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Originally published In Press as doi:10.1074/jbc.M102781200 on August 22, 2001

J. Biol. Chem., Vol. 276, Issue 43, 39872-39878, October 26, 2001
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Accelerated S-Nitrosothiol Breakdown by Amyotrophic Lateral Sclerosis Mutant Copper,Zinc-Superoxide Dismutase*

Michael A. JohnsonDagger , Timothy L. MacdonaldDagger , Joan B. Mannick§, Mark R. Conaway, and Benjamin Gaston||**

From the Dagger  Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, the § Department of Medicine, University of Massachusetts Medical School, Rose Reed Gordon Building, Shrewsbury, Massachusetts 01545, the  Department of Health Evaluation Sciences, Division of Biostatistics and Epidemiology, University of Virginia Health System, Charlottesville, Virginia 22908, and the || Department of Pediatrics, Division of Pulmonary Medicine, University of Virginia Health System, Charlottesville, Virginia 22908

Received for publication, March 29, 2001, and in revised form, July 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutations in copper,zinc-superoxide dismutase (SOD) have been implicated in familial amyotrophic lateral sclerosis (FALS). We have investigated the breakdown of S-nitrosothiols by wild-type (WT) SOD and two common FALS mutants, alanine-4 valine (A4V) SOD and glycine-37 arginine (G37R) SOD. In the presence of glutathione, A4V SOD and G37R SOD catalyzed S-nitrosoglutathione breakdown three times more efficiently than WT SOD. Indeed, A4V SOD catabolized GSNO more efficiently than WT SOD throughout the physiological range of GSH concentrations. Moreover, a variety of additional S-nitrosothiols were catabolized more readily by A4V SOD than by WT SOD. Initial rate data for fully reduced WT SOD and A4V SOD, and data using ascorbic acid as the reductant, suggest that FALS mutations in SOD may influence the efficiency of reduction of the copper center by glutathione. We have identified a potentially toxic gain of function of two common FALS mutations that may contribute to neurodegeneration in FALS.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amyotrophic lateral sclerosis (ALS)1 is a fatal disorder characterized by the degradation of motor neurons in the cerebral cortex, brain stem, and spinal cord (1). The average age of onset is 55 years with complete paralysis and death resulting two to five years after the appearance of symptoms (2). Although the cause of ALS remains unknown, recent breakthroughs have linked ~20% of familial ALS (FALS) cases to any one of more that 90 different autosomal dominant mutations in sod1, the gene encoding copper,zinc-superoxide dismutase (SOD) (3).

Since this discovery, considerable effort has gone into determining the underlying mechanisms of motor neuron damage caused by ALS mutant SOD. In 1994, Gurney and co-workers (4) found that mice expressing both FALS mutant SOD and their own SOD developed ALS symptoms, suggesting that FALS mutant SODs cause motor neuron damage by a toxic gain of function. Proposals for this gain of function include aberrant oxidative reactions (5, 6), tyrosine nitration (7-11), and polymerization of mutant SODs (12, 13). Recently, Jourd'heuil and co-workers (14) demonstrated that wild-type bovine CuZn-SOD catalyzes the decomposition of S-nitrosoglutathione (GSNO) and other low molecular weight S-nitrosothiols in the presence of a reductant such as reduced glutathione (GSH). It is therefore possible that a fourth gain of function of mutant SOD involves increased S-nitrosothiol breakdown.

S-Nitrosothiols exhibit a wide range of physiological functions. These include immune functions (15), inhibition of oxidant response enzymes (16-23), modulation of ion channel conductivity (24-27), and antimicrobial effects (17, 29, 30). Additionally, S-nitrosothiols have been proposed to have a role in bronchodilation (31-33) and vasodilation (34-38).

Under certain conditions (24), low molecular weight S-nitrosothiols have also been shown to be neuroprotective (39, 40). Indeed, GSNO appears to be concentrated in the central nervous system on the order of 6-8 µM (41). One potential mechanism of neuroprotection by S-nitrosothiols involves S-nitrosylation of free thiol(s) at the redox modulatory site of the N-methyl-D-asparatate receptor by S-nitroso-L-cysteine (24). Additionally, recent advances have implicated S-nitrosylation of the active-site cysteine of caspases 1, 3, and 8 in the inhibition of apoptosis (42-50). Because the activation of caspases 1 and 3 by denitrosylation has been linked to familial ALS in mouse models (51), it is possible that inhibition of the caspase cascade by S-nitrosylation may protect motor neurons from apoptosis. In fact, many of the biological actions of nitric oxide (NO) may be mediated through S-transnitrosation reactions in which low molecular weight S-nitrosothiols transfer NO to other low molecular weight thiols or to protein-bound free thiols (45). It is possible that a sufficient level of low molecular weight S-nitrosothiols may be required to maintain an adequate state of S-nitrosylation of proteins involved in apoptosis. Therefore, we are interested in pathways of S-nitrosothiol degradation that may be associated with neurodegenerative conditions.

Recent evidence has suggested that a number of mutations associated with FALS may increase the openness of the active site channel, improving access to the copper active site by larger molecules (5). We hypothesized that GSNO breakdown would be accelerated by these mutations, inhibiting its protective effect. Here, we show that A4V SOD and G37R SOD catalyze the breakdown of GSNO with greater efficiency than WT SOD under physiological conditions. Our data also suggest that this increased decomposition is influenced by reduction of the copper active site as well as by access of the S-nitrosothiol to the active site. This study identifies an additional, potentially toxic, gain of function of human A4V SOD and suggests that S-nitrosothiol depletion may contribute to motor neuron death in FALS.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- All chemicals used were of the highest grade and purity available. Sodium nitrite, glutathione, ascorbic acid, cysteine, and cysteinyl glycine were purchased from Aldrich (Milwaukee, WI). All other chemicals were purchased from Sigma. Phosphate-buffered saline (PBS) was treated with Chelex-100 resin (Bio-Rad) prior to use to remove trace metals.

SOD Expression, Purification, and Characterization-- SOD was expressed and purified from yeast cells as reported previously (5). The culture medium was supplemented with 2.5 mM CuSO4 and 0.5 mM ZnSO4. Plasmids for human WT SOD and human A4V SOD, EG-118 yeast transformed to express G37R SOD and non-transformed EG-118 yeast were the kind gifts of J. S. Valentine, Department of Chemistry and Biochemistry, University of California, Los Angeles (5). The purity and identity of the isolated protein was assayed by gel electrophoresis with silver staining and Western blot. All enzymes were dialyzed against Chelex-treated PBS prior to use. The copper and zinc content of all enzymes were determined by atomic absorption spectroscopy and 4-pyridylazaresorcinol assay (52). The zinc content of both mutants was equal to that of WT SOD, while of copper content of both mutants was slightly less than WT SOD. A nondenaturing 4-pyridylazaresorcinol assay was conducted on all enzymes to confirm the absence of adventitiously bound copper and zinc (52). Crow and co-workers (52) have established that A4V SOD has a 30-fold less binding affinity for zinc compared with WT SOD. However, it is worth noting that, to facilitate the determination of the binding constants, they incubated WT SOD and A4V SOD, expressed in Escherichia coli, at 37 °C in the presence of 2 M urea to accelerate the metal loss. This led to a half-life of zinc release by A4V SOD of 13.5 h. In the absence of a denaturant, it took more than 2 weeks to establish a zinc loss equilibrium between bovine CuZn-SOD and 2-pyridinecarboxylate at pH 6.25 (52). Throughout the course of our preparations of WT SOD, A4V SOD, and G37R SOD, the temperature was maintained at 0-4 °C and the pH was maintained at 7.0. The entire procedure was completed within a week, and the samples were stored at -80 °C. This likely kept metal loss from WT SOD and both mutant SODs to a minimum, as reflected in the similar metallations of the enzymes. The identities of WT SOD, A4V SOD, and G37R SOD were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (W. M. Keck Biological Mass Spectrometry Center, University of Virginia) by comparison of the measured mass to the corresponding theoretical mass. For all enzymes tested, the observed mass fell within 4 atomic mass units of the predicted mass, and the difference in mass between WT SOD and each mutant agreed with the corresponding mutation. We isolated 15-70 mg of pure protein from each 10-liter culture.

Synthesis of S-Nitrosothiols-- Commercially available GSNO and S-nitroso-N-acetyl-D,L-penicillamine (Toronto Research Chemicals, North York, Ontario) was used. S-Nitroso-bovine serum albumin (SNO-BSA), S-nitroso-N-acetylcysteine (NAcSNO), and S-nitroso-L-cysteine (CysNO) were synthesized according to previously published procedures (53). SNO-BSA was passed through a G-25 (Sigma) column to remove salts prior to use. The concentrations of all prepared S-nitrosothiols were determined as previously reported (14).

Decomposition of S-Nitrosothiols-- In a typical experiment, 40-µl reaction volumes of glutathione and S-nitrosothiol were incubated with 10 µM SOD in 10 mM PBS at 37 °C. The reductant concentration, S-nitrosothiol concentration, incubation time, and reduction time were varied. For incubations analyzed by high performance liquid chromatography-mass spectrometry (LC-MS), the reactions were quenched prior to analysis by adding 40 µl of a solution containing 1 mM neocuproine, 200 µM EDTA, and 20 µM tryptamine (used as an internal standard) dissolved in 80% formic acid (0.2%) and 20% methanol. Incubations analyzed by chemiluminescence were injected at the appropriate time for analysis. Incubations in which the concentration of SNO-BSA was measured by Saville assay (55) sat for 30 min at 37 °C prior to analysis. All S-nitrosothiols were protected from light.

S-Nitrosothiol Quantitation by LC-MS-- For LC-MS analysis, 5 µl of the incubation sample was injected onto a Waters Symmetry C18 microbore column (1.0 × 150 mm) coupled to a Finnigan LCQ Duo mass spectrometer (Thermoquest Corp., San Jose, CA) fitted with an electrospray ionization (ESI) source. The samples were eluted isocratically at 50 µl/min using 90% formic acid (0.1%) and 10% methanol. The mass spectrometer was configured to isolate and monitor protonated tryptamine at m/z = 161.0 and protonated GSNO at m/z = 336.9. The peaks were integrated using the Excaliber software package (Thermoquest Corp., San Jose, CA). GSNO concentrations were determined using tryptamine as an internal standard.

S-Nitrosothiol Quantitation by Chemiluminescence-- S-Nitrosothiol content was determined by reductive chemiluminescence as previously described (56).

Statistics-- All experiments were performed in triplicate. Potential outliers were either accepted or rejected using the Student's t test. Data are presented as mean ± S.E. Nonlinear regression methods (57) were used for fitting models. Standard errors for the parameters in the models were computed using the covariance matrix of Zhang et al. (58). F-tests were used for the overall comparisons of control, WT SOD, A4V SOD, and G37R SOD.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Decomposition of GSNO by A4V SOD, G37R SOD, and WT SOD-- To compare the relative rates of GSNO decomposition, 7 µM GSNO (41) was incubated with 125 µM glutathione and 10 µM SOD in PBS at pH 7.4. The GSNO content was measured over the course of 80 min by both chemiluminescence (Fig. 1A) and LC-MS (Fig. 1B). The samples assayed by chemiluminescence and LC-MS indicate that, compared with WT SOD, A4V SOD, and G37R SOD catabolized about two and three times as much GSNO, respectively, in relation to the no enzyme control after 80 min (p < 0.01, F2,60 = 4.54, A4V SOD compared with WT SOD, Fig. 1A; p < 0.001, F2,60 = 31.4, G37R SOD compared with WT SOD, Fig. 1A; p < 0.01, F2,60 = 4.20, A4V SOD compared with WT SOD, Fig. 1B; p < 0.001, F2,60 = 15.8, G37R SOD compared with WT SOD, Fig. 1B).


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  		Fig. 1.   Decomposition of GSNO is accelerated in the presence of A4V and G37R mutant SODs. Decomposition of 7 µM GSNO in the presence of 125 µM glutathione and 10 µM WT SOD (squares), 10 µM A4V SOD (diamonds), 10 µM G37R SOD (triangles), or 0 µM SOD control (circles) in 10 mM phosphate-buffered saline at pH 7.4 and 37 °C. GSNO content was analyzed by: A, chemiluminescence; and B, LC-MS. The curves representing A4V SOD and G37R SOD derived from chemiluminescence measurements (A4V SOD, F2,60 = 4.54, p < 0.01; G37R SOD, F2,60 = 31.4, p < 0.001) as well as from LC-MS data (A4V SOD, F2,60 = 4.20, p < 0.02; G37R SOD, F2,60 = 15.8, p < 0.001) are significantly different from the curves representing WT SOD.

Chemiluminescence measurements indicated that ~2 µM GSNO remained after an 80-min incubation with A4V SOD, while analysis by LC-MS suggested the breakdown of all GSNO after 80 min. Given that chemiluminescence measures total S-nitrosothiol content while LC-MS measures only GSNO content, this discrepancy is likely due to the transnitrosation of NO from GSNO to free thiol sites of A4V SOD. The fact that the chemiluminescence signal disappeared upon removal of the protein from the incubation mixture further supported this notion (data not shown). Incubation with G37R SOD resulted in complete GSNO breakdown, as measured by chemiluminescence and LC-MS. The decomposition of GSNO by WT SOD and A4V SOD produced 3.5 µM NO over a 35-min period, as measured by chemiluminescence (data not shown), which is in agreement with the NO production results of Jourd'heuil and co-workers (14). To ensure that the GSNO signal detected by LC-MS arose from GSNO that was present in the incubation mixture rather than from GSNO that formed on the column by reaction with acidified nitrite and glutathione, we injected 5 µl of a solution containing 5 µM sodium nitrite and 500 µM glutathione. The absence of a detectable signal at m/z = 336.9 indicated that any GSNO detected by LC-MS analysis had been present in the incubation mixture (data not shown).

Kinetics of GSNO Breakdown by A4V SOD, G37R SOD, and WT SOD-- To obtain Km and Vmax for A4V SOD, G37R SOD, and WT SOD, we measured the initial rates of decomposition of GSNO at different GSNO concentrations in the presence 10 µM SOD and 125 µM glutathione (Fig. 2, A and B). All three enzymes obeyed Michaelis-Menten kinetics. A4V SOD and G37R SOD catalyzed GSNO breakdown about three times as efficiently as WT SOD. The kinetic parameters of WT SOD, A4V SOD, and G37R SOD are summarized in Table I. From the above studies, it is evident that both the A4V and G37R mutations enhance the catabolic activities of both enzymes.


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  		Fig. 2.   Kinetics of GSNO decomposition in the presence of A4V SOD, G37R SOD, and WT SOD. A, plot of initial rate of decomposition versus GSNO concentration in the presence of WT SOD (squares), A4V SOD (diamonds), or G37R SOD (triangles) in 10 mM phosphate-buffered saline at 37 °C and pH 7.4 in the presence of 10 µM WT SOD, A4V SOD, or G37R SOD, 125 µM glutathione and GSNO concentrations ranging from 2 to 300 µM. B, Lineweaver-Burk plots of GSNO breakdown by A4V SOD (circles), G37R SOD (squares) and WT SOD (diamonds). The two curves representing A4V SOD and G37R SOD in Fig. 2A are significantly different from WT SOD (A4V SOD, F2,38 = 126, p < 0.001; G37R SOD, F2,38 = 126, p < 0.001). The slopes of the lines representing A4V SOD and G37R SOD in Fig. 2B are significantly different from WT SOD (A4V SOD, F1,25 = 132, p < 0.001; G37R SOD, F1,25 = 132, p < 0.001). The inverse rates of A4V SOD and G37R SOD are significantly different from WT SOD at inverse concentrations 0.0625 µM-1, 0.100 µM-1, and 0.250 µM-1 (p < 0.05).

                              
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Table I

Effect of Glutathione Concentration on GSNO Breakdown-- Given that the kinetics of GSNO breakdown of A4V SOD and G37R SOD were similar (p = 0.18, Fig. 2B), we used A4V SOD as a model system to further characterize the differences in function between ALS mutant SODs and WT SOD. We incubated GSNO in the presence of 0 to 5 mM glutathione and either A4V SOD or WT SOD for 30 min at 37 °C and at pH 7.4 (Fig. 3). The maximum GSNO decomposition by A4V SOD and WT SOD occurred between 60 and 250 µM glutathione. The GSNO breakdown curves for A4V SOD and WT SOD were markedly different throughout the range of glutathione concentrations tested (p < 0.005, F2,77 = 5.9). Within the glutathione concentration range of 60-125 µM, A4V SOD broke down over twice the amount of GSNO as WT SOD after 30 min. Interestingly, maximum GSNO breakdown by A4V SOD occurred at 125 µM glutathione while the maximum GSNO decomposition by WT SOD occurred within a glutathione concentration range of 250-500 µM. Decreased GSNO breakdown occurred at glutathione concentrations greater than 250 µM. A similar decrease in GSNO breakdown by bovine CuZn-SOD at high glutathione concentrations was observed by Jourd'heuil and co-workers (14). This study indicates that A4V SOD catabolizes GSNO significantly more efficiently than WT SOD throughout the physiologically relevant range of glutathione concentrations.


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  		Fig. 3.   The A4V mutation results in accelerated GSNO breakdown over a wide range of glutathione concentrations. 7 µM GSNO was incubated for 30 min at 37 °C and pH 7.4 in the presence of 10 µM WT SOD (squares), 10 µM A4V SOD (diamonds), or 0 µM SOD control (circles) in 10 mM phosphate-buffered saline. The curves for WT SOD and A4V SOD are significantly different with respect to the effect of glutathione concentration on mean GSNO concentration (F2,77 = 5.9, p < 0.005).

Decomposition of S-Nitroso Bovine Serum Albumin by SOD-- Inorganic copper causes the breakdown of S-nitrosothiols, particularly in the presence of cysteine or glutathione (56). To demonstrate that GSNO decomposition occurs by reaction of the S-nitrosothiol with copper embedded in the SOD subunit, we incubated 5 µM SNO-BSA with 125 µM glutathione and either 10 µM WT SOD or 10 µM A4V SOD (Fig. 4). Only slight breakdown of the S-N bond of SNO-BSA was observed after incubation with WT SOD or A4V SOD (p = 0.89), while incubation with 125 µM glutathione and 5 µM CuSO4 resulted in complete SNO-BSA breakdown (p < 0.001, when CuSO4 is compared with WT SOD, A4V SOD, and PBS incubations). Collectively, these data suggest that the observed GSNO breakdown occurs by reaction with protein-bound copper rather than free copper released from the enzyme.


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  		Fig. 4.   The S-N bond of SNO-BSA is not broken by SOD. 5 µM SNO-BSA was incubated with either 10 µM WT SOD or 10 µM A4V SOD and 125 µM glutathione in PBS at 37 °C for 30 min. SNO-BSA concentrations were determined by the method of Saville (55). As controls, SOD was substituted with either PBS or 5 µM CuSO4 (far right column). SNO-BSA levels for PBS, WT SOD, and A4V SOD differed significantly from CuSO4 (p < 0.001). SNO-BSA levels did not differ significantly among PBS, WT SOD, and A4V SOD (p = 0.89).

Decomposition of Various S-Nitrosothiols by SOD-- To compare the efficiencies of WT SOD and A4V SOD in catabolizing S-nitrosothiols of various sizes, we incubated five different S-nitrosothiol substrates with WT SOD or A4V SOD in the presence of 125 µM glutathione for 30 min (Fig. 5). The values are represented as a percentage of the no-enzyme control for each S-nitrosothiol. With the exception of NAcSNO, the overall decomposition of S-nitrosothiols increased with decreasing size. The difference in decomposition between WT SOD and A4V SOD, given as a percentage of the control, ranged from 13 to 22% for all substrates except SNO-BSA (0%). Of note, CysNO, a critical neuroprotective molecule (24), was completely depleted in the presence of A4V SOD under physiological conditions. The above data: 1) show that the A4V mutation increases the ability of SOD to catalyze the breakdown of a variety of S-nitrosothiols and 2) imply that steric considerations and access to the catalytic site may be critical to SOD-mediated S-nitrosothiol catabolism.


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  		Fig. 5.   All low molecular weight S-nitrosothiols are preferentially catabolized by A4V SOD. 7 µM of each substrate was incubated with either 10 µM WT SOD or 10 µM A4V SOD and with 125 µM glutathione in PBS at 37 °C for 30 min and measured by chemiluminescence. Values are reported in terms of percentage of S-nitrosothiol remaining in no-enzyme control incubations. Abbreviations: CysNO, S-nitroso-L-cysteine; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; GSNO, S-nitrosoglutathione. Significance comparisons of WT SOD and A4V SOD: CysNO, SNAP, and GSNO, p < 0.001; NAcSNO, p < 0.005; SNO-BSA, p < 0.56.

Effect of Various Ascorbic Acid Concentrations on GSNO Breakdown-- Next, we sought to investigate the role of copper reduction in catalyzing S-nitrosothiol breakdown. If the reduction step were an important factor in determining the difference in catalytic rates, then replacing glutathione with a different reductant should alter how WT SOD and A4V SOD break down GSNO. Therefore, we incubated GSNO with either WT SOD or A4V SOD in the presence of various concentrations of ascorbic acid, ranging from 0 to 20 mM (Fig. 6). The experiment was conducted in the same manner as the glutathione concentration dependence experiment described above. The incubation samples were analyzed by chemiluminescence. Although high ascorbic acid concentrations (up to 20 mM) did not inhibit GSNO breakdown, higher concentrations of ascorbic acid than glutathione were required to catalyze the same amount of GSNO degradation. Despite the fact that a region of increased GSNO breakdown by A4V SOD over WT SOD was observed in the ascorbic acid concentration range of 0 to 500 µM, the overall curves are not statistically different (p = 0.11). It has been established that high concentrations of ascorbic acid react with S-nitrosothiols (59), and this is evident from the no-enzyme control in Fig. 6. However, even when we account for this increased breakdown in the WT SOD and A4V SOD curves by subtracting GSNO broken down in the control (data not shown), we find that the catalytic ability of both enzymes are significantly altered when ascorbic acid is substituted for glutathione. Therefore, it is evident that S-nitrosothiol catabolism in the presence of SOD is dependent upon the reductant used.


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  		Fig. 6.   GSNO Catabolism by WT SOD and A4V SOD is altered in the presence of ascorbic acid. 7 µM GSNO was incubated for 30 min in the presence of ascorbic acid and 10 µM A4V SOD (diamonds), 10 µM WT SOD (squares), or 0 µM SOD (circles) in 10 mM phosphate-buffered saline at pH 7.4 and 37 °C. The curves for WT SOD and A4V SOD are not significantly different (F4,65 = 2.0, p = 0.11).

Pre-reduction of WT SOD and A4V SOD-- To assess the effect of reduction state of WT SOD and A4V SOD on GSNO catabolism, we pre-reduced WT SOD and A4V SOD by incubation with glutathione for various amounts of time and measured the initial rate of GSNO catabolism (Fig. 7). The overall curves differed significantly over the entire time course (p < 0.001). The maximum difference in the initial rate occurred when SOD was not pre-reduced by glutathione. This difference approached about 0.02 µM min-1 as the pre-reduction time approached 120 min. The initial rate after 120 min of pre-reduction reached a maximum value of ~0.23 µM min-1. These data imply that the initial rate of catalysis is highly dependent upon the initial oxidation state of the copper active site.


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  		Fig. 7.   Pre-reduction of SOD affects the differential initial rate of S-nitrosothiol breakdown by A4V SOD and WT SOD. 125 µM glutathione was incubated with either 10 µM WT SOD (squares) or A4V SOD (diamonds) at pH 7.4 and 37 °C for various amounts of time prior to the addition of 7 µM GSNO. Samples were analyzed by chemiluminescence to determine initial rate of GSNO breakdown. The curves are significantly different (F3,24 = 11.4, p < 0.001).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

S-Nitrosylation of caspases as well as the N-methyl-D-asparatate receptor are thought to inhibit cellular apoptosis (24, 42-51). A pathway of protein S-nitrosylation appears to involve transnitrosation from low molecular weight S-nitrosothiols to protein thiol sites (45). S-Nitrosoglutathione is present in the central nervous system at micromolar levels (41) and, along with other low molecular weight S-nitrosothiols, is biologically active (60). Therefore, sufficient concentrations of low molecular weight S-nitrosothiols may be important in maintaining a proper balance between neuronal survival and death.

It is striking that the GSNO-catabolic efficiency of SOD increased 3-fold with either the A4V or G37R mutation (Table I). If WT SOD is necessary to regulate the total amount of S-nitrosothiol present in the cellular environment, it is possible that increases in the catalytic rate of this magnitude will alter the balance of S-nitrosothiol present. Although the normal CuZn-SOD concentration in the motor neuron has not been clearly defined, the intracellular concentration of CuZn-SOD has been reported to be 10 µM in yeast cells and (54) 10-30 µM in erythrocytes and hepatocytes (14). CuZn-SOD levels are thought to be exceptionally high in motor neurons (61). Higher enzyme concentrations may further exaggerate S-nitrosothiol breakdown by WT SOD and mutant SODs. Over time, this could have a detrimental effect on neuronal survival given the neuroprotective effects of low molecular weight S-nitrosothiols (24, 39, 40).

The catalytic breakdown of S-nitrosothiols by A4V SOD and WT SOD may be extended to other low molecular weight S-nitrosothiols as well (Fig. 5). In general, for both WT SOD and A4V SOD, the smaller S-nitrosothiols were broken down more readily than the larger S-nitrosothiols. This is likely a substrate access issue: smaller S-nitrosothiols in general should be better able to access the copper active site than larger S-nitrosothiols. The exception to this trend is NAcSNO. Although it is not clear why this difference is observed, it is thought that the electrostatic loop surrounding the active site channel plays a significant role in promoting substrate access to the copper active site (62). Therefore, one possibility is that, because NAcSNO is not a zwitterion, its access to the copper active site is hindered by an electrostatic mechanism. Another possibility is that NAcSNO breakdown is suppressed by steric factors.

The presence of S-nitrosothiols other than GSNO may also be critically relevant to neuronal survival. For example, Lipton and co-workers (24) have demonstrated that CysNO S-nitrosylates the N-methyl-D-asparatate receptor as a mechanism of modulating the flow of Ca2+ into the neuron. Therefore, the fact that CysNO was completely catabolized by A4V SOD after 30 min (Fig. 5) may have implications for modulating the passage of Ca2+ through the N-methyl-D-asparatate receptor channel. Inhibition of Ca2+ flow into the cell may reduce oxidative stress and favor cell survival.

GSNO breakdown by A4V SOD was greater than WT SOD throughout the physiological range of glutathione concentrations (Fig. 3). However, it is interesting that the most pronounced difference between A4V SOD and WT SOD occurs at ~125 µM. Indeed, lowered glutathione levels have been associated with a number of neurodegenerative diseases, including ALS (63). Based on our kinetics data (Fig. 2; Table I), a decreased concentration of glutathione in the cytoplasm of the motor neuron would be expected to accelerate S-nitrosothiol breakdown by WT SOD. These data also suggest that, in the presence of A4V SOD or G37R SOD, lowered glutathione concentrations could have an even more profound effect, resulting in critically low intracellular levels of S-nitrosothiols. Given that SOD is particularly abundant in the motor neurons (61), this may have a negative effect on motor neuron survival.

Mutations in human SOD have been shown to alter the metal ion-binding sites (64). In fact, the loss of copper in several ALS mutant SODs isolated from familial ALS patients has been demonstrated (65). It has also been well established that free copper in the presence of a reductant such as glutathione is known to catalyze S-nitrosothiol breakdown (28). With this in mind, it was our first inclination that increased GSNO breakdown by mutant SODs over WT SOD occurred due to copper release from the mutants. However, WT SOD and A4V SOD did not catalyze cleavage of the S-N bond of SNO-BSA, while incubation with 5 µM free copper in the presence of glutathione resulted in almost complete S-nitrosothiol decomposition (see Fig. 4). This result suggests that the observed increase in GSNO breakdown by A4V SOD is not mediated by free copper released by the enzyme. Moreover, the concentration of free copper in the cell is thought to be very low, less than one atom per cell (54). This suggests that intracellular S-nitrosothiol breakdown should be mediated by enzyme-bound copper rather than free copper. Therefore, we sought to gain insight into the mechanism of SOD-mediated GSNO breakdown.

It has been suggested that familial ALS mutations in SOD not only increase access to the copper active site (5) but may also alter the redox behavior of the protein (64). Steric factors as well as reduction potential may play a role in the reduction of the copper active site. The fact that the breakdown profile of GSNO changes significantly when ascorbic acid (Fig. 6) is substituted for glutathione (Fig. 3) implies that reduction of enzyme-bound Cu2+ is critical to its catalytic activity. Therefore, we sought to explore the link between reduction state and catalysis. If the difference in S-nitrosothiol catabolic efficiency were dependent solely upon access of the substrate to the copper catalytic site, then the initial rate of GSNO breakdown should not be affected by the oxidation state of the enzyme. However, as shown in Fig. 7, this is not the case. As the time of reduction of both WT SOD and A4V SOD is increased, the initial rate of GSNO breakdown by WT SOD increases up to a limiting value of about 0.21 µM min-1 while that of A4V SOD increased up to about 0.23 µM min-1. In fact, as the pre-reduction time increases, the difference in the initial rates of GSNO breakdown decreases, indicating that the difference in the initial rate of GSNO breakdown may be partially mediated by reduction of the copper active site. Presently, the degree to which the reduction step of SOD-mediated GSNO breakdown affects the reaction rate is not known. However, we are actively pursuing this question.

To summarize, we have found that the A4V and G37R mutations enhance the ability of the SOD to break down low molecular weight S-nitrosothiols in physiologically relevant conditions. Our data suggest that these mutations in SOD may facilitate reduction of the copper active site. This, in turn, may contribute to the increased efficiency in S-nitrosothiol catabolism. We have uncovered a previously unknown and potentially toxic gain of function caused by the A4V and G37R mutations of SOD that may have implications in the development of therapies for FALS.

    ACKNOWLEDGEMENT

We are grateful to Professor Donald J. Kirwan, Chemical Engineering Department, University of Virginia, for the use of his 10L fermentor.

    FOOTNOTES

* This work was supported by the National Institutes of Health Grants NS34678 (to T. L. M.) and HL59337 (to B. G.). Additional support was provided by the Henry B. Wallace Foundation and the University of Virginia Children's Medical Center (to B. G.).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.

** To whom correspondence should be sent. Tel.: 804-924-1820; Fax: 804-243-6618; E-mail: BMG3G@Virginia.edu.

Published, JBC Papers in Press, August 22, 2001, DOI 10.1074/jbc.M102781200

    ABBREVIATIONS

The abbreviations used are: ALS, amyotrophic lateral sclerosis (Lou Gehrig's disease); FALS, familial amyotrophic lateral sclerosis; SOD, superoxide dismutase; WT, wild type; A4V, alanine 4 valine; G37R, glycine 37 arginine; GSNO, S-nitrosoglutathione; CysNO, S-nitroso-L-cysteine; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; NAcSNO, S-nitroso-N-acetylcysteine; SNO-BSA, S-nitroso-bovine serum albumin; PBS, phosphate-buffered saline; LC-MS, liquid chromatography-mass spectrometry.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J. P., Deng, H.-X., Rahmani, Z., Krizus, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S. M., Berger, R., Tanzi, R. E., Halperin, J. J., Herzfeldt, B., Van de Bergh, R., Hung, W.-Y., Bird, T., Deng, G., Mulder, D. W., Smyth, C., Laing, N. G., Soriano, E., Pericak-Vance, M. A., Haines, J., Rouleau, G. A., Gusella, J. S., Horvitz, H. R., and Brown, R. H. (1993) Science 362, 59-62
2. Brown, R. H., Jr. (1997) Arch. Neurol. 54, 1246-1250[Abstract/Free Full Text]
3. Rowland, L. P., and Shneider, N. A. (2001) N. Engl. J. Med. 344, 1688-1700[Free Full Text]
4. Gurney, M. E., Pu, H., Chiu, A. Y., Dal Canto, M. C., Polchow, C. Y., Alexander, D. D., Caliendo, J., Hentati, A., Kwon, Y. W., Deng, H.-X., Chen, W., Zhai, P., Sufit, R. L., and Siddique, T. (1994) Science 264, 1772-1775[Abstract/Free Full Text]
5. Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. B., Roe, J. A., Lee, M. K., Valentine, J. S., and Bredesen, D. E. (1996) Science 271, 515-518[Abstract]
6. 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[Abstract/Free Full Text]
7. Estévez, A. G., Crow, J. P., Sampson, J. B., Reiter, C., Zhuang, Y., Richardson, G. J., Tarpey, M. M., Barbeito, L., and Beckman, J. S. (1999) Science 286, 2498-2500[Abstract/Free Full Text]
8. Bruijn, L. I., Beal, M. F., Becher, M. W., Schulz, J. B., Wong, P. C., Price, D. L., and Cleveland, D. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7606-7611[Abstract/Free Full Text]
9. Beal, M. F., Ferrante, R. J., Browne, S. E., Matthews, R. T., Kowall, N. W., and Brown, R. H., Jr. (1997) Ann. Neurol. 42, 646-654
10. Ferrante, R. J., Shinobu, L. A., Schulz, J. B., Matthews, R. T., Thomas, C. E., Kowall, N. W., Gurney, M. E., and Beal, M. F. (1997) Ann. Neurol. 42, 326-334[CrossRef][Medline] [Order article via Infotrieve]
11. Crow, J. P., Ye, Y. Z., Strong, M., Kirk, M., Barnes, S., and Beckman, J. S. (1997) J. Neurochem. 69, 1945-1953[Medline] [Order article via Infotrieve]
12. Chou, S. M., Wang, H. S., and Taniguchi, A. (1996) J. Neurol. Sci. 139, 16-26
13. Bruijn, L. I., Houseweart, M. K., Kato, S., Anderson, K. L., Anderson, S. D., Ohama, E., Reaume, A. G., Scott, R. W., and Cleveland, D. W. (1998) Science 281, 1851-1854[Abstract/Free Full Text]
14. Jourd'heuil, D., Laroux, F. S., Miles, A. M., Wink, D. A., and Grisham, M. B. (1999) Arch. Biochem. Biophys. 361, 323-330[CrossRef][Medline] [Order article via Infotrieve]
15. Park, J. (1996) Biochem. Biophys. Res. Commun. 220, 31-35[CrossRef][Medline] [Order article via Infotrieve]
16. Clancy, R., Levartovsky, D., Leszczynska-Piziak, J., Yegudin, J., and Abramson, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3680-3684[Abstract/Free Full Text]
17. Hausladen, A., Privalle, T., Keng, T., DeAngelo, J., and Stamler, J. S. (1996) Cell 86, 719-729[CrossRef][Medline] [Order article via Infotrieve]
18. Han, J., Stamler, J. S., Li, H.-L., and Griffith, O. (1996) in Biology of Nitric Oxide (IV) (Stamler, J. S. , Gross, S. , Moncada, S. , and Higgs, A., eds) , Portland Press, London
19. Mohr, S., Stamler, J. S., and Brüne, B. (1994) FEBS Lett. 348, 223-227[CrossRef][Medline] [Order article via Infotrieve]
20. Nikitovic, D., and Holmgren, A. (1996) J. Biol. Chem. 271, 19180-19185[Abstract/Free Full Text]
21. Clark, A. G., and Degnam, P. (1988) Biochem. Pharmacol. 37, 3199-3201[CrossRef][Medline] [Order article via Infotrieve]
22. Asahi, M., Fujii, J., Suzuki, K., Seo, H. G., Kuzuya, T., Hori, M., Tada, M., Fujii, S., and Taniguchi, N. (1995) J. Biol. Chem. 270, 21035-21039[Abstract/Free Full Text]
23. Becker, K., Guy, M., and Schirmer, R. (1995) Eur. J. Biochem. 234, 472-478[Medline] [Order article via Infotrieve]
24. Lipton, S. A., Choi, Y.-B., Pan, Z.-H., Lei, S. Z., Chen, H.-S. V., Sucher, N. J., Loscalzo, J., Singel, D. J., and Stamler, J. S. (1993) Nature 364, 626-632[CrossRef][Medline] [Order article via Infotrieve]
25. Kamosinska, B., Radomski, M., Duszyk, M., Radomski, A., and Man, S. (1997) Am. J. Physiol. 272, 1098-1104
26. Campbell, D., Stamler, J. S., and Strauss, H. (1996) J. Gen. Physiol. 108, 277-293[Abstract/Free Full Text]
27. Botolina, V., Najibi, S., Palacino, J., Pagano, P., and Cohen, R. (1994) Nature 368, 850-853[CrossRef][Medline] [Order article via Infotrieve]
28. Stubauer, G., Giuffrè, A., and Sarti, P. (1999) J. Biol. Chem. 274, 2128-28133
29. Morris, S., Walsh, R., and Hansen, J. (1984) J. Biol. Chem. 259, 13590-13594[Abstract/Free Full Text]
30. Degroote, M., Testerman, T., Xu, Y., Stauffer, G., and Fang, F. (1996) Science 272, 414-417[Abstract]
31. Gaston, B., Reilly, J., Drazen, J. M., Fackler, J., Ramdev, P., Arnelle, D., Mullins, M. E., Sugarbaker, D. J., Chee, C., Singel, D. J., Loscalzo, J., and Stamler, J. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10957-10961[Abstract/Free Full Text]
32. Gaston, B., Drazen, J. M., Jansen, A., Sugarbaker, D. A., Loscalzo, J., Richards, W., and Stamler, J. S. (1994) J. Pharmacol. Exp. Ther. 268, 978-984[Abstract/Free Full Text]
33. Bannenberg, G., Xue, J., Engman, L., Cotgreave, I., Moldéus, P., and Ryrfeldt, A. (1995) J. Pharmacol. Exp. Therap. 272, 1238-1245[Abstract/Free Full Text]
34. Davisson, R., Travis, M., Bates, J., and Lewis, S. (1996) Circ. Res. 79, 256-262[Abstract/Free Full Text]
35. Kowaluk, E. A., and Fung, H. L. (1990) J. Pharmacol. Exp. Therap. 255, 1256-1264[Abstract/Free Full Text]
36. Stamler, J. S., Jia, L., Eu, J., McMahon, T., Demchenko, I., Bonaventura, J., Gernert, K., and Piantadosi, C. (1997) Science 276, 2034-2037[Abstract/Free Full Text]
37. Funai, E., Davidson, A., Seligman, S., and Finlay, T. (1997) Biochem. Biophys. Res. Commun. 239, 875-877[CrossRef][Medline] [Order article via Infotrieve]
38. Emery, C. J. (1995) Physiol. Res. 44, 19-24[Medline] [Order article via Infotrieve]
39. Rauhala, P., Parameswarannay, K. P., Sziraki, I., Lin, A. M.-Y., and Chiueh, C. C. (1996) Synapse 23, 58-60[CrossRef][Medline] [Order article via Infotrieve]
40. Rauhala, P., Lin, A. M.-Y., and Chiueh, C. C. (1998) FASEB J. 12, 165-173[Abstract/Free Full Text]
41. Kluge, I., Gutteck-Amsler, U., Zollinger, M., and Do, K. Q. (1997) J. Neurochem. 69, 2599-2607[Medline] [Order article via Infotrieve]
42. Kim, Y.-M., de Vera, M. E., Watkins, S. C., and Billar, T. R. (1997) J. Biol. Chem. 272, 1402-1411[Abstract/Free Full Text]
43. Kim, Y.-M., Talanian, R. V., and Billiar, T. R. (1997) J. Biol. Chem. 272, 31138-31148[Abstract/Free Full Text]
44. Mohr, S., Zech, B., Lapetina, E. G., and Brune, B. (1997) Biochem. Biophys. Res. Commun. 238, 387-391[CrossRef][Medline] [Order article via Infotrieve]
45. Liu, Z., Rudd, M. A., Freedman, J. E., and Loscalzo, J. (1998) J. Pharmacol. Exp. Therap. 284, 526-534[Abstract/Free Full Text]
46. Rössig, L., Fichtscherer, B., Breitschopf, K., Haendeler, J., Zeiher, A. M., Mulsch, A., and Dimmeler, S. (1999) J. Biol. Chem. 274, 6823-6826[Abstract/Free Full Text]
47. Kim, Y.-M., Talanian, R. V., Li, J., and Billiar, T. R. (1998) J. Immunol. 161, 4122-4128[Abstract/Free Full Text]
48. Kim, Y.-M., Kim, T. H., Chung, H. T., Talanian, R. V., Yin, X. M., and Billiar, T. R. (2000) Hepatology 32, 770-778[CrossRef][Medline] [Order article via Infotrieve]
49. Dimmeler, S., Haendeler, J., Nehls, M., and Zeiher, A. M. (1997) J. Exp. Med. 185, 601-607[Abstract/Free Full Text]
50. Mannick, J. B., Hausladen, A., Liu, L., Hess, D. T., Zeng, M., Miao, Q. X., Kane, L. S., Gow, A. J., and Stamler, J. S. (1999) Science 284, 651-654[Abstract/Free Full Text]
51. Li, M., Ona, V. O., Guégan, C., Chen, M., Jackson-Lewis, V., Andrews, L. J., Olszewski, A. J., Stieg, P. E., Lee, J.-P., Przedborski, S., and Friedlander, R. M. (2000) Science 288, 335-339[Abstract/Free Full Text]
52. Crow, J. P., Sampson, J. B., Zhuang, Y., Thompson, J. A., and Beckman, J. S. (1997) J. Neurochem. 69, 1936-1944[Medline] [Order article via Infotrieve]
53. Hart, T. W. (1985) Tetrahedron Lett. 26, 2013-2026[CrossRef]
54. Rae, T. D., Schmidt, P. J., Pufahl, R. A., Culotta, V. C., and O'Halloran, T. V. (1999) Science 284, 805-808[Abstract/Free Full Text]
55. Saville, B. (1958) Analyst 83, 670-672[CrossRef]
56. Fang, K., Ragsdale, N. V., Carey, R. M., Macdonald, T. L., and Gaston, B. (1998) Biochem. Biophys. Res. Commun. 252, 535-540[CrossRef][Medline] [Order article via Infotrieve]
57. Seber, G., and Wild, C. (1989) Nonlinear Regression , pp. 325-340, John Wiley and Sons, New York
58. Zhang, J., Peddada, S., and Rogol, A. (2000) in Statistics for the 21st Century, Methodologies for Applications of the Future (Rao, C. R. , and Szekeley, G., eds) , Marcel-Dekker, New York
59. Kashiba-Iwatsuki, M., Yamaguchi, M., and Inoue, M. (1996) FEBS Lett. 389, 149-152[CrossRef][Medline] [Order article via Infotrieve]
60. Lipton, A. J., Gaston, B., Czapala, M. A., and Gozal, D. (2000) Am. J. Crit. Care Med. 161, A483
61. Pardo, C. A., Xu, Z., Borchelt, D., Price, R., Sisodia, S. S., and Cleveland, D. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 954-958[Abstract/Free Full Text]
62. Polticelli, F., Battistoni, A., O'Neill, P., Rotilio, G., and Desideri, A. (1998) Protein Sci. 7, 2354-2358[Medline] [Order article via Infotrieve]
63. Pocernich, C. B., La Fontaine, M., and Butterfield, D. A. (2000) Neurochem. Int. 36, 185-191[CrossRef][Medline] [Order article via Infotrieve]
64. Lyons, T. J., Liu, H., Soto, J. J., Nersissian, A., Roe, J. A., Café, C., Ellerby, L. M., Bredesen, D. E., Gralla, E. B., and Valentine, J. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12240-12244[Abstract/Free Full Text]
65. Ogawa, Y., Kosaka, H., Nakanishi, T., Shimizu, Ohoi, N., Shouji, H., Yanagihara, T., and Sakoda, S. (1997) Biochem. Biophys. Res. Commun. 241, 251-257[CrossRef][Medline] [Order article via Infotrieve]

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