COMMUNICATION:
A Familial Amyotrophic Lateral Sclerosis-associated A4V Cu,Zn-Superoxide Dismutase Mutant Has a Lower Km for Hydrogen Peroxide
CORRELATION BETWEEN CLINICAL SEVERITY AND THE Km VALUE*

(Received for publication, December 19, 1996, and in revised form, January 23, 1997)

Hyung-Soon Yim , Jung-Hoon Kang Dagger , P. Boon Chock , Earl R. Stadtman and Moon B. Yim §

From the Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


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 (Obardot 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.

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

Site-directed Mutagenesis for the A4V

Human Cu,Zn-SOD cDNA was isolated previously in our laboratory from human placental cDNA library in lambda 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).

Other Methods

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 epsilon 258 = 1.03 × 104 M-1 cm-1 (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).

A spin trap, DMPO, was used to convert transient free radicals (R·) to stable free radical adducts according to the Reaction R1.
<UP>DMPO</UP>+<UP>R<SUP>·</SUP></UP>→<UP>DMPO−R<SUP>·</SUP></UP>
<UP><SC>Reaction</SC></UP><UP> 1</UP>
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

Characterization and Dismutation Activity of the Recombinant Cu,Zn-SOD

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.


Fig. 1. SDS-PAGE and immunoblot analysis of purified recombinant wild-type and FALS mutants A4V and G93A Cu,Zn-SOD. Purified proteins were analyzed by SDS-PAGE on 4-20% gradient gel and visualized with Coomassie Blue (A, 5-µg proteins) or subjected to immunoblot analysis with antibodies to human Cu,Zn-SOD (B, 0.2-µg proteins). Lanes in A and B are as follows: lane 1, wild-type Cu,Zn-SOD; lane 2, G93A mutant Cu,Zn-SOD; lane 3, A4V mutant Cu,Zn-SOD; and lane 4, human erythrocyte, Cu,Zn-SOD.
[View Larger Version of this Image (29K GIF file)]


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.


Fig. 2. EPR spectra of DMPO-OH radical adducts formed in solutions containing H2O2 and the human Cu,Zn-SOD. Solutions contained 100 mM DMPO and 5 mM H2O2 in 23.5 mM NaHCO3/CO2 buffer at pH 7.6. In addition, the reaction mixture contained 0.8 µM wild-type Cu,Zn-SOD (spectrum A), 0.8 µM G93A mutant Cu,Zn-SOD (spectrum B), 0.8 µM A4V mutant Cu,Zn-SOD (spectrum C), and 0.8 µM heat-inactivated wild-type Cu,Zn-SOD (spectrum D). The spectra were recorded at 5 min after initiating the reaction with H2O2.
[View Larger Version of this Image (16K GIF file)]


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).


Fig. 3. Double-reciprocal plots of DMPO-OH radical adducts formation catalyzed by the Cu,Zn-SOD as a function of H2O2 concentration. The data were obtained in the presence of 100 mM DMPO in 23.5 mM NaHCO3/CO2 buffer at pH 7.6. In addition, the solutions contained 0.8 µM wild-type (line A), 0.8 µM G93A (line B), and 0.8 µM A4V (line C) Cu,Zn-SOD.
[View Larger Version of this Image (17K GIF file)]



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

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address: Dept. of Genetic Engineering, Chongju University, Chongju 360-764, Korea.
§   To whom all correspondence should be addressed. Tel.: 301-496-9494; Fax: 301-496-0599.
1   The abbreviations used are: FALS, familial amyotrophic lateral sclerosis; Cu,Zn-SOD, copper,zinc-superoxide dismutase; G93A, Gly-93 right-arrow Ala substitution; A4V, Ala-4 right-arrow 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.


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