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(Received for publication, December 19, 1996, and in revised form, January 23, 1997)
From the Laboratory of Biochemistry, NHLBI, National Institutes of
Health, Bethesda, Maryland 20892
ABSTRACT Point mutations of Cu,Zn-superoxide
dismutase (Cu,Zn-SOD) have been linked to familial amyotrophic lateral
sclerosis (FALS). We reported that Cu,Zn-SOD can catalyze free radical
generation and a FALS mutant, G93A, exhibits an enhanced free
radical-generating activity, while its dismutation activity is
identical to that of the wild-type enzyme (Yim, M. B., Kang, J.-H.,
Yim, H.-S., Kwak, H.-S., Chock, P. B., and Stadtman, E. R. (1996)
Proc. Natl. Acad. Sci. U. S. A. 93, 5709-5714). The A4V
mutation is both the most commonly detected of FALS-associated
SOD1 mutations and among the most clinically severe (Rosen,
D. R., Bowling, A. C., Patterson, D., Usdin, T. B., Sapp, P., Mezey,
E., McKenna-Yasek, D., O'Regan, J. P., Rahmani, Z., Ferrante, R. J.,
Brownstein, M. J., Kowall, N. W., Beal, M. F., Horvitz, H. R., and
Brown, R. H., Jr. (1994) Hum. Mol. Genet. 3, 981-987). We
cloned the cDNA for the FALS A4V mutant, overexpressed the protein
in Sf9 insect cells, purified the protein, and studied its enzymic
activities. Our results show that the mutant and wild-type enzymes
contain one copper ion per subunit and have identical dismutation
activities. However, the free radical-generating activity of the
mutant, as measured by the spin trapping method at low
H2O2 concentration, is enhanced relative to
that of the wild-type and G93A enzyme (wild-type < G93A < A4V). This is due to the decrease in the Km value for H2O2, wild-type > G93A > A4V,
while the kcat is identical for these enzymes.
Thus, the FALS symptoms are not associated with the reduction in the
dismutation activity of the mutant enzyme. The fact that the A4V mutant
has the lowest Km for H2O2 is correlated to the clinical severity observed with the A4V patients, if FALS is associated with a differential gain of the free
radical-generating function of the Cu,Zn-SOD mutant.
INTRODUCTION Familial amyotrophic lateral sclerosis
(FALS)1 is an autosomal dominant disorder
of motor neurons of cortex, brainstem, and spinal cord (1). Recent
studies showed that FALS cases have missense mutations in the coding
regions in SOD1, the gene for Cu,Zn-superoxide dismutase
(Cu,Zn-SOD) (2, 3) that catalyzes the dismutation of superoxide radical
anions (O Clinical studies have shown that the A4V mutation is the most commonly
detected in all FALS (3, 10). In addition, this mutation is found to be
associated with the most clinically severe, in terms of reduced
survival time after the onset of the disease: 1.2 years, as compared
with 2.5 years for all other FALS patients (10). We therefore
investigated to find out whether a correlation exists between severity
of the disease and enhancement of the free radical generating function
of Cu,Zn-SOD mutants. Our results showed that under low
H2O2 concentration, the A4V mutant has a higher
free radical-generating activity than those of the G93A and the
wild-type enzyme (A4V > G93A > wild-type), due to the decrease in the value of Km for
H2O2 (A4V < G93A < wild-type).
EXPERIMENTAL PROCEDURES Human Cu,Zn-SOD
cDNA was isolated previously in our laboratory from human placental
cDNA library in The identities and purities of the
recombinant human Cu,Zn-SOD, both the wild-type enzyme and its mutant
A4V, were analyzed by SDS-PAGE, immunoblotting, activity staining, and
assay of enzymic activity. Protein concentration was determined
spectrophotometrically using the extinction coefficient
A spin trap, DMPO, was used to convert transient free radicals
(R·) to stable free radical adducts according to the Reaction
R1.
Volume 272, Number 14,
Issue of April 4, 1997
pp. 8861-8863
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
CORRELATION BETWEEN CLINICAL SEVERITY AND THE
Km VALUE*
,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
2) to hydrogen peroxide and oxygen molecules (4).
Cu,Zn-SOD also catalyzes free radical generation using
H2O2 and small anions as substrates (5-7).
Most of the FALS mutants have point-mutation sites in conserved
interaction regions critical to the subunit fold and dimer contact,
rather than residues in the active-site or in the electrostatic active
channel (3). Initial studies of Cu,Zn-SOD activity in erythrocytes and
brain tissues of FALS patients carrying mutations at the
SOD1 locus demonstrated reduced Cu,Zn-SOD dismutation activity compared with that of normal individuals (3, 8-11). This
reduction in SOD dismutation activity may facilitate the pathway of
oxidative damage to cause FALS symptoms. However, several studies with
transgenic mice (12, 13), transfected cells (14, 15), and lymphoblasts
of patients (16) also demonstrated that levels of total Cu,Zn-SOD
dismutation activities remain high or higher than normal, which
suggests that the FALS mutations in SOD1 may act through a
dominant cytotoxic gain-of-function (12-16). Recently, we have shown
that a FALS mutant G93A and the wild-type Cu,Zn-SOD prepared by the
recombinant method have identical dismutation activity of superoxide
anions (17). However, the free radical-generating function (5, 6) of
the G93A mutant measured by the spin trapping method is enhanced
relative to that of the wild-type enzyme (17). We found that this
enhancement is due to a small decrease in the value of
Km for H2O2. Wiedau-Pazos
et al. (18) have also shown that several FALS mutants
produced higher levels of DMPO-OH adducts relative to the wild-type
enzyme.
gt11 by using the polymerase chain reaction
technique. For the site-directed mutagenesis, two flanking primers were
used: the forward flanking primer,
5
-ATCGGATCCATGGCGACGAAGGTCGTGTGC-3, in which the
BamBI restriction and the mismatched (bold italic letter)
sites are located, and the reverse flanking primer,
5-CGAGAATTCTTATTGGGCGATCCCAATTAC-3, in which the EcoRI
site is located. Other experimental procedures, including construction
of recombinant baculovirus carrying human Cu,Zn-SOD cDNA,
overproduction of the enzymes in Sf9 insect cells, and purification of
proteins, were described previously (17).
258 = 1.03 × 104
M
1 cm1 (4). The dismutation
activity was measured by monitoring the capacity of the enzyme to
inhibit the reduction of ferricytochrome c by
xanthine/xanthine oxidase as described by McCord and Fridovich (4). The
identities of purified wild-type and A4V proteins were verified by
electrospray mass spectroscopy, and the content of copper ions in
these proteins was determined by atomic absorption spectroscopy
(Perkin-Elmer, model 4100ZL).
The nature of the trapped free radical can be identified by
hyperfine coupling constants of the spin adduct measured by EPR spectroscopy. The EPR spectrometer and the procedure for sample transfer were described previously (5, 6). Spectral acquisitions began
30 s after initiation of the reaction by the injection of H2O2. The conditions for the acquisition of
spectral data were as follows: temperature, 25 °C; microwave power,
20 milliwatts; modulation amplitude, 1 G; conversion time, 10.24 ms;
time constant, 82 ms; sweep time, 21 s; sweep width, 70 G with
2048-point resolution. The 3-carbamoylproxyl spin label was used as a
standard to estimate the concentration of the DMPO-free radical
adducts. All buffers used were pretreated with Chelex 100 resin.
RESULTS
The expression system, which uses Sf9 insect cells
infected with the recombinant baculoviruses, was described previously
(17). The recombinant enzymes were purified using a combination of
ammonium sulfate precipitation, gel filtration, and ion exchange
chromatography and were dialyzed against phosphate buffer (2.5 mM, pH 7.8) in the presence of Chelex 100 (17). The
SDS-PAGE and immunoblot of the purified recombinant enzyme shown in
Fig. 1 do not exhibit any other protein band. The masses
of the subunits measured using electrospray mass spectroscopy were
determined to be 15,845.2 Da for the wild-type, 15,859.6 Da for G93A,
and 15,874.0 Da for A4V proteins. These results indicate that the
recombinant proteins contain acetylated N-terminals, and the glycine
and the alanine are substituted by the alanine and the valine in the
G93A and A4V mutants, respectively. The concentration of the copper
ions in these proteins determined by atomic absorption spectroscopy is
almost identical, showing that 1 mol of each subunit contains 0.95 ± 0.05 mol of copper ions. Superoxide dismutation activities of the
recombinant Cu,Zn-SOD were measured by monitoring their ability to
inhibit the reduction of cytochrome c by xanthine/xanthine oxidase (4). The specific activities of the recombinant G93A and A4V
Cu,Zn-SOD so determined are 95 ± 4% of that exhibited by the
wild-type enzyme.
Free Radical-generating Activity of Cu,Zn-SOD
We have
described previously (5, 6) that Cu,Zn-SOD has a free
radical-generating function, which catalyzes the formation of
·OH radicals with H2O2 as substrate and
the formation of scavenger-derived radicals with anionic radical
scavengers and H2O2 as substrates. Results
obtained from spin trapping experiments with the recombinant enzymes
are shown in Fig. 2. Spectrum A originates
from the ·OH radical adduct of DMPO (DMPO-OH) generated in a
solution containing 100 mM DMPO, 5 mM
H2O2, and 0.8 µM recombinant
wild-type Cu,Zn-SOD in 23.5 mM
NaHCO3/CO2 buffer at pH 7.6. The
heat-inactivated Cu,Zn-SOD fails to catalyze the formation of free
radical adducts (spectrum D), indicating that active enzyme
is required. When the experiments were repeated under the identical
conditions using the recombinant mutant enzymes, the EPR signal of the
DMPO-OH adduct was enhanced and showed the relative amplitude in the
following order: wild-type < G93A (spectrum B) < A4V
(spectrum C). The results obtained with varying
concentration of H2O2 consistently show that
the A4V mutant is a better catalyst for ·OH radical production
than the G93A and the wild-type enzyme.
To examine the cause of this enhancement, we measured the initial rates
of the DMPO-OH adduct formation as a function of
H2O2 concentrations. The double-reciprocal plot
of these results is shown in Fig. 3. It shows that the
wild-type, G93A, and A4V enzymes have an identical
Vmax value (3.1 µM/min,
kcat = 4.0/min) for the formation of the adducts
at pH 7.6. However, the Km for
H2O2 is consistently lower with the A4V mutant
relative to those with the G93A and the wild-type enzyme. The
Km so determined are 44, 25, and 13 mM
for the wild-type, G93A, and A4V, respectively. The
Km values of the wild-type and the G93A and their
kcat values are very similar to the values reported previously (17).
DISCUSSION
The results obtained in this study with the purified recombinant human wild-type Cu,Zn-SOD and its FALS mutants, A4V and G93A, revealed that these enzymes contain one copper ion per subunit, and they exhibit similar superoxide dismutation activities. However, the free radical-generating activity of the A4V and the G93A mutants, as measured by spin trapping and EPR methods, is consistently enhanced in comparison with that of the wild-type enzyme (A4V > G93A > wild-type) (Fig. 2). This enhancement is caused by the lower Km values for H2O2 found with the A4V and the G93A mutants relative to that of the wild-type enzyme (A4V (13 mM) < G93A (25 mM) < wild-type (44 mM)) (Fig. 3). The lowest Km value found with the A4V mutant is less than one-third of the value obtained with the wild-type enzyme.
The enhanced free radical-generating activity of the FALS mutants may, in part, be responsible for the cause of FALS. The high capacity of the mutant enzymes to catalyze the generation of ·OH radicals will lead to following consequences: (i) the higher concentration of ·OH radicals generated by the mutants accelerates their direct damaging reactions against the biological environments, which include the Cu,Zn-SOD mutants themselves. The damaging reaction will accelerate the inactivation of the mutant Cu,Zn-SOD and causes the release of its metal ions. The released copper ions, when bound to proper ligand molecules or proteins, will also enhance Fenton-type site-specific damaging reactions. The mutant Cu,Zn-SOD and perhaps the metal-free inactive SOD may also participate in other damaging reactions, such as enhancement of peroxynitrite-mediated tyrosine nitration, and lead to permanent impairment of the signal transduction pathway by blocking phosphorylation (19-21). (ii) The lower Km for H2O2 of the mutants will enhance the production of anionic scavenger-derived radicals from the scavengers such as neurotransmitters, glutamate and taurine (6), and cellularly abundant glutathione (22). These radicals may exert more specific deleterious effects in motor neurons (17).
In view of the fact that the intracellular concentration of the H2O2 is in the low or submillimolar range, the differential Km values observed with the mutant enzymes should play a dominant role in the severity of the various FALS. In this regard, one expects the much reduced Km value of the A4V mutant obtained in this study to correlate with the severity of A4V FALS patients. Rosen et al. (10) have shown in their clinical study that the A4V mutation is both the most commonly detected and the most clinically aggressive form associated with FALS patients. They found that about 40% of FALS families subjected for their study have the A4V mutation, and these patients survive only an average of 1.2 years after the onset of the disease, as compared with 2.5 years for the average survival of all other FALS patients. Therefore, our results together with those reported by Rosen et al. (10) indicate that the Km values for H2O2 of different FALS mutants are associated with aggressiveness of the disease progress.
FOOTNOTES
Present address: Dept. of Genetic Engineering, Chongju University,
Chongju 360-764, Korea.
Ala substitution; A4V, Ala-4 Val
substitution; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; PAGE,
polyacrylamide gel electrophoresis.
ACKNOWLEDGEMENT
We thank Dr. Henry M. Fales for performing electrospray mass spectroscopy.
REFERENCES
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