Invited Symposium: Oxidative Stress and the CNS


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Enhanced Free Radical Generation by FALS-associated Cu,Zn-SOD Mutants

Contact Person: Moon B Yim (yimm@gwgate.nhlbi.nih.gov)

Association of FALS with Cu,Zn-SOD Mutants

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is an age-dependent degenerative disorder of motor neurons of cortex, brainstem, and spinal cord. About 10% of all ALS patients are familial cases, and approximately 20% of FALS cases are associated with dominantly inherited missense mutations in the coding regions in SOD1, the gene for Cu,Zn superoxide dismutase (1, 2) which catalyzes the dismutation of superoxide radical anions O2-. to hydrogen peroxides and oxygen molecules (3). 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 (1, 2). 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 to that of normal individuals (2). This reduction in SOD dismutation activity may facilitate the pathway of oxidative damage to cause FALS symptoms. However, several studies with transgenic mice (4, 5), transfected cells (6, 7), and lymphoblasts of patients (8) revealed 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 (4-8).

The nature of the cytotoxic gain-of-function caused by FALS mutants is in debate. The proposed mechanisms include that FALS Cu,Zn-SOD mutants possess: (i) enhanced free radical-generating activity with H2O2 as substrate(9-11); (ii) enhanced peroxynitrite-mediated tyrosine nitration which caused nitration of tyrosine residues (12, 13); and (iii) induced aggregate formation at the motoneurons (14, 15). Here we summerize our findings obtained from the studies with purified FALS Cu,Zn-SOD mutants.

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Free Radical-Generating Activity

In addition to the usual superoxide dismutation activity, Cu,Zn-SOD is known to exhibit anion binding capacity (16, 17), inactivation by its own reaction product H2O2 (18-20), the purported peroxidase activity (21), and capacity to enhance nitration of protein tyrosine residues by peroxinitrite (22). We also found that Cu,Zn-SOD, but not Mn-SOD, has free radical-generating activity (23, 24). The active Cu,Zn-SOD can catalyze the generation of free (. OH) radicals from H2O2. In the presence of H2O2 and radical scavengers, especially with anionic character which include neurotransmitters glutamate and taurine, both free (. OH) radicals and scavenger-derived radicals are produced (22, 23). In addition, we found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (9, 10). We describe our findings of the free radical-generating activity of Cu,Zn-SOD itself in this section, which will be followed on FALS mutants in the next section.

To monitor the production of free radicals, two spin traps, 5,5-dimethyl-1-pyrroline N -oxide (DMPO) and N-tert -butyl--phenylnitrone (PBN), were used in our studies (23, 24) to convert transient free radicals to stable free radical adducts according to the following reactions:

DMPO + R. ---> DMPO-R.

PBN + R. ---> PBN-R.

The nature of the trapped free radical was identified by EPR spectroscopy. When O2-. (dissolved KO2 in dried dimethylsulfoxide) was added to the NaHCO3/CO2 buffer at pH 7.6 containing 100 mM DMPO, EPR resonance lines of DMPO-superoxide adduct (DMPO-OOH) were observed. When similar experiments were carried out in the presence of Cu,Zn-SOD, DMPO-hydroxyl radical adduct (DMPO-OH) signals were detected in place of the DMPO-OOH signals. Addition of both Cu,Zn-SOD and catalase inhibited the formation of DMPO-OH adducts, indicating that H2O2 is needed for the formation of DMPO-OH adducts. The direct addition of H2O2 in place of O2-. produced similar results. DMPO-OH formation also required active Cu,Zn-SOD, because heat-inactivated Cu,Zn-SOD or Mn-SOD failed to produce these radical adducts. Addition of chelating agents EDTA or DTPA (0.1 mM) did not give any effect on the radical adduct formation, indicating that free Cu ion was not the cause of this formation. In the presence of . OH radical scavengers with anionic character such as HCO3 - or N3 -, DMPO adducts of the scavenger-derived radicals, DMPO-CO2 -. or DMPO-N3 ., were observed in addition to DMPO-OH. The concentration ratio between DMPO-OH and DMPO-scavenger-derived radical adducts observed in these experiments reflects competition reactions between DMPO and scavengers of free . OH radicals. Surprisingly, with neutral . OH radical scavengers, such as ethanol, the formation of DMPO-hydroxyethyl radical adducts was insignificant and the concentration of DMPO-OH formed was unaffected. In contrast, with a different spin trap, PBN, in place of DMPO, ethanol yields PBN-hydroxyethyl radical adducts as expected in the competition reaction for free . OH radicals. These seemingly contradicting results may be caused by the different affinities of these spin traps for the positively charged active channel leading to the active site of Cu,Zn-SOD, where the . OH radicals are generated. To prove this hypothesis, we carried out experiments to measure binding constants of the spin traps to the active site by using a negatively charged chromogen, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) (24).

The ABTS is known to react with . OH radical with a diffusion-controlled rate (Reaction 3) to produce a stable oxidized free radical, ABTS+., which has strong absorption at 415 nm (E415 = 3.6 x 104 M-1 cm-1) and 660 nm (E660 = 1.2 x 104 M-1 cm-1).

ABTS + . OH ---> ABTS +. + OH- ; k = 1.2 x 1010 M-1 s-1 (3)

Addition of active Cu,Zn-SOD also produced ABTS +. in the presence of H2O2 and ABTS. The formation of ABTS +. (monitored at 415 or 660 nm) followed first order kinetics with respect to Cu,Zn-SOD and H2O2. However, it showed a binding isotherm with ABTS that yielded a dissociation constant for ABTS-Cu,Zn-SOD of K d = 7.1 ± 0.5 µM. The formation of ABTS +. was inhibited by the presence of DMPO or PBN in the reaction mixture. By performing a competition study, K d values for DMPO and PBN were obtained as 0.63 and 11 mM, respectively. A radical scavenger, formate anion, also inhibits the formation of ABTS +., whereas ethanol does not. The results together indicate that DMPO and anionic scavengers have easy access inside the positively charged active site channel of Cu,Zn-SOD, whereas PBN and neutral ethanol stay outside of the channel. Consequently, DMPO or anionic scavengers are in a position to intercept the newly formed .OH radicals and also drastically reduce the probability for the reaction of .OH radicals with ethanol. Therefore, DMPO-hydroxyethyl radical adducts could not be detected in the Cu,Zn-SOD-catalyzed reaction. PBN and ethanol, however, are able to compete in the bulk solution for free .OH radicals released from the active site of Cu,Zn-SOD and thus both PBN-hydroxyethyl radical and PBN-OH can be detected. Conversely, these results indicate that free .OH radicals and scavenger-derived radicals can reach the bulk solution and may cause oxidative damage to the biological environments.

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Enhanced Free Radical-Generating Activity

We investigated whether there are any differences in the free radical-generating function between the wild-type and FALS Cu,Zn-SOD mutants (9, 10). For this purpose, we constructed recombinant baculovirus carrying wild-type, G93A, and A4V cDNA of human Cu,Zn-SOD and overproduced the enzymes in insect cell Sf9 infected with the recombinant virus stocks. The subunit mass of the purified enzymes measured using electrospray mass spectroscopy indicate that the recombinant proteins contain acetylated N-terminals and one glycine and one alanine have been substituted by one alanine and one valine in the G93A and A4V mutants, respectively. The copper content in these proteins determined by atomic absorption spectroscopy is almost identical, showing that each subunit contains 0.93 ± 0.05 equivalent 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 (3). The specific activities of the recombinant G93A and A4V Cu,Zn-SOD so determined are also almost identical, 93 ± 4 %, to that exhibited by the wild-type enzyme. The free radical-generating activities of the mutants are, however, enhanced relative to that of the wild-type enzyme in the following order: wild-type < G93A < A4V. To examine the cause of this enhancement, we measured the initial rates of DMPO-OH adduct formation at various H2O2 concentrations. The double reciprocal plots of these results showed that the Km values for H2O2 are consistently lower with the mutants;wild-type (44 mM) > G93A (25 mM)> A4V (13 mM) (9, 10). Thus, the enhanced free radical-generating activity of mutants was due to the increase in affinity for H2O2. It should be emphasized that the enzymes used in this study are as purified, not reconstituted. A number of investigators have used reconstituted enzymes by replacing the Cu and Zn ions in active site.

We also investigated whether FALS Cu,Zn-SOD mutants have enhanced capacity for tyrosine nitration mediated by peroxinitrite (unpublished results). In this study, we used two substrates, free tyrosine and a synthetic pentadecameric peptide patterned after the tyrosine phosphorylation site of p34(cdc2) kinase, which was used in our previous work (25). The reaction products obtained by rapid quench-flow method were analyzed by HPLC to determine the time course of the nitrated-products formation. The results showed that the nitration activity of the FALS mutants is virtually identical to that of wild-type enzyme. The enzymes inactivated by H2O2 do not promote nitration of tyrosine. Previous reports, however, indicated that FALS mutants have lower affinity for zinc (13, 26), and the zinc-deficient enzyme shows enhanced capacity to accelerate tyrosine nitration by peroxinitrite (12, 13).

The gain-of-function of FALS Cu,Zn-SOD mutants observed so far from studies with purified active enzymes is the enhanced activity for free radical generation. In view of the fact that the intracellular concentration of the H2O2 is in the low or submillimolar range, the differential in 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.  (27) and Juneja et al. (28) 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 to 2.4 years for the average survival of G93A FALS patients. Therefore, our results together with those reported by Rosen et al. (27) may indicate that the Km values for H2O2 of different FALS mutants are associated with the aggressiveness of the disease progression.

Recently, Singh, et.al. (29) reexamined our spin-trapping investigations. They reported that there is no difference in free radical-generating activity or purported peroxidase activity (21) between wild-type and FALS mutants and, thus, concluded that free radicals are not involved in FALS disease. There are differences in the experimental design and enzymes used in this study. Singh, et.al. prepared proteins by expressing in bacterial system and enzymes were reconstituted by incubating the proteins in the presence of equal moles of cupric citrate and zinc sulfate. Our enzymes used are fully active as purified. Our data were obtained by measuring the time-courses (the first data point at 30 sec. after injecting H2O2 in situ set up) of the DMPO-OH formation in the presence of varying concentration of H2O2. The data obtained in this way are consistently reproducible, while the data of Singh, et.al. were obtained with a single point measurement immediately after addition of H2O2 at an undefined time. In addition, they concluded that free hydroxyl radical is not the source of their observed DMPO-OH adducts, but rather, it is entirely due to the peroxidase reaction between the DMPO and the active-site Cu2+-.OH. If true, DMPO should inhibit the inactivation of the enzyme induced by H2O2, because removal of the metal-bound .OH by DMPO should prevent the enzyme inactivation by the metal-bound .OH. However, they pointed out that DMPO, up to 200 mM, could not prevent the inactivation, an experimental fact which we also found and discussed in our previous publication (24). Indeed, their observation support our conclusion that a large portion of the copper-bound .OH are released to yield free .OH radicals.

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Concluding Remarks

We have compared the enzymic activities of FALS Cu,Zn-SOD mutants, G93A and A4V, to those of wild-type. Our results obtained with intact active enzymes indicate that the capacity to activate tyrosine nitration and superoxide dismutation activity of FALS mutants are virtually identical to those of the wild-type. The free radical-generating activity of the FALS mutants is, however, consistently enhanced in comparison with that of wild-type enzyme. This enhancement is attributed to reduced Km values for H2O2 [wild-type (44 mM) > G93A (25 mM)> A4V (13 mM)]. An apparent correlation between the Km values for H2O2 and the clinical severity of the disease for the two FALS mutants suggest that free radical may play a role in the progression of ALS.

Recent in vivo studies demonstrated the involvement of free radicals in ALS. Bogdanov, et.al. observed elevated hydroxyl radical generation in vivo in G93A transgenic mice (30). Andrus, et.al. demonstrated in similar transgenic mice that lipid peroxidation and protein carbonyl levels, marker of protein oxidative damage, were elevated in comparison with nontransgenic mice (31, 32). Also, the fibroblasts obtained from FALS patients showed an increased sensitivity to H2O2 stress, which was explained by the increased affinity of the mutants for H2O2 (33). Bruijn, et.al. (14), however, suggested that poorly or unstably folded mutants mitigate Cu,Zn-SOD-containing aggregate that are toxic to motor neurons. They suggested that this process is induced by a yet-unidentified chemical reaction rather than the involvement of free radicals. Chou, et.al, (15) observed advanced glycation endproducts in neurofilament conglomeration of motor neurons in ALS patients. It has been shown that glycation endproducts (some are protein cross-linked products) are formed by oxidative stress. In addition, we have shown that the protein cross-linked product serve as an active site for the generation of free radicals (34, 35). Thus, the aggregation may be mediated by free radicals initially and these cross-linked proteins will then function as free radical generators to create an autocatalytic phenomenon for the progression of the disease.

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Yim, M.B.; Yim, H.-S.; Chock, P.B.; Stadtman, E.R.; (1998). Enhanced Free Radical Generation by FALS-associated Cu,Zn-SOD Mutants. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/juurlink/yim0183/index.html
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