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Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis¹

Department of Biochemistry, Osaka University Medical School, Suita, Osaka 565-0871, Japan; and
^* Department of Biochemistry, Yamagata University School of Medicine, Yamagata 990-9585, Japan

²Correspondence: Department of Biochemistry, Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: proftani{at}biochem.med.osaka-u.ac.jp

SPECIFIC AIMS

Although nearly 100 types of different mutations in Cu, Zn-superoxide (Cu, Zn-SOD) have been found in familial ALS (FALS) patients, the mechanisms by which those mutants of Cu, Zn-SOD lead to neurodegenerative disorder have so far not been fully elucidated. To elucidate the pathological role of mutated Cu, Zn-SOD in FALS, the susceptibility of mutated Cu, Zn-SODs to glycation was examined.

PRINCIPAL FINDINGS

1. Mutated Cu, Zn-SODs are susceptible to glycation
Wild-type (WT) and three mutated Cu, Zn-SODs—G37R, G93A, and I113T—were produced in the baculovirus/Sf21 insect cell system. To examine the susceptibility of mutated Cu, Zn-SODs to glycation, purified WT and mutated Cu, Zn-SODs were incubated with 100 mM glucose at 37°C for 14 days, and the glycated proteins were quantified by Western blot using the anti-hexitol lysine antibody, which reacts with the reduced Amadori product, an early glycation product. The mutated enzymes (G37R, G93A, I113T) were more highly glycated than in the WT enzyme. Moreover, glycated forms were detected in the mutants even before incubation with glucose, suggesting that mutated Cu, Zn-SODs were glycated during the 4 days incubation with 4 mM glucose present in the culture medium. We next separated glycated and nonglycated Cu, Zn-SODs using a boronate affinity column and the nonglycated fractions were incubated with various concentrations of glucose. Figure 1 A shows data on the dose-dependent glycation of nonglycated fractions of Cu, Zn-SODs, as quantified by Western blot using the anti-hexitol lysine antibody. Before incubating with glucose, it was confirmed that neither the WT nor the mutated Cu, Zn-SODs (G37R, G93A) were glycated. After incubation with 1 or 10 mM glucose at 37°C for 7 days, the glycation reaction was dose dependent and the mutated Cu, Zn-SODs (G37R, G93A) were more susceptible to glycation than the WT. Figure 2 indicates that glycation occurred in a time-dependent manner and that G93A was more susceptible to glycation than the WT.

View larger version (38K): [in this window] [in a new window]   Figure 1. Dose- and time-dependent glycation of mutated Cu, Zn-SODs. After separating glycated and nonglycated Cu, Zn-SOD by boronate affinity column, nonglycated fractions were incubated with glucose. Glycated proteins were detected by Western blot using an anti-hexitol lysine antibody. A) Dose-dependent glycation of wild-type (WT) and mutated Cu, Zn-SODs (G37R, G93A). Proteins (0.5 mg/mL) were incubated with the indicated concentration of glucose at 37°C for 7 days. B) Time-dependent glycation of G93A and WT. Proteins (0.5 mg/mL) were incubated with 10 mM glucose. Each lane contains 0.5 µg of protein.

View larger version (25K): [in this window] [in a new window]   Figure 2. The production of hydrogen peroxide by incubation of wild-type and G93A Cu, Zn-SOD with glucose or fructose. A) 0.5 mg/mL WT or G93A was incubated with 100 mM glucose or 1 mM fructose for 7–14 days and hydrogen peroxide was measured. *P < 0.05 compared with WT incubated with 100 mM glucose for 7 days. #P < 0.05 compared with WT incubated with 100 mM glucose for 14 days. $P < 0.05 compared with WT incubated with 1 mM fructose for 7 days. B) After 0.5 mg/mL WT or G93A was incubated with 100 mM glucose or 1 mM fructose for 7 days, the samples were dialyzed against 50 mM potassium phosphate buffer (pH 7.4) to remove free glucose and fructose. The dialyzed Cu, Zn-SODs were incubated at 37°C for an additional 7 days. Data indicate mean ± SD of 3–4 separate experiments in each group. *P < 0.05 compared with WT incubated with 100 mM glucose for 7 days. #P < 0.05 compared with WT incubated with 1 mM fructose for 7 days.

2. Mutated Cu, Zn-SODs are susceptible to fructation
Since fructose is a more potent glycating agent than glucose, we also examined the effects of fructose. After separation of glycated and nonglycated Cu, Zn-SODs using a boronate affinity column, the nonglycated proteins were incubated with 1 or 10 mM fructose for 3 days. G93A and G37R were more susceptible to fructation than the WT enzyme, and fructation occurred in dose-dependent manner.

3. Mutated Cu, Zn-SOD (G93A) produces more hydrogen peroxide from glycation and fructation
As we previously reported, a site-specific fragmentation of Cu, Zn-SOD is caused by ROS formed from the glycation reaction. To identify the radical species produced during glycation, ESR spectra were measured using DMPO as a trapping agent. When 1 mg/mL of WT or mutated Cu, Zn-SODs were incubated with 100 mM glucose at 37°C for 7 days, no signal was observed. We next examined the production of hydrogen peroxide, since superoxide anion is thought to be generated from glycation reaction, then the hydrogen peroxide could be formed by a dismutase activity of Cu, Zn-SOD. As shown in Fig. 2 A, when G93A and WT Cu, Zn-SODs were incubated with 100 mM glucose or 1 mM fructose, hydrogen peroxide was produced in time-dependent manner; the level of hydrogen peroxide produced from G93A was almost twice that of the WT. In this system, ROS produced by glucoseautoxidation was included; therefore, we next excluded free glucose or fructose by dialysis against 50 mM potassium phosphate buffer (pH 7.4). After dialysis, these samples were further incubated at 37°C and the extent of hydrogen peroxide production was measured. As seen in Fig. 2B , glycated and fructated G93A produced more hydrogen peroxide than the glycated and fructated WT, respectively.

CONCLUSIONS AND SIGNIFICANCE

Glycation of FALS-related mutants of Cu, Zn-SOD was examined. When the purified proteins were incubated with glucose, all the mutated Cu, Zn-SODs were found to be more susceptible to glycation than the WT. The mutated Cu, Zn-SODs were also more susceptible to fructation than the WT. When glycated Cu, Zn-SOD were incubated, hydrogen peroxide was produced, and the mutated Cu, Zn-SODs produced high levels in proportion to the extent of glycation. Production of hydrogen peroxide was also observed when fructated proteins were incubated; again, the mutants produced more hydrogen peroxide than the WT. It has been reported that Lewy body-like hyaline inclusions characteristically found in FALS patients are immunopositive both Cu, Zn-SOD and AGEs such as CML, pyrraline and pentosidine. From our data using purified Cu, Zn-SOD, the origin of these immunopositive lesions could be Cu, Zn-SOD itself, and it is likely that the glycation of mutated Cu, Zn-SODs is accelerated in vivo as well.

The primary characteristic of ALS is the selective degeneration of motor neurons, initiated in mid-adulthood. Since many different types of mutations in Cu, Zn-SOD have been observed in FALS, certain common characteristics of these mutants should explain the pathogenesis of the disease. Glycation proceeds through the formation of a Schiff base between reducing sugars and amine groups of lysine residues and a subsequent rearrangement to yield Amadori products, and finally produce AGEs, which have been shown to accumulate during aging. Glycation has been implicated in the chronic complications of diabetes mellitus and has been suggested to play an important role in the pathogenesis of neurodegenerative diseases. In this study, we hypothesize that the susceptibility of mutated Cu, Zn-SODs to glycation is a factor in the pathogenesis of FALS. An increased glycation has also been reported in sporadic ALS; Amadori products are present in axonal spheroids in the anterior horn of the spinal cord. The target protein of glycation in sporadic ALS is not known, but it is possible that some type of ROS produced during glycation is involved in the pathogenesis of sporadic ALS as well.

We previously reported that mutant enzymes form aggregates at a higher rate when incubated with copper ion and aggregates of mutant enzymes retain their enzymatic activity. Although glycation decreases the enzymatic activity of Cu, Zn-SOD, some activity should remain, and accelerated aggregation might play an important role in the microenvironment. Therefore, hydrogen peroxide could be produced by the aggregated forms of glycated Cu, Zn-SOD. We and other groups have also reported that the copper binding affinities were decreased in mutated Cu, Zn-SOD. This could be due to the instability of the mutated proteins, but it also could be due to glycation: we previously indicated that glycation causes the fragmentation of Cu, Zn-SOD.

These collective findings suggest that the glycation of mutated Cu, Zn-SODs can 1) produce superoxide anion, and 2) hydrogen peroxide is formed by the enzymatic activity of the aggregates, then 3) hydroxyl radicals can be formed via the Fenton reaction involving the free copper ion released from the mutated Cu, Zn-SOD. In this study, hydroxyl radicals were not observed, but we assume that detection of this radical is dependent on the scale of the experiment; in the case of this study, the amount of free copper might not have been sufficient to accomplish this. Both hydrogen peroxide and hydroxyl radical are toxic to neuronal cells; thus, it is possible that the increased level of glycation of the mutated Cu, Zn-SOD (which requires a long time) induces toxic oxidative stress especially in the anterior horn of the spinal cord, which contains high levels of Cu, Zn-SOD. It is also possible that 3-deoxyglucosone, methylglyoxal, or AGEs produced from glycated Cu, Zn-SOD directly affect neuronal cell death, as has been suggested by other groups.

The mechanisms by which the mutation of Cu, Zn-SOD affects glycation are currently under investigation. Since it has been observed from many aspects that mutation of Cu, Zn-SODs leads to instability of the protein, it is possible that the exposure status or electronic environment of the lysine residues is altered in the case of mutants. Abnormal glucose tolerance has been reported in 40% of ALS patients. Crystallographic studies are now under way, and we expect that structural information will provide us with clues for elucidating the mechanism whereby susceptibility to glycation is commonly observed in mutated Cu, Zn-SODs.

View larger version (17K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the pathogenesis of FALS based on the susceptibility of mutated Cu, Zn-SOD to glycation. 1) Mutated Cu, Zn-SODs are highly susceptible to glycation (Fig. 1) . 2) Superoxide anion is generated from the glycation reaction. 3) The mutated Cu, Zn-SODs form aggregates at a higher rate. 4) The aggregates of Cu, Zn-SODs retain their enzymatic activity. 5) The mutated Cu, Zn-SOD produced higher levels of hydrogen peroxide by glycation than wild-type (Fig. 2) . 6) Copper binding affinities were decreased in mutated Cu, Zn-SOD.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0768fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0768fje

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