qi‡,
John P. Crow¶, Neil R. Cashman§储**, Leslie H. Kondejewski§, and Avijit Chakrabartty‡ ‡‡
From the ‡Departments of Medical Biophysics and Biochemistry, Ontario Cancer Institute, University of Toronto, Toronto,
Ontario M5G 2M9, Canada, §Caprion Pharmaceuticals, Inc., St. Laurent, Quebec H4S 2C8, Canada, ¶Departments of
Anesthesiology, Pharmacology/Toxicology, and Biochemistry and Molecular Genetics, University of Alabama,
Birmingham, Alabama 35294, and 储Centre for Research in Neurodegenerative Diseases and Sunnybrook and Women’s
College Health Sciences Centre, University of Toronto, Toronto, Ontario M5S 3H2, Canada
The presence of intracellular aggregates that contain
Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic
lateral sclerosis (ALS). Although SOD1 is abundant in all
cells, its half-life in motor neurons far exceeds that in
any other cell type. On the basis of the premise that the
long half-life of the protein increases the potential for
oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and
SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type
holo-protein. Oxidation of select histidine residues that
bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain
the accumulation of SOD1 aggregates in motor neurons
affected in ALS.
ALS1 is a fatal neuromuscular disease that presents as
weakness, spasticity, and muscle atrophy. The disease is
caused by selective degeneration of motor neurons in the brain,
brainstem, and spinal cord. Although ALS presents mostly as a
sporadic disease, a familial form of ALS is seen in ⬃10% of
cases. Twenty percent of familial ALS (FALS) cases are caused
by point mutations in the SOD1 gene. More than 90 distinct
amino acid mutations spread throughout the sequence of this
153-residue protein have been identified (1). The finding that
many FALS-associated SOD1 mutants possess full specific enzyme activity (2) suggests that the disease is not caused by loss
of normal dismutase activity. Further support for this idea has
come from transgenic mice studies. Transgenic mice that harbor FALS-associated SOD1 mutations develop ALS-like symptoms despite having greater than normal levels of SOD1 activity, including the normal complement of endogenous mouse
SOD1 enzyme (3). Furthermore, SOD1 knockout mice do not
* This work was supported by grants from Caprion Pharmaceuticals
Inc., Temerty Family Foundation (to N. R. C.), and Canadian Institutes
of Health Research (to A. C.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
** N. R. C. holds the Jeno and Ilona Diener Chair of Neurodegenerative Diseases.
‡‡ To whom correspondence should be addressed. E-mail: chakrab@
uhnres.utoronto.ca.
1
The abbreviations used are: ALS, amyotrophic lateral sclerosis;
FALS, familial amyotrophic lateral sclerosis; SOD1, Cu/Zn superoxide
dismutase; CCS, copper chaperone protein; ANS, 8-anilino-1-napthalene-sulfonic acid; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide.
This paper is available on line at http://www.jbc.org
develop ALS-like symptoms. Thus, it has been proposed that
mutations in SOD1 cause FALS by a gain, rather than a loss,
of function (reviewed in Ref. 1).
One proposed gain of function involves free radical generation by SOD1. Because the dismutase action of SOD1 runs in a
reversible catalytic cycle with a number of different possible
substrates (4 – 6), under some conditions, SOD1 may catalyze
the reverse reaction and generate radical species. It has been
proposed that certain FALS-associated SOD1 mutants have
lower Km values for hydrogen peroxide in the reverse reaction
and therefore possess greater free radical generating activity
than do wild-type enzymes. This makeup ultimately allows a
greater number of cytotoxic peroxidation reactions to occur in
these mutants (4, 5).
The exact species responsible for oxidative damage, however,
has recently come under question. Fridovich and co-worker (7)
showed that the production of hydroxyl radicals would be negligible because of competition with bicarbonate ions for hydroxyl radicals bound to copper in SOD1.
Another possible gain of function implicates the formation of
zinc-deficient enzyme as the common toxic entity derived from
all mutants. One property shared by many FALS-associated
SOD1 mutants is a decreased affinity for Zn2⫹ (8, 9). It has
been proposed that reduced Zn2⫹ binding destabilizes the
structure of SOD1, increasing the rate of abnormal reduction of
bound Cu2⫹ to Cu⫹ by intracellular reducing agents. This
reduced form of SOD1 could then catalyze the reverse enzymatic reaction and become a net producer of superoxide anion.
In the absence of a well defined protein fold, the electrostatic
gradient that is normally present in SOD1 (10) does not exist to
prevent diffusion of the resultant radical anion. Therefore, in
the presence of nitric oxide, which reacts five times faster with
superoxide than does SOD1 itself, zinc-deficient SOD1 becomes
a net producer of peroxynitrite (11). Thus, the zinc-deficient
SOD1 hypothesis maintains that peroxynitrite is the final mediator of oxidative neuronal injury and works by either nitrating and/or oxidizing critical cellular targets.
Active site copper plays a critical role in both of the proposed
mechanisms for a gain of function of FALS-associated SOD1
mutants described above. A recent study that used transgenic
mice that expressed FALS-associated SOD1 mutants but lacked
the gene for the copper chaperone protein (CCS) investigated
whether alterations in copper loading would affect disease pathobiology (12). CCS facilitates the incorporation of Cu2⫹ into SOD1
in vivo (13, 14), and copper is essential for normal dismutase
activity as well as for any gained functions that are oxidantmediated. The transgenic study found that knocking out the CCS
gene reduced copper incorporation into FALS-associated SOD1
47551
47552
Oxidation-induced Aggregation of SOD1
TABLE I
Comparison of relative amino acid composition of oxidized to
control SOD1
Ratio oxidized/control
Amino acid
Aspartic acid
Threonine
Serine
Glutamic acid
Glycine
Valine
Isoleucine
Leucine
Phenylalanine
Histidine
Lysine
Arginine
Proline
Alanine
FIG. 1. Zinc-deficient and mutant SODs form visible aggregates on oxidation, whereas the wild-type protein does not. A,
comparison of right-angle scattering signals from various SOD1 solutions on oxidation with 4 mM ascorbate and 0.2 mM CuCl2 (black) versus
control (gray) at 37 °C, pH 7.0, for 48 h. Dotted line indicates scattering
produced by 10 mM Tris acetate buffer with 4 mM ascorbate and 0.2 mM
CuCl2. B, pH dependence of oxidation-induced aggregation of SOD.
Zinc-deficient SOD1 forms visible aggregates over a large pH range
(5.0 –7.5) on oxidation (triangles). Wild-type SOD1 does not form visible
aggregates under similar conditions (circles). Zinc-deficient SOD1 controls also yielded greater than base-line scattering (squares). C, lightscattering signal of zinc-deficient SOD1 treated with copper/ascorbate
oxidation under various inhibition conditions (for details, see “Experimental Procedures”). Anaerobic conditions and copper removal by
EDTA chelation prevents aggregation, whereas free radical scavengers
(mannitol and DMPO) do not. Light-scattering measurements were
made with a PTI QM-1 fluorimeter. Excitation and emission wavelengths were set to 350 nm (bandpass ⫽ 2 nm).
mutants; however, disease onset and progression in the mouse
model was largely unaffected. The fact that 20 –30% of total
SOD1 activity remained in the absence of CCS prevents this
study (12) from completely ruling out copper-mediated mechanisms of toxicity in SOD1 transgenic mice, but it does suggest
that other mechanisms such as protein aggregation may play an
important role in the overall cytotoxicity.
Another dramatic gain of function exhibited by SOD1 mutants is a very high propensity to aggregate (3, 15). COS7 cells
transfected with FALS-associated SOD1 mutants produce cytoplasmic aggregates composed of the SOD1 mutant protein;
transfections of wild-type SOD1, on the other hand, do not
cause such cellular alterations (15). A number of transgenic
mice, all of which expressed a particular FALS-associated
SOD1 mutant and co-expressed different amounts of wild-type
SOD1, were shown to uniformly exhibit intracellular SOD aggregation in neural tissue as well as ALS-like symptoms regardless of whether wild-type SOD1 expression was elevated or
eliminated (3). SOD1 aggregates have been proposed to produce toxicity by interference with normal proteasome function
(16) or by altering chaperone (e.g. heat shock protein 70) (17,
18) activity.
In the present study, we sought to elucidate physiologically
relevant environmental factors that may trigger aggregation of
Wild-type SOD1
Zinc-deficient SOD1
1.00
1.02
1.01
1.06
1.00
1.00
0.95
0.99
0.94
0.63
0.95
0.97
0.91
1.07
1.01
1.03
1.00
1.00
1.03
1.03
1.02
0.95
0.94
0.62
0.84
0.87
0.93
1.00
SOD1 in motor neurons. SOD1 aggregates seen in ALS patients and transgenic mouse models are limited to neural tissue
(motor neurons and, occasionally, neighboring astrocytes) and
are not seen in other cell types. Given that SOD1 is present in
high concentrations in all cells, an environmental factor must
exist within motor neurons that induces aggregation specifically in this cell type. Two differences between SOD1 molecules
in motor neurons and other cells are its long half-life and
higher concentration. Concentration of SOD1 is greater in motor neurons than in other neurons and glial cells, and it is found
not only in the cell body of motor neurons but also within axons
and nerve termini (19). To reach the nerve termini, SOD1 is
transported through the axon by using the slow component b of
the anterograde axonal transport system (20), which has a rate
of 2– 8 mm/day. Thus, the transport time for motor neurons
with a meter-long axon could approach 500 days, and the life
span of the protein must exceed the transport time. The long
life span of this protein increases the chances of oxidative
modification by reactive oxygen species; one possible byproduct
of oxidative modification is induction of protein aggregation.
The greater life span of SOD1 in motor neurons means that it
would have more opportunity to accumulate oxidative modifications and to be altered in ways that could increase its own
production of abnormal oxidants (i.e. to become zinc-deficient
and catalyze the formation of peroxynitrite).2 Oxidative damage to SOD1, either self-induced or the result of other oxidant
sources, in turn may trigger aggregation. In support of this
hypothesis, markers of oxidative damage were shown to be
significantly elevated in neural tissue of ALS patients as compared with controls (21, 22). To explore the possibility that
oxidation triggers SOD1 aggregation, we examined the effects
of oxidation on fully metallated wild-type SOD1 (holo-SOD1),
on zinc-deficient SOD1, and on four SOD1 mutants.
EXPERIMENTAL PROCEDURES
In Vitro Aggregation of SOD1—Wild-type Cu-Zn SOD1 from human
erythrocytes was obtained from Sigma. Mutant and zinc-deficient SODs
were prepared as described previously (9). Oxidation reactions consisted of 10 ␮M SOD1, 4 mM ascorbic acid, and 0.2 mM CuCl2 in 10 mM
Tris, 10 mM acetate buffer, whereas control reactions were 10 ␮M SOD1
in buffer. Reactions were incubated at 37 °C for 48 h. The pH was 7.0
unless stated otherwise.
Inhibition of in Vitro Aggregation—To readily recognize inhibition of
SOD1 aggregation, the most aggregation-prone SOD1 species (zincdeficient SOD1) was used. SOD1 aggregation mixtures (10 ␮M SOD1, 4
mM ascorbate, 0.2 mM CuCl2, 10 mM Tris acetate, pH 7) were incubated
with 2 mM EDTA, 10 mM mannitol, or 10 mM DMPO as probes for the
reactive oxygen species. Anaerobic conditions were achieved by degas2
M. J. Strong, W. L. Strong, B. P. He, M. M. Sopper, and J. P. Crow,
personal communication.
Oxidation-induced Aggregation of SOD1
47553
TABLE II.
Summary of mass spectroscopic analysis of tryptic fragments of oxidized SOD1
Tryptic peptide sequence
HVGDLGNVTADK
TLVVHEK
Experimental mass
Theoretical mass
[M ⫹ H]⫹
Control
Oxidized
80–91
1225.6
1225.6
116–122
825.5
825.5
1225.6
1241.6
825.5
841.5
Position
Modified amino acid
His80
His80 ⫹ His16
His120
His120 ⫹ His16
Congo Red Spectral Shift Assay—SOD1 aggregates were diluted to a
final concentration of 3 ␮M (⬃100 ␮g/ml) and incubated with 6 ␮M
Congo red for 30 min before measuring near-UV and visible absorbance.
Circular Dichroism (CD)—Zinc-deficient SOD1 aggregates were centrifuged for 5 min at 13,000 ⫻ g, and the supernatant was removed and
replaced with 20 mM sodium phosphate buffer, pH 7.0. Aggregates were
then resuspended by vortex and sonication before CD spectra were
recorded on an Aviv CD spectrometer model 62 DS at 25 °C.
RESULTS AND DISCUSSION
FIG. 2. Oxidative modification sites of SOD1 revealed by tryptic digestion and mass spectrometry. SOD (30 ␮M) was incubated
with 2 mM ascorbate, 25 ␮M copper, 10 mM sodium acetate, pH 5.0, at
37 °C for 24 h. The protein was reduced and alkylated with dithiothreitol and iodoacetamide in 6 M guanidine hydrochloride and then digested with trypsin (25:1 substrate to enzyme ratio) at 38 °C for 50 h
and analyzed by capillary LC-MS/MS. The ribbon diagram was created
from the PDB coordinates 1SPD with use of the program PYMOL
(Delano Scientific). Side chains of modified His residues (80 and 120)
are orange, the copper ion is blue, and the zinc ion is gray.
sing all solutions and oxidizing them under vacuum (37 °C) in a vacuum
hydrolysis tube (Pierce).
Right Angle Light Scattering—Light scattering measurements were
made with a Photon Technology International QM-1 fluorescence spectrophotometer. Excitation and emission wavelengths were set to 350
nm (bandpass ⫽ 4 nm).
Atomic Force Microscopy—All images were obtained by using a Digital Instruments NanoScope III© atomic force microscope. Samples
were deposited and dried onto freshly cleaved mica under positive
pressure. Contact-mode images were obtained by using a Si3N4 tip
(Digital Instruments) with a nominal spring constant of 0.12 N/m.
Electron Microscopy—Electon microscopy grids (Canemco, Quebec,
Canada) were floated on 10 ␮l drops of SOD1 samples, negative stained
with uranyl acetate (MecaLab Inc., Quebec, Canada), and examined in
an FEI Tecnai 12 transmission electron microscope (80 kV accelerating
voltage).
Amino Acid Analysis—Amino acid compositions of oxidized and control SOD1 were determined by using the Waters Picotag Amino Acid
Analysis system, which uses gas phase acid hydrolysis (6N HCl, 120 °C),
and either precolumn derivitization with phenylisothiocyanate or postcolumn derivitization with ninhydrin.
Capillary Liquid Chromatography/Tandem Mass Spectroscopy—
Peptides were analyzed by using a Q-TOF Ultima mass spectrometer
(Micromass, Manchester, UK) coupled to a capillary high-pressure liquid chromatography. Peptides eluted by acetonitrile were ionized by
electrospray, and peptide ions were automatically selected and fragmented in a data-dependent acquisition mode. Data base searching was
done with Mascot (Matrix Science).
ANS/Thioflavin T Binding—10 ␮M SOD1 in 10 mM Tris acetate (pH
7.0) was incubated for 30 min with 20 ␮M ANS or 20 ␮M thioflavin T
before measuring emission spectrum (excitation at 372 and 450 nm,
respectively).
Metal-catalyzed Oxidation of SOD1—We used metal-catalyzed
oxidation with CuCl2 and ascorbic acid to generate reactive oxygen species because of the physiological relevance of this system.
Metal-catalyzed oxidation is the principal source of hydroxyl
radicals under normal physiological conditions (23), and it is
especially important under conditions of oxidative stress (24).
The concentrations of ascorbic acid used in this study (2– 4 mM)
are well within the normal concentration range (0.5–10 mM)
found in neurons and glial cells (25). We examined the effects of
oxidation on three different ALS-associated mutants of SOD1:
A4V, D90A, and G93A, as well as a site-directed mutant (D124N)
that has decreased zinc-binding affinity (26) and serves as a
model of zinc-deficient SOD1. A4V is the most common mutation
that causes FALS, D90A causes a rare autosomal recessive form
of FALS (1), and G93A is the mutant most widely used for the
transgenic mouse model of ALS. We examined the effect of oxidation on the zinc-deficient form of wild-type SOD1, because this
species has been implicated in neurotoxicity associated with ALS
(11) and because it can use ascorbate to produce superoxide and
hydrogen peroxide directly.
We find that at a neutral pH, oxidation of each of the three
SOD1 mutants and zinc-deficient wild-type SOD1 induces the
formation of large aggregates that scatter light (Fig. 1A). The
zinc-deficient protein displayed the most robust aggregation
reaction and, interestingly, D90A, the mutation that causes an
autosomal recessive form of FALS, displayed the least amount
of aggregate formation. Oxidation of wild-type SOD1 under
identical conditions did not induce the formation of aggregates
detectable by right-angle light scattering (i.e. visible aggregates ⬎350 nm in diameter). With the exception of zinc-deficient SOD1, aggregates did not form in control samples that
lacked oxidants. The small amount of aggregate observed in
control samples of zinc-deficient protein suggests that this form
of the protein has an intrinsic aggregation tendency. The aggregation reaction displays distinct pH dependence, with reduced aggregation at pH ⬍ 5.5 (Fig. 1B). Similar pH dependence has been observed in the oxidation-induced aggregation of
human relaxin, in which oxidation of a single His residue
apparently accounts for the pH dependence (27). Performance
of the oxidation reaction under anaerobic conditions or in the
presence of EDTA inhibited aggregation and revealed that
copper and oxygen are an absolute requirement for oxidationinduced aggregation (Fig. 1C). On the other hand, the addition
of the free radical scavengers mannitol and DMPO did not
inhibit aggregation (Fig. 1C). Similar results have been obtained with copper-catalyzed, oxidation-induced aggregation of
both human relaxin (28) and hamster prion protein (29). The
insensitivity to free radical scavengers and the pH dependence
of the oxidation-induced aggregation are consistent with the
47554
Oxidation-induced Aggregation of SOD1
FIG. 3. A, atomic force microscope height image of an aggregate formed by zinc-deficient SOD1. Aggregates are large and amorphous. Horizontal
scale bar ⫽ 10 ␮m, vertical scale ⫽ 2 ␮m. Inset: close-up of protein aggregate shows that the aggregate is made up of smaller particles. Horizontal
scale bar ⫽ 2 ␮m, vertical scale ⫽ 1 ␮m. B, transmission electron micrograph of SOD1 incubated in the presence of 25 ␮M copper and 2 mM ascorbate
in 10 mM sodium acetate buffer, pH 5.0, for 48 h at 37 °C. Scale bar ⫽ 400 nm.
site-specific metal-catalyzed oxidation mechanism. This mechanism requires a metal ion binding site that is in close spatial
proximity to the modification sites (23). In this type of oxidation
reaction, very few residues are modified.
Characterization of Oxidative Modification Sites—Amino
acid analysis was performed on oxidized wild-type protein and
on oxidized and aggregated zinc-deficient SOD1 (Table I ). The
most striking feature of the amino acid analysis of both types of
oxidized protein was the loss of histidine residues. Amino acid
analysis suggested that three of the eight histidine residues of
the SOD1 subunit were modified. It is known that metalcatalyzed oxidation of proteins leads to conversion of histidine
residues to 2-oxohistidine, 4-hydroxy-glutamate, aspartate, or
asparagine (23). Because the glutamate and aspartate contents
do not appear to be altered by oxidation, it is likely that histidines have been largely converted to 2-oxohistidines. Further
support for the conversion to 2-oxohistidine was obtained by
sequencing tryptic peptides of oxidized wild-type SOD1 by LCMS/MS (Table II). The masses of two tryptic peptides were
increased by 16 mass units, which is consistent with the formation of 2-oxohistidine. Peptide sequencing revealed that
both His 80 and His 120 contain an additional 16 mass units;
these residues are located at the zinc and copper binding sites,
respectively, of SOD1 (Fig. 2).
Morphology and Structure of SOD1 Aggregates—The results
presented here demonstrate that oxidation of select His residues induces misfolding and aggregation of SOD1. However,
the question remains, do these in vitro aggregates represent
aggregates seen in ALS? Examination of ALS inclusion bodies
by light, electron, and immunoelectron microscopy have shown
them to be a unique feature of ALS and distinct from the
amyloid plaques and neurofibrillary tangles seen in Alzheimer’s disease and the intracellular deposits seen in Parkinson’s
disease (30 –32). In particular, ALS inclusion bodies are not
stained by the amyloid dye Congo red (30). Instead, the inclusion bodies seen in COS7 cells that express ALS mutants of
SOD1 (15), transgenic mouse models of ALS (3, 33), and ALS
patients (34 –37) are all composed of a mixture of granular
aggregates and some thick fibers as compared with the thin
fibrils seen in amyloid diseases (38).
Our atomic force microscopy examination of aggregates
formed by oxidation of zinc-deficient SOD1 revealed large
amorphous aggregates (⬍10 ␮m diameter) that were composed
of smaller globular particles (0.2– 0.5 ␮m diameter) (Fig. 3A)
reminiscent of in vivo inclusion bodies (34 –37). Incubation of
oxidized wild-type protein at pH 5 produced a scant number of
aggregates that could be detected by negative staining electron
microscopy. These heterogeneous aggregates were composed of
amorphous aggregates along with fibrous aggregates that were
40 nm in diameter and several micrometers long (Fig. 3B).
These fibrous aggregates are thicker than the amyloid fibrils
formed by the Alzheimer amyloid peptide, which are 60 –90 Å
in diameter (38).
Dye binding experiments with thioflavin T and Congo red, as
well as CD, were also used to determine whether the SOD1
aggregates possessed amyloid characteristics. A 2-fold enhancement of thioflavin T fluorescence was observed with the
aggregates produced from zinc-deficient SOD1 (Fig. 4A); however, the fluorescence enhancement seen with amyloid fibrils is
usually 3 orders of magnitude higher (39). On binding to Congo
red, very little, if any, increase was seen in absorbance or
spectral shift (Fig. 4B), which would have been expected had
the aggregates in fact been amyloid (40). This lack of increase
is in keeping with the failure of Congo red to bind SOD inclusion bodies in vivo (30).
The CD spectrum of SOD1 undergoes a large change on oxidation-induced aggregation (Fig. 4C). However, the CD spectrum
of SOD1 aggregates indicates random coil rather than the characteristic ␤-sheet spectrum of amyloid. Thus, although it appears
that oxidative damage of SOD1 results in misfolding and aggregation, the resultant aggregates do not appear to be amyloid.
Indeed, the morphology of the aggregates observed in vitro in this
study compare favorably with that of granular SOD1 inclusions
observed in ALS models and patients.
Structural Changes to SOD1 before Aggregation—To determine whether susceptibility to oxidation-induced aggregation
of zinc-deficient SOD1 and SOD1 mutants results from an
altered conformation, ANS dye binding experiments were performed on untreated unoxidized protein samples. ANS binding
is a probe of exposed hydrophobic surfaces in proteins. Zincdeficient SOD1 bound the most ANS, wild-type SOD1 did not
show any ANS binding, and the SOD1 mutants displayed vary-
Oxidation-induced Aggregation of SOD1
47555
FIG. 5. Comparison of ANS-binding of wild-type (filled circles)
to mutant (dashed lines) and zinc-deficient SOD1 (open circles).
Ten ␮M SOD1 in 10 mM Tris acetate (pH 7.0) was incubated with 20
ANS before the emission spectrum was measured (excitation at 372
nm). Blue shift and increased intensity of ANS fluorescence in mutants
and zinc-deficient SOD1 indicates increased exposure of hydrophobic
domains. Inset: integrated ANS fluorescence signal from mutant and
zinc-deficient SODs compared with ANS fluorescence of wild-type
SOD1 (dashed line).
FIG. 4. A, comparison of thioflavin T binding of zinc-deficient SOD
aggregates (thin line) and wild-type SOD (thick line). Ten ␮M SOD1 in
10 mM Tris acetate (pH 7.0) was incubated with 20 ␮M thioflavin T
before the emission spectrum was measured (excitation at 450 nm).
Although some increase occurred in observed fluorescence intensity, it
is far less than the increase typically seen on thioflavin T binding to
amyloid fibrils. The increase observed is attributed to sequestering of
the fluorophore from quenchers. B, spectral shift assay of aggregates
with the use of Congo red. Six ␮M Congo red (solid line) had comparable
absorbance to 3 ␮M SOD and 6 ␮M Congo red (dotted line). C, CD spectra
of SOD aggregates (solid line) and native SOD (dotted line). SOD
aggregates do not contain a high proportion of ␤-sheet structures and,
with the dye binding experiments, this indicates that these aggregates
are likely not amyloid.
ing intermediate degrees of ANS binding (Fig. 5). It is known
that the bound zinc in SOD1 helps maintain the structure of
the active site and is not directly involved in catalysis, and
removal of zinc destabilizes the enzyme (41). The ANS binding
experiments indicate that in addition to general destabilization, an alteration in conformation that leads to exposure of
hydrophobic surface is also associated with zinc removal. The
intermediate levels of ANS binding observed with the SOD1
mutants may have resulted from an altered looser conformation of the protein in solution, as has been suggested by the
crystal structures of mutant SOD1 (42). Alternatively, the in-
termediate ANS binding may result from heterogeneity in the
metallation status of the mutants, in which mutant preparations that show the greatest ANS binding contain significant
quantities of incompletely metallated protein, much of which
could be zinc-deficient.
Concluding Remarks—We have shown that zinc-deficient
SOD1, a site-directed mutant with low zinc binding affinity,
low zinc content (D124N), and three FALS-associated SOD1
mutants, is much more susceptible to oxidation-induced aggregation than the fully metallated wild-type protein. These findings, coupled with the long half-life of SOD1 in motor neurons
and the high levels of oxidative damage that are known to occur
in neural tissues of ALS patients (21), provide a possible explanation for the SOD1 aggregates observed in ALS. Although
it still remains to be established whether the SOD1 aggregates
are intrinsically toxic, evidence is mounting that protein aggregates exhibit a general toxicity that is independent of the
function of the protein in its native state (43). Our data are also
consistent with the recent model put forward by OkadoMatsumoto and Fridovich (18) in which antiapoptotic factors
such as heat shock proteins are sequestered by abundant
misfolded/aggregated proteins, such as SOD1 or other misfolded proteins induced by oxidation/nitration, leading to
apoptosis.
Acknowledgments—We thank Dr. Harry Ledebur, Dr. Irene
Mazzoni, Dr. Jennifer Griffin, and Eric Thibaudeau for helpful discussions and Dr. Clarissa Desjardins and Lloyd Segal for encouragement
and support. We thank Dr. Yingxin Zhuang for rigorous and meticulous
efforts to produce consistently high-quality purified SOD1 preparations
that proved vital to our studies. We also thank Dr. I. Fridovich for
critical evaluation of this work.
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