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Common molecular signature in SOD1 for both

sporadic and familial amyotrophic lateral sclerosis

Arie Gruzman*, William L. Wood†, Evgenia Alpert*, M. Dharma Prasad*, Robert G. Miller‡, Jeffery D. Rothstein§,
Robert Bowser¶, Ronald Hamilton¶, Troy D. Wood†, Don W. Cleveland储**, Vishwanath R. Lingappa*, and Jian Liu**††
*Bioconformatics Laboratory and ††Department of Neuroscience, California Pacific Medical Center Research Institute, San Francisco, CA 94107;
†Department of Chemistry, Natural Sciences Complex, University at Buffalo, State University of New York, Buffalo, NY 14260; §Department

of Neurology, Johns Hopkins University, Baltimore, MD 21287; ‡Department of Neurology, California Pacific Medical Center, San Francisco, CA 94115;
储Ludwig Institute for Cancer Research, Departments of Medicine and Neuroscience, University of California at San Diego, La Jolla, CA 92093;

and ¶Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

Contributed by Don W. Cleveland, June 4, 2007 (sent for review August 25, 2006)

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron noreactivity (12). The extent of a chemical modification reaction
degenerative disease whose etiology and pathogenesis remain is strongly affected by the degree of the accessibility of a side
poorly understood. Most cases of ALS (⬇90%) are sporadic (SALS), group to a modifying agent. This, in turn, is influenced by the
occurring in the absence of genetic associations. Approximately overall conformational structure of the protein, thereby provid-
20% of familial ALS (FALS) cases are due to known mutations in the ing a basis by which different conformers react differently to a
copper, zinc superoxide dismutase (SOD1) gene. Molecular evi- covalent modifying agent (13). Thus, a conformational differ-
dence for a common pathogenesis of SALS and FALS has remained ence in a small proportion of molecules or a subtle difference in
elusive. Here we use covalent chemical modification to reveal an structure may be magnified. Using this approach, we now
attribute of spinal cord SOD1 common to both SOD1-linked FALS provide direct evidence for a common underlying pathogenesis
and SALS, but not present in normal or disease-affected tissues of SALS and FALS through the identification of a unique,
from other neurodegenerative diseases, including Alzheimer’s, crosslinked protein species of SOD1 that is common to both.
Parkinson’s, and Huntington’s diseases and spinal muscular atro-
phy, a non-ALS motor neuron disease. Biotinylation reveals a Results
32-kDa, covalently cross-linked SOD1-containing protein species A 32-kDa SOD1-Immunoreactive (IR) Protein Species Revealed by
produced not only in FALS caused by SOD1 mutation, but also in Biotinylation of Spinal Cord Extracts of ALS Patients. Conventional
SALS. These studies use chemical modification as a novel tool for immunoblotting after protein denaturation under strongly re-
the detection of a disease-associated biomarker. Our results iden- ducing conditions of spinal cord tissue extracts from ALS
tify a shared molecular event involving a known target gene and patients or normal samples revealed only the expected 16-kDa
suggest a common step in the pathogenesis between SALS and SOD1 polypeptide recognized by an antibody generated to a
FALS. peptide from human SOD1 (Fig. 1B, “⫺” lanes). After testing 12
commercially available chemical compounds that can modify
copper, zinc superoxide dismutase 兩 motor neuron 兩 neurodegeneration side-chains of amino acids of lysine, cysteine, aspartic acid,
glutamic acid, and arginine [see supporting information (SI)
Table 1], side-chain modification by covalent reaction with
A myotrophic lateral sclerosis (ALS) pathology and symptoms
in copper, zinc superoxide dismutase (SOD1)-linked famil-
ial ALS (FALS) patients closely resemble those of sporadic ALS
N-hydroxysulfosuccinimide-long chain-biotin (Sulfo-NHS-LC-
Biotin), which covalently adds biotin to conformationally acces-
(SALS) patients. In light of the linkage of the SOD1 gene to sible lysine side chains (Fig. 1 A), revealed a new 32-kDa species
FALS (1), it has long been speculated that there may be a in the SOD1-linked FALS sample [from a patient with a
common molecular basis for both forms of the disease, but no dominant alanine to valine mutation in SOD1 at position 4
such feature has been identified. A plethora of evidence has (SOD1A4V)], but not a normal sample (Fig. 1B). Surprisingly, a
demonstrated that mutant SOD1 causes motor neuron death by similar biotinylation-dependent 32-kDa species was also seen in
gain of toxic properties (2, 3). However, mechanisms of mutant a SALS sample (Fig. 1B).
SOD1-mediated toxicity to motor neurons remain elusive. Many Further characterization of the biotinylation reaction revealed
studies have investigated the possibility of structural differences that the intensity of the 32-kDa species depended on time of
in SOD1 caused by mutations as a basis for such toxic effects (4, incubation (Fig. 1C), concentration of Sulfo-NHS-LC-Biotin,
5). Although several common features of mutant SOD1 proteins the amount of the initial extract and incubation temperature
such as instability, higher sensitivity to denaturing conditions, (Fig. 1 D–F). The reaction was highly SOD1 protein conforma-
and propensity to form aggregates are found to be associated
with mutant SOD1-linked FALS disease course and severity
Author contributions: A.G., W.L.W., T.D.W., V.R.L., and J.L. designed research; A.G., W.L.W.,
(6–11), no single biochemical feature has been found to be E.A., M.D.P., and J.L. performed research; R.G.M., J.D.R., R.B., R.H., and D.W.C. contributed
shared by all identified mutant SOD1s, nor has there been a new reagents/analytic tools; A.G., W.L.W., T.D.W., D.W.C., V.R.L., and J.L. analyzed data;
single molecular feature of SOD1 identified to be associated with and A.G., D.W.C., V.R.L., and J.L. wrote the paper.
SALS. Conflict of interest statement: V.R.L. is a founder and J.L. a consultant to Prosetta Corpo-
We propose that SOD1 is more conformationally heteroge- ration, an early stage biotechnology company.
neous in vivo and that these ‘‘conformers’’ are relevant to ALS Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; FALS, familial
but difficult to distinguish by conventional biochemical analyses. amyotrophic lateral sclerosis; HD, Huntington’s disease; Hip, anterior hippocampus; IR,
immunoreactive; PD, Parkinson’s disease; SALS, sporadic amyotrophic lateral sclerosis;
To reveal such conformational differences especially those in- SMA, spinal muscular atrophy; SOD1, copper, zinc superoxide dismutase; Sulfo-NHS-LC-
trinsic to minor subspecies of SOD1 we applied a new approach Biotin, N-hydroxysulfosuccinimide-long chain-biotin.
involving biotinylation of accessible lysine residues in proteins, **To whom correspondence may be addressed. E-mail: or liuj@
followed by SDS/PAGE and immunoblot analysis for SOD1.
Covalent chemical modification of amino acid side groups has This article contains supporting information online at
the advantage of rapidly proceeding to completion, being readily 0705044104/DC1.
quenched, and capable of potentially affecting antibody immu- © 2007 by The National Academy of Sciences of the USA

12524 –12529 兩 PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0705044104
and four FALS with mutations in the SOD1 gene were exam-
ined. After biotinyation, an increased intensity of the 32-kDa
protein species IR to SOD1 antiserum was found in all of the
ALS samples (Fig. 2A). In 14 of 16 normal samples, this species
was absent or at much reduced level, although the expected 16
kDa species representing monomeric SOD1 was found in com-
parable amounts before and after biotinylation (Fig. 2 A). The
signal intensities of the 32-kDa species were significantly higher
in spinal cord samples from ALS versus normal subjects when
determined by either combining FALS and SALS samples (Fig.
2B; 118.0 ⫾ 46.5, n ⫽ 23 for all ALS and 25.1 ⫾ 34.2, n ⫽ 16,
for normal; P ⬍ 0.0001) or treating them separately (105.6 ⫾
37.8, n ⫽ 19; P ⬍ 0.0001 for SALS and 176.7 ⫾ 81.0, n ⫽ 4; P ⬍
0.0001 for FALS). Taken together, there is an ⬇5-fold increase
in the 32-kDa signal intensity in all ALS compared with normal
control (Fig. 2B). In addition, when a sample that contains a
mutation in another ALS-linked gene, Senetaxin (14) (also
known as ALS4), was examined, the 32-kDa signal was also
present (Fig. 2C).
To further determine the area within the spinal cord that gives
rise to this 32-kDa signal, we separated the spinal cord into gray
and white matter and analyzed them separately. As shown in Fig.
2D, the 32-kDa signal was exclusively associated with the gray
matter where motor neuron, glia, and other cells reside in
contrast to the white matter containing different axonal tracks,
including the cortical-spinal track. Because these assays compare
constant amounts of protein before and after biotinylation, the
increased immunoreactivity is likely due to enhanced antigen
availability to the antiserum as a result of biotinylation.
SOD1 mutations are also observed in SALS, albeit at a very
low frequency (⬍5%) (15–18). To eliminate the possibility that
the 32-kDa signals from some SALS subjects could come from
Fig. 1. A 32-kDa SOD1-IR protein species revealed by biotinylation of spinal subjects that might harbor SOD1 mutations, the coding se-
cord extracts of ALS patients. (A) Schematic diagram of the detection of a 32-kDa quences were determined for the SOD1 genes from spinal cord
SOD1-IR protein species by immunoblot analysis. Sulfo-NHS-LC-Biotin reacts with
autopsy samples from SALS-10, -11, -14, -15, and -17, samples
structurally available NH2 groups (outside the rings) from the lysine residues in the
SOD1 protein resulting in the modification of the protein at the sites of the
which had signal intensities of the 32 kDa that were comparable
side-chains (indicated by *). The biotinylated SOD1 proteins were separated on to those from FALSs. No SOD1 mutations were found (data not
SDS/PAGE gel and immunorecognized by the SOD1 antibody (darker ring). (B–G) shown) in these five samples (Fig. 2 A). Although we cannot rule
Spinal cord cytosolic proteins were incubated with (⫹) or without (⫺) Sulfo-NHS- out that there is no SOD1 mutation in the remaining 14 SALS

LC-Biotin, separated on SDS/PAGE gel, and immunoblotted with the SOD1 anti- (we have 19 total, minus 5 sequenced) samples analyzed, the
body. (B) The FALS sample was from an A4V mutation. Cytosolic proteins from 32-kDa signals from the SALS samples represent a typical
SALS-4 subject were used for C–G. (C) Time course of the reaction. One hundred population of non-SOD1 mutant-linked SALS, rather than
percent value was taken for the density of the 32 kDa at 150-min reaction time. SOD1-linked FALS.
(D) Sulfo-NHS-LC-Biotin dose responses. One hundred percent value was taken
for the density of the 32 kDa at 100 mM biotin concentration. (E) Dose–response
of the total amount of proteins used. One hundred percent value was taken at a
The 32-kDa SOD1 Species Is Specific for Disease-Affected Tissues in
total amount of 20 ␮g of proteins. (F) Temperature dependence of the reaction. ALS but Not Those in Alzheimer’s Disease (AD), Parkinson’s Disease
The optimal temperature for this reaction lies around 25°C. (G) Conformation- (PD), Huntington’s Disease (HD), and Spinal Muscular Atrophy (SMA).
specificity of the reaction. NHS-digoxigenin (1 mM) was added before the reac- To understand whether the 32 kDa is unique to ALS pathology
tion can block the biotinylation reaction resulting in the disappearance of the as opposed to a reflection of a process derived simply from a
32-kDa protein species (two middle lanes). All error bars represent SD from three prolonged agonal state, we further examined this signal in similar
independent experiments. samples from three additional non-ALS neurodegenerative dis-
eases, including AD, PD, and HD. Two spinal cord samples were
initially examined from PD subjects. The intensities of the 32-
tion-specific, because substrates containing partial or related kDa signal were comparable to those from normal subjects and
structural groups to Sulfo-NHS-LC-Biotin including sulfo-NHS, were significantly lower than those from ALS (Fig. 3B). As spinal
biotin, long chain (LC)-biotin, iodoacetyl-LC-biotin, NHS- cord is not the primary site of pathology in PD, we also examined
Squarin carboxilate (data not shown), and NHS-digoxigenin. affected brain regions in AD, PD, and HD together with the
(Fig. 1G) did not result in ALS-specific change of SOD1 motor cortical area as the common central nervous tissue for all
proteins. Maximum intensity of the 32-kDa SOD1 species was diseases. Hippocampus is one of the areas severely affected in
reached after 25 min at an optimal temperature of 25°C in the AD. When examined, there was no significant increase of the 32-
presence of 10 mM biotin. This condition was used in all efforts kDa signal in this brain area compared with the much increased
below. level in ALS spinal cord (P ⬍ 0.0001, Fig. 3 A and D). Similar
results were found for putamen, an affected brain region in both
The 32-kDa SOD1 Adduct Is Common to Both SALS and FALS. To PD and HD (Fig. 3D, P ⬍ 0.0001 and P ⬍ 0.02, respectively,
determine whether the appearance of the 32-kDa SOD1-IR compared with ALS spinal cord). For the motor cortexes, the
species in the initial SALS and SOD1-linked FALS samples after intensities of the 32-kDa signal in ALS, AD, PD, and HD were
reaction with Sulfo-NHS-LC-Biotin was a general feature of significantly lower when compared with those from ALS spinal
FALS and SALS, a series of spinal cord extracts from 19 SALS cords (Fig. 3D, P ⬍ 0.001, P ⬍ 0.0001, P ⬍ 0.0001, and P ⬍ 0.001,

Gruzman et al. PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12525
Fig. 2. Prevalence of the 32-kDa SOD1-IR protein species in both SALS and FALS spinal cords after biotinylation. Immunoblot analysis of cytosolic proteins from
human spinal cord autopsy samples (A and B) incubated with (⫹) or without (⫺) Sulfo-NHS-LC-Biotin. The density of the 32-kDa SOD1-IR signal from SALS-1 spinal
cord autopsy sample (indicated as SALS-std) was chosen as a 100%, which was run on all gels as the standard for spinal cord samples. (A) Spinal cords are from
normal, SALS, and SOD1-linked FALS autopsy samples. Mutations in SOD1 are G93C, A4V, G127X, D101G for FALS-1,2,3,4, respectively. (B) Bar graphs of intensities
of the 32-kDa signal from spinal cords for all ALS (SALS ⫹ SOD1-linked FALS, n ⫽ 23) and normal (n ⫽ 16). Error bars represent SD. *, P ⬍ 0.0001. (C) Subject with
a mutation in the Senetexin gene (ALS4). (D) The spinal cord samples from one normal and one SALS subject were separated as the white and gray matter before
the analysis.

respectively). Furthermore, the difference in the intensity of the the selective pathological role of the 32 kDa in ALS. Lastly, to
32-kDa signal in the motor cortex between ALS and AD or PD gain some insight on how widely the 32 kDa might be involved
is also significant (Fig. 3D, P ⬍ 0.02). This motor cortex value in motor neuron damage in other motor neuron diseases, we
between ALS and HD did not quite reach statistical difference examined spinal cord tissues from subjects with Type 1 form of
(P ⫽ 0.05) because of the large variation and small sample size SMA. The 32 kDa was not present in the four SMA spinal cords
(n ⫽ 4) for HD (Fig. 3D). The higher level of the 32 kDa in the examined (Fig. 3C and D, P ⬍ 0.0001). Motor neuron death in
motor cortex of ALS but not AD or PD is consistent with the Type I SMA differs from that of ALS by the fact that it is a much
known upper motor neuron pathology in ALS, demonstrating accelerated process and devoid of aging factors. Together, these

Fig. 3. The 32-kDa SOD1-IR protein species is specific for ALS pathology. Immunoblot analysis of cytosolic proteins from different brain region autopsy samples
were incubated with (⫹) or without (⫺) Sulfo-NHS-LC-Biotin. The density of the 32-kDa SOD1-IR signal from spinal cord autopsy sample SALS-1 (indicated as
SALS-std) was chosen as a 100%, which was run on all gels as the standard for all samples. (A) The anterior hippocampus (Hip) and primary motor cortex (MC)
from AD subjects were analyzed. The spinal cord from two PD subjects (B) and four SMA patients (C) was analyzed. (D) Bar graphs of the relative intensities the
32-kDa signal from ALS spinal cords (SC) and MC, AD Hip and MC, PD putamen (PU) and MC, HD PU and MC, and SMA SC.

12526 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0705044104 Gruzman et al.

Fig. 4. Antigen-specific peptide eliminates observed immunoreactivity to
SOD1 in the 32 kDa. Biotinylation and immunoblot were done as described.
Samples used are cytosolic proteins from muscle (SALS-2) and spinal cords
(SALS-7 and FALS-4). Peptides were incubated with the primary antibody for
30 min at room temperature in 500 ␮l of 5% milk in PBST before incubation
with the membranes. Antigen-specific peptide (DDLGKGGNEESTK) was used
at 1 ␮g and 4 ␮g, respectively. Nonspecific peptide (KESNGPVKVWGSIKGGC)
was used at 1 ␮g.

data demonstrate that the 32 kDa appears in the pathologic Fig. 5. The 32 kDa is a modified SOD1 protein species. (A) The commercial
process of age-related motor neuron degeneration such as that purified hSOD1 protein (Std, 40 ␮g) and SALS spinal cord cytosolic proteins
in ALS but not involved in pathology of AD, PD, HD, or SMA. (SALS-4, 300 ␮g) were subjected to gel filtration without biotinylation (⫺Bi-
otin). Molecular mass standards were used during elution, and only the
The 32 kDa Is a Modified SOD1 Protein Species. To confirm that the fractions corresponding to ⬇32-kDa range (between ⬇45 kDa and 28 kDa, as
observed 32 kDa is an altered form of SOD1, peptide compe- indicated) were shown in fractions 1–10. Fractions from the SALS-4 samples
were further subjected to biotinylation (⫹Biotin). (B) SALS muscle cytosolic
tition studies were performed. Antigen-specific peptide elimi-
proteins (300 ␮g) were applied to the Q-type ion exchange column, and the
nated reactivity to the 32-kDa (and the 16-kDa SOD1) signal in flow-through (fractions 1–3) and eluted fractions (4 –9) were analyzed. An
a dose-dependent manner (Fig. 4). In contrast, a different aliquot of the same sample (total input, 1/30) before the Q-Column is also
peptide from SOD1 had no such effect on the SOD1 immuno- shown. (C) The Q-column flow-through material was subjected to gel filtra-
reactrivity when used at similar a concentration (Fig. 4). To tions and analyzed directly (⫺Biotin) or after biotinylation (⫹Biotin). All
corroborate this conclusion, nanoelectrospray mass spectrome- proteins were separated on SDS/PAGE and immunoanalyzed with the SOD1
try was used to determine whether the 32 kDa in fact contained antibody.
SOD1 protein. The gel slice containing the 32 kDa was purified
as described (see SI Methods). Peptide sequence analysis based
on mass spectra acquired and peptide mapping (see SI Fig. 6 and immunoreactivity of this 32- kDa species with the anti-peptide
SI Table 2, respectively) provided 100% coverage of the human SOD1 antibody was markedly enhanced after biotinylation (Fig.
SOD1 protein sequence. These results demonstrate that the 32 5C Lower, fractions 2–7). These findings clearly demonstrate that
kDa does indeed contain the human SOD1 protein. the 32-kDa protein species is an endogenously modified SOD1
To further demonstrate that the SDS-resistant 32 kDa is a that is covalently linked to another SOD1 polypeptide or to a
polypeptide of similar size and that this species has substantially

modified SOD1 molecule in complex with itself or a similarly
sized protein (19), gel filtration and ion exchange chromatog- divergent biochemical properties relative to the natural non-
raphy experiments were performed to characterize the pro- covalently linked SOD1 homodimer.
tein(s) that gave rise to the 32-kDa species during biotinylation.
As expected, under nonreducing, nondenaturing conditions,
most SOD1 polypeptides in spinal cord cytosolic proteins were One mystery associated with not only ALS, but many other
present in dimers that eluted in gel filtration fractions corre- neurodegenerative diseases, is how to reconcile disease mecha-
sponding to ⬇32-kDa molecular mass, a range similar to the nisms at the molecular level between involvement of mutations
elution position of commercially purified human SOD1 protein in a few molecules that are responsible for a small percentage of
(Fig. 5A Top and Middle). Biotinylation of gel filtration fractions familial forms of the disease and unknown molecules that
revealed that the 32-kDa species was observed exclusively in the underlie the majority of the diseases that are of sporadic nature.
same molecular mass range (Fig. 5A Bottom), demonstrating the An assumption frequently made is that there is a common path
substrate for biotinylation behaved as a 32-kDa protein species. shared by both familial and sporadic forms of ALS that ulti-
Although biotinylation was required to observe the 32-kDa mately leads to a common clinical manifestation of mixed lower
SOD1 species in spinal cord extracts, it was observable even and upper motor neuron degeneration. Evidence supporting this
without biotinylation in muscle extracts from FALS or SALS view has remained elusive until now. Our study demonstrates at
patients (Fig. 5B). We exploited this property to determine the least one component of a shared pathway is an already known
native characteristics of this 32-kDa SOD1 species without molecule linked to ALS: SOD1.
biotinylation and to eliminate the possibility of noncovalent Like other neurodegenerative diseases, ALS has been considered
dimeric SOD1 becoming covalently linked during biotinylation. a disease of protein misfolding (20). Demonstrations that mecha-
Cytosolic ALS protein extracts were fractionated before bioti- nisms of mutant SOD1-mediated toxicity are due to gain of toxic
nylation by Q-column ion exchange chromatography. The nor- properties (2) suggest that altered conformations of SOD1 proteins
mally folded, non-covalently associated SOD1 dimers bound to because of mutation have different behaviors than the WT SOD1
the matrix and eluted only at higher ionic strength (Fig. 5B, (SOD1WT) protein. This is fully consistent with the more than 110
eluted in fractions 5–7). A small amount of IR SOD1 did not bind mutations in the SOD1 molecule linked to ALS (21) for which a
to the column. Subsequent immunoblotting after denaturation normally folded ‘‘WT’’ state and a misfolded one linked to toxicity
under reducing conditions revealed that this unbound SOD1 have frequently been found. Our observation of a covalently
exclusively migrated at a position corresponding to 32 kDa (Fig. crosslinked, biotinylation-dependent, SOD1-IR species (32 kDa)
5 B, fractions 1–3; C Upper, fractions 3–6). As before, the within the spinal cord demonstrates that SOD1 exists in at least two

Gruzman et al. PNAS 兩 July 24, 2007 兩 vol. 104 兩 no. 30 兩 12527
forms, one of which is disease-associated and common to both was carried out in identical procedure except omitting Sulfo-
FALS and SALS. The increased level of the 32-kDa species in ALS NHS-LC-Biotin in the reaction.
patients must originally come from the normally folded SOD1WT.
Our data demonstrate that an aberrant SOD1 species is present (or Gel Filtration. Sephacryl 300 column (Amersham Biosciences, Pis-
associated with) human ALS pathology, suggesting a possible cataway, NJ) was equilibrated with PBS buffer, pH 7.4, containing
involvement in that pathology. This conclusion extends previous 150 mM sodium chloride. The protein sample was applied onto the
results from in vitro cellular and animal models of ALS indicating column at a ratio of 60 ␮g of cytosolic proteins per 1 ml of Sephacryl
that SOD1WT or its oxidized form can be toxic in ALS disease 300. Proteins were eluted under gravity in the same buffer at 4°C,
mechanisms (22–28). Interestingly, the WT normal ␣-synuclein and a volume of 500 ␮l was collected for each fraction. All fractions
proteins, when produced by an additional locus in human, can also were dialyzed against deionizated water for 24 h at 4°C followed by
cause PD (29). lyopholization. Lyopholized samples were resuspended in PBS for
Our observation that the 32-kDa species is not present in biotinylation and/or immunoblot analysis.
normal and three non-motor neuron diseases including AD, PD
and HD, nor in Type I SMA which involves motor neuron death Column Chromatography. Fast-flow Q Sepharose anion exchange
somewhat different from that in ALS, strongly suggests that the column was equilibrated with 20 mM triethanolamine buffer, pH
32 kDa is ALS pathology-related. Compared with the vast 7.8. The protein sample was applied onto the column at a ratio
majority of SOD1 molecules that are almost certainly irrelevant of 320 ␮g of cytosolic proteins per 1 ml of Q-Sepharose. Proteins
to disease, the low levels of this disease-specific SOD1-IR species were eluted with the same buffer containing 0, 0.025, 0.05, 0.075,
(⬇1% of total SOD1) are usually below detection limits, but can 0.1, 0.15, 0.2, 0.25, 0.35, and 0.4 M NaCl (corresponding to
be revealed after biotinylation. Thus, although chemical modi- fractions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively) at a flow rate
fication has been used to study overall differences in protein of 0.5 ml/min. All fractions were dialyzed against deionized
conformation, the approach we have used here has uncovered a water for 24 h followed by concentration in Speed Vac (Savant,
molecular signature common to both familial and sporadic ALS. Holbrook, NY) at 25°C to a final volume of ⬇40 ␮l.
SOD1 IR protein species of molecular weight approximately
the size of human SOD1 dimer are reported in refs. 30–32. More Immunoblot Analysis. Proteins were separated on 12% SDS/PAGE
recently, studies from transgenic mouse models of FALS have gels and transferred onto PVDF membranes. Membranes were
suggested that the intermolecular disulfide-linked SOD1 aggre- incubated with a rabbit polyclonal anti-SOD1 antiserum (34)
gates are associated with disease toxicity (8, 23). One study used followed by an HRP-conjugated anti-rabbit IgG secondary anti-
immunoprecipitation to conclude an apparent SOD1 species of body before visualization with ECL plus solution (GE Biosciences,
⬇35 kDa was a complex between SOD1 and Bcl-2 (19), albeit our Little Chalfont, U.K.). Protein signals were quantified on Typhoon
mass spectrometric analysis offered no support for any linkage 9410, using Image Quant program (GE Biosciences).
to Bcl-2, at least in SALS. The covalently crosslinked, partially
misfolded species we have identified here is different from these Analysis of Human SOD1 cDNA Sequence. Sequence analysis of
previously proposed complexes. Its identification suggests a
RT-PCR-generated SOD1 cDNAs from the spinal cords of five
common underlying pathogenic mechanism in inherited and
individuals was performed. Total RNA was extracted by using
sporadic ALS, a finding that carries a substantial implication for
TRI-Reagent (Sigma–Aldrich, St. Louis, MO). Primers for human
design of therapeutic approaches for sporadic disease.
SOD1 RT-PCR were synthesized based on GenBank cDNA com-
Materials and Methods plete nucleotide sequence (GenBank accession no. AY049787)
as follows: forward 5⬘-ATGGCGACGAAGGCCGTGT-
Obtainment of Human Samples. Human autopsy samples were
obtained as described in ref. 24, from The Brain and Tissue Bank GCGTGC-3⬘ and reverse 5⬘-TTATTGGGCGATCCCAATTA-
for Developmental Disorders of the National Institute of Child CACCACAAGCC-3⬘. The RNA was reverse-transcribed and
Health and Human Development (Baltimore, MD), Forbes PCR-amplified by using the ImProm-II Reverse Transcription
Norris MDA/ALS Research Center (San Francisco, CA), and System and Pfu DNA polymerase (Promega, Madison, WI). Syn-
ALS Tissue Bank at University of Pittsburgh School of Medicine thesis of SOD1 cDNA and PCR were carried in a PTC-100
(Pittsburgh, PA). Informed consents were obtained from all programmable thermal controller. The PCR products were puri-
subjects before sample collections. Handling of all human sam- fied, the sequencing was performed on both strands, and the
ples is in accordance with the approved protocols by California sequence was determined at the DNA Analysis Unit of Integrated
Pacific Medical Center Internal Board Review, University of DNA Technologies (Coralville, IA).
Pittsburgh Internal Board Review, and Committee for Oversight
of Research Involving the Dead. Patients’ information is listed Statistical Analysis. Statistical significance was assessed between
in SI Table 3. groups, using a two-tailed Student’s t test. Significance is set at
P ⬍ 0.05.
Subcellular Fractionation and Biotinylation. Cytosolic proteins from
the autopsy samples were prepared as described in ref. 33. We thank William J. Hansen, Yelena Mikityansky, William J. Santoni,
and David Ovadia for technical support and help with preparation of the
Protein concentrations were measured by using the BCA method
article and Jason Mass for coordinating tissue collections at The Forbes
(Pierce Chemical, Rockford, IL). Biotinylation reaction, unless Norris MDA/ALS Research Center. This work has been supported by
indicated otherwise, was performed with a total of 10 ␮g of the California Pacific Medical Center Research Institute (V.R.L. and
cytosolic proteins incubated with 10 mM Sulfo-NHS-LC-Biotin J.L.), The Forbes Norris MDA/ALS Research Center (J.L.), the ALS
(Pierce Chemical) in PBS buffer, pH 7.4, for 25 min at 25°C. The Association (J.L.), the Center for Excellence in Bioinformatics at
reaction was stopped by adding free lysine-HCl at a final University at Buffalo (T.D.W.), and the National Institutes of Health
concentration of 20 mM for 20 min at 25°C. Control treatment (R.B. and D.W.C.).

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