Mol. Cells, Vol. 15, No. 2, pp. 194-199 M olecules and Cells KSMCB 2003 Modification and Inactivation of Human Cu,Zn-Superoxide Dismutase by Methylglyoxal Jung Hoon Kang* Department of Genetic Engineering, Chongju University, Chongju 360-764, Korea. (Received November 15, 2002; Accepted February 11, 2003) Methylglyoxal (MG) has been identified as an intermediate in non-enzymatic glycation, and increased levels have been reported in patients with diabetes. In this study, the effect of MG on the structure and function of human Cu,Zn-superoxide dismutase (SOD) was investigated. MG modifies Cu,Zn-SOD, as indicated by the formation of fluorescent products. When Cu, Zn-SOD was incubated with MG, covalent crosslinking of the protein increased progressively. MG-mediated modification of Cu,Zn-SOD led to loss of enzymatic activity and release of copper ions from the protein. Radical scavengers inhibited the crosslinking of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to MG was analyzed, glycine, histidine, lysine, and valine residues were found to be particularly sensitive. It is suggested that oxidative damage to Cu,Zn-SOD by MG may perturb cellular antioxidant defense systems and damage cells. This effect may account, in part, for organ deterioration in diabetes. Non-enzymatic glycation, an early stage of the Maillard reaction (Monnier et al., 1992), is a post-translational modification that involves free reducing sugars and the free amino groups of proteins. Several dicarbonyl compounds have been proposed as intermediates in the Maillard reaction (Glomb and Monnier, 1995). One is methylglyoxal (MG), an α-oxoaldehyde that can arise from various precursors, including glycolytic intermediates, amino acetone and threonine (Thornalley, 1999). MG reacts strongly with the amino groups of proteins to form crosslinks, which are stable end products called advanced glycation end products (AGEs) (Brownlee et al., 1988). MG readily reacts with lysine and arginine residues in proteins to produce high molecular weight, cross-linked products (Nagaraj et al., 1996). AGE formation is irreversible and is found to increase with age, as well as in atherosclerosis, and diabetes mellitus; it is especially associated with long-lived proteins such as collagens (Monnier et al., 1986), lens crystallines (Monnier and Cerami, 1981), and nerve proteins (Vlassara et al., 1981). The reaction between amines and oxoaldehyde is thought to be catalyzed by oxygen free radicals (SzentGyörgyi and Mclaughlin, 1975). AGEs have been identified as pentosidines, pyrrole derivatives, pyrazine derivatives, and N∈-(carboxymethyl)lysine (CML) (Yim et al., 1995). In the presence of molecular oxygen, the formation of these products from sugars is catalyzed by transition metal ions via glycoxidation, which oxidizes Amadori products to CML, and autoxidation of glucose, which produces superoxide anions, H2O2, and α-ketoaldehyde (Hunt et al., 1990; Jiang et al., 1990). Moreover, free radicals induced by MG reduce the activity of antioxidant enzymes and accelerate peroxidative damage (Choudhary et al., 1997). Taken together these findings suggest that the toxic effects of MG are mediated by free radical processes. In this report, the modification and inactivation of Cu,ZnSOD by MG is described. It is shown that exposure of Cu,Zn-SOD to MG leads to covalent crosslinking of the protein. The crosslinking is associated with enzyme inactivation and the release of copper ions. In addition, it was found that glycine, histidine, lysine, and valine residues of Cu,Zn-SOD are particularly sensitive to modification by MG. * To whom correspondence should be addressed. Tel: 82-43-229-8562; Fax: 82-43-229-8432 E-mail: email@example.com Abbreviations: MG, methylglyoxal; PAGE, polyacrylamide gel electrophoresis; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase. Keywords: Crosslinking; Cu,Zn-SOD; Inactivation; Methylglyoxal. Introduction Demo Jung Hoon Kang Materials and Methods Materials Methylglyoxal (MG), N-acetyl cysteine, glutathione, and thiourea were purchased from Sigma. Chelex 100 resin was from Bio-Rad. Recombinant human Cu,Zn-SOD was prepared as described previously (Kang et al., 1997). Commercial human erythrocyte Cu,Zn-SOD was further purified by gel filtration chromatography using a Superose 6 FPLC column (Pharmacia, Sweden). All solutions were treated with Chelex 100 to remove traces of transition metal ions. Protein modification Protein concentration was determined by the BCA method (Smith et al., 1985). Treatment of Cu,Zn-SOD (0.25 mg/ml) was carried out by incubating the enzyme in 10 mM potassium phosphate buffer (pH 7.4) in the presence and absence (control) of MG at 37°C. After incubation the mixtures were placed into Microcon filters (Amicon) and centrifuged at 13,000 rpm for 1 h to remove MG. They were then washed with Chelex 100- treated water and centrifuged for 1 h at the same speed to further remove MG. This was repeated four times. The filtrate was freeze dried and dissolved in water. Protection by radical scavengers was assayed by preincubating the enzyme in the presence of a given radical scavenger at room temperature for 5 min followed by incubating with 30 mM MG for 24 h at 37°C. Unreacted reagent was removed with a Microcon filter (Amicon). Characterization of MG-modified proteins After treatment with various concentrations of MG for various periods of time, samples of the reaction mixtures were diluted with concentrated sample buffer (0.25 mM Tris, 8% SDS, 40% glycerol, 20% βmercaptoethanol, 0.01% bromophenol blue) and heated at 100°C for 5 min. An aliquot of each sample was subjected to SDS-PAGE as described by Laemmli (1970), using a 15% acrylamide slab gel. The gels were stained with 0.15% Coomassie Brilliant Blue R-250. The activity of Cu,Zn-SOD was measured by monitoring its ability to inhibit the reduction of ferricytochrome c by xanthine/xanthine oxidase, as described by McCord and Fridovich (1969). The fluorescence of MGmodified Cu,Zn-SOD was recorded at an excitation wavelength of 334 nm with a BIO-TEK SFM 25 spectrofluorometer. Amino acid analysis Aliquots of modified and native Cu,ZnSOD preparations were hydrolyzed at 110°C for 24 h after addition of 6 N HCl. Since acid hydrolysis destroys tryptophan, the tryptophan content of oxidized and native Cu,Zn-SOD preparations was determined by alkaline hydrolysis as described previously (Hugli and Moore, 1972). The amino acid content of acid and alkaline hydrolysates was determined by separation of their phenylisothiocyanate-derivatives by HPLC using a Pico-tag free amino acid analysis column and 996 photodiode array detector (Waters, USA). Determination of free copper ions Protein samples were incubated with MG for various times and subjected to ultrafiltration 195 using a Microcon filter (Amicon) with a molecular mass cut-off of 3 kDa. The concentration of copper ions in the filtrates was determined by atomic absorption spectrophotometry (Shimadzu, AA-6601F). Replicates Unless otherwise indicated, each result described in this paper is representative of at least three separate experiments. Results and Discussion To establish whether MG can modify Cu,Zn-SOD, the fluorescence characteristics of MG-treated Cu,Zn-SOD were examined. MG-treated Cu,Zn-SOD showed a fluorescence emission maximum at 410 nm with excitation at 334 nm (Fig. 1). This signal gradually increased with concentration of MG. Native Cu,Zn-SOD did not fluoresce at this wavelength. The MG-induced fluorescence shows that reaction has taken place between MG and Cu,Zn-SOD. As shown in Fig. 2A, when Cu,Zn-SOD was incubated with 30 mM MG, covalent crosslinking of the protein increased with time. The extent of crosslinking also increased with MG concentration (Fig. 2B). Incubation of Cu,Zn-SOD with MG at 37°C resulted in a time-dependent decrease of enzymatic activity as defined by the cytochrome c reduction assay (see Materials and Methods) (Fig. 2C). Thus crosslinking by MG is associated with inactivation of the enzyme. During incubation of Cu,Zn-SOD with MG the release of free copper ions gradually increased (Fig. 3). Cellular metabolism has been shown to generate oxygen species such as hydrogen peroxide, hydroxyl radicals, and superoxide anions. Trace metals such as copper and iron, which are present in bio- Fig. 1. Fluorescence spectra of MG-modified and control Cu,Zn-SOD. The reaction mixture contained Cu,Zn-SOD (0.25 mg/ml) in 10 mM potassium phosphate buffer (pH 7.4) at 37°C for 24 h. MG was added as follows: a, control; b, 0.1 mM; c, 1 mM; d, 10 mM; e, 30 mM. 196 Modification of Human Cu, Zn-SOD by Methylglyoxal A B C Fig. 2. Modification and inactivation of Cu,Zn-SOD by MG. A. Cu,Zn-SOD (0.25 mg/ml) was incubated with 30 mM MG in 10 mM potassium phosphate buffer (pH 7.4) at 37°C for the indicated times and aliquots were fractionated by SDS-PAGE. B. Cu,Zn-SOD was incubated with the indicated concentrations of MG at 37°C for 24 h. C. After incubation of Cu,Zn-SOD with 30 mM MG, its enzymatic activity was measured by monitoring inhibition of the reduction of ferricytochrome c by xanthine/ xanthine oxidase. Closed squares, activity in the absence of MG; closed circles, activity in the presence of MG. logical systems, may interact with such reactive oxygen species, or with ionizing or microwave radiation, to damage macromolecules (Halliwell and Gutteridge, 1999). Modification of metalloproteins by oxidative damage may raise the level of metal ions in cells (Kang and Kim, 1997; Kim and Kang, 1997). It has been reported that Fig. 3. The release of copper ions from Cu,Zn-SOD by MG. Cu,Zn-SOD was incubated with 30 mM MG for various times. Aliquot were withdrawn, and free copper ions determined by atomic absorption spectrophotometry. Closed squares, copper ions released from Cu,Zn-SOD in the absence of MG; closed circles, copper ions released from Cu,Zn-SOD in the presence of MG. Data represent the means ± S.D. (n = 3−5). protein fragmentation occurred when Cu,Zn-SOD was treated with hydrogen peroxide, and that the oxidative damage to SOD then caused the release of copper ions from the enzyme (Choi et al., 1999). Release of copper ions has also been observed as a result of peroxyl radicalmediated human cerulloplasmin oligomerization (Kang et al., 2001). Thus the release of copper ions by MGmodified Cu,Zn-SOD may induce a pro-oxidant condition. The effect of radical scavengers on the oligomerization of Cu,Zn-SOD by MG was also investigated (Fig. 4), and N-acetylcysteine, glutathione, thiourea, mannitol and ethanol were found to be able to protect SOD against crosslinking. Radical scavengers also inhibited the inactivation of Cu,Zn-SOD (Fig. 5) and release of copper ions from the enzyme (Fig. 6). Oxidative stress is believed to modulate glycation. Wolff and co-workers (Hunt et al., 1990; Jiang et al., 1990) demonstrated that reducing sugars can undergo oxidation in the presence of oxygen and transition metal ions, with generation of H2O2, oxygen radicals, and α-ketoaldehydes. This reaction leads to protein browning, conformational changes, and fragmentation. Therefore, AGEs in vivo are the combined products of glycation and oxidative modification. It has been reported that free radicals are generated during the reaction of methylglyoxal with amino acids (Yim et al., 1995). Hence free radicals may participate in MG-mediated Cu,Zn-SOD modification. In order to identify target residues, Cu,Zn-SOD exposed to MG for 24 h at 37°C was analyzed by amino acid analysis following acid hydrolysis. Glycine, histidine, Jung Hoon Kang Fig. 4. The effects of radical scavengers on the modification of Cu,Zn-SOD by MG. Cu,Zn-SOD was incubated with 30 mM MG in the presence of radical scavengers at 37°C for 24 h. Lane 1, Cu,Zn-SOD control; lane 2, MG; lane 3, MG plus 20 mM Nacetyl cysteine; lane 4, MG plus 20 mM glutathione; lane 5, MG plus 20 mM thiourea; lane 6, MG plus 200 mM mannitol; lane 7, MG plus 200 mM ethnol. 197 Fig. 6. The effects of radical scavengers on the release of copper ions from Cu,Zn-SOD by MG. Cu,Zn-SOD was incubated with 30 mM MG in the presence of radical scavengers at 37°C for 24 h. Lane 1, Cu,Zn-SOD control; lane 2, MG; lane 3, MG plus 20 mM N-acetyl cysteine; lane 4, MG plus 20 mM glutathione; lane 5, MG plus 20 mM thiourea; lane 6, MG plus 200 mM mannitol; lane 7, MG plus 200 mM ethanol. Aliquots were withdrawn, and free copper ions determined by atomic absorption spectrophotometry as described in Materials and Methods. Data represent means ± S.D. (n = 3−5). Fig. 7. Modification of amino acid residues in Cu,Zn-SOD by MG. Cu,Zn-SOD was incubated with 30 mM MG in 10 mM potassium phosphate buffer (pH 7.4) at 37°C. After incubation for 0 h (black bar) and 24 h (gray bar), the amino acid composition of acid hydrolysates was determined as described in Materials and Methods. Fig. 5. The effects of radical scavengers on the inactivation of Cu,Zn-SOD by MG. Cu,Zn-SOD was incubated with 30 mM MG in the presence of radical scavengers at 37°C for 24 h. Lane 1, Cu,Zn-SOD control; lane 2, incubation with MG; lane 3, MG plus 20 mM N-acetyl cysteine; lane 4, MG plus 20 mM glutathione; lane 5, MG plus 20 mM thiourea; lane 6, MG plus 200 mM mannitol; lane 7, MG plus 200 mM ethanol. Enzyme activity was measured as described in Materials and Methods. Data represent means ± S.D. (n = 3−5). lysine, and valine residues were found to be particularly sensitive to modification. As shown in Fig. 7, 6 of the 25 glycine residues, 2 of the 8 histidine residues, 4 of the 11 lysine residues, and 4 of the 14 valine residues were lost. MG is reactive towards amino and guanidino groups in protein (Lo et al., 1994). Reaction of MG with lysine and arginine residues in protein produces well-characterized 198 Modification of Human Cu, Zn-SOD by Methylglyoxal compounds such as N∈-(carboxymethyl)lysine (CML) (Ahmed et al., 1986) and imidazolones (Lo et al., 1994). Another product of the reaction of MG with lysine, the lysine-lysine cross-link, imidazollysine, was originally characterized in reactions involving MG (Nagaraj et al., 1996). The results of the present study suggest that MG may interact directly with lysine residues, leading to covalent cosslinking of Cu,Zn-SOD. They also indicate that the inactivation of Cu,Zn-SOD may be closely associated with the loss of histidine residues, since this amino acid is essential for Cu,Zn-SOD activity (Maria et al., 1995). Cu,Zn-SOD contains a binuclear cluster, with the active copper and zinc bridged by a common ligand (His-63). Copper is bound to the ligand, coordinated with His-63, His-46, His-48, and His-120 in the active site of Cu,Zn-SOD (Tainer et al., 1983). Thus, it has been suggested that copper binding sites are modified during the reaction of Cu,Zn-SOD with MG, with the result that copper is freed from the ligand, released from the oxidatively damaged enzyme, and activity is lost. The finding that copper ions were released from the MGmodified Cu,Zn-SOD supports this mechanism. Glycine and valine residues may not react strongly with MG because they do not have amino and guanidino groups in their side chains. Therefore, it may be assumed that the modification of glycine and valine residues is due to oxidative damage by free radicals. Cu,Zn-SOD is a metalloenzyme that is essential for the dismutation of O2- to H2O2. Thus it is a very important component of cellular defenses against oxygen toxicity. Its inactivation by MG may perturb the antioxidant system. In addition the copper ions released from the oxidatively damaged enzyme by these radicals can enhance metal-catalyzed reactions producing ROS and causing oxidative damage to macromolecules. This mechanism may contribute to the increased peroxidation of lipids when glycated protein was added in vitro, and may also accelerate oxidative modification of vascular wall lipid in diabetic complications. References Ahmed, M. U., Thorpe, S. R., and Baynes, J. W. (1986) Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. J. Biol. Chem. 261, 4889−4894. Brownlee, M., Cerami, A., and Vlassara, H. (1988) Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N. Eng. J. Med. 318, 1315− 1321. Choi, S. Y., Kwon, H. Y., Kwon, O. B., and Kang, J. H. (1999) Hydrogen peroxide-mediated Cu,Zn-superoxide dismutase fragmentation: protection by carnosine, homocarnosine and anserine. Biochim. Biophys. Acta 1472, 651−657. Choudhary, D., Chandra, D., and Kale, R. K. (1997) Influence of metylglyoxal on antioxidant enzymes and oxidative damage. Toxicol. Lett. 93, 141−152. Glomb, M. A. and Monnier, V. M. (1995) Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction. J. Biol. Chem. 270, 10017− 10026. Halliwell, B. and Gutteridge, J. M. C. (1999) Free Radicals in Biology and Medicine, Oxford, New York. Hugli, T. E. and Moore, S. (1972) Determination of the tryptophan content of proteins by ion exchange chromatography of alkaline hydrolysates. J. Biol. Chem. 247, 2828−2834. Hunt, J. V., Smith, C. C., and Wolff, S. P. (1990) Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabete 39, 1420−1424. Jiang, Z.-Y., Woollard, A. C., and Wolff, S. P. (1990) Hydrogen peroxide production during experimental protein glycation. FEBS Lett. 268, 69−71. Kang, J. H., Choi, B. J., and Kim, S. M. (1997) Expression and characterization of recombinant human Cu,Zn-superoxide dismutase in Escherichia coli. J. Biochem. Mol. Biol. 30, 60− 65. Kang, J. H., Kim, K. S., Choi, S. Y., Kwon, H. Y., and Won, M. H. (2001) Oxidative modification of human ceruloplasmin by peroxyl radicals. Biochim. Biophys. Acta 1568, 30−36. Kang, J. H. and Kim, S, M. (1997) Fragmentation of human Cu,Zn-superoxide dismutase by peroxidative reaction. Mol. Cells 7, 553−558. Kim, S. M. and Kang, J. H. (1997) Peroxidative activity of human Cu,Zn-superoxide dismutase. Mol. Cells 7, 120−124. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227, 680−685. Lo, T. W., Westwood, M. E., McLellan, A. C., Selwood, T., and Thornalley, P. J. (1994) Binding and modification of proteins by methylglyoxal and physiological conditions. A kinetics and mechanistic study with N alpha-acetylarginine, N alphaacetylcysteine, and N alpha-acetyllysine, and bovine serum albumin. J. Biol. Chem. 269, 32299−32305. Maria, C. S., Revilla, E., Ayala, A., de la Cruz, C. P., and Machado, A. (1995) Changes in the histidine residues of Cu/Zn superoxide dismutase during aging. FEBS Lett. 374, 85−88. McCord, J. M. and Fridovich, I. (1969) Superoxide dismutase. J. Biol. Chem. 244, 6049−6055. Monnier, V. M. and Cerami, A. (1981) Nonenzymatic browning in vivo: possible process for aging of long-lived proteins. Science 211, 491−493. Monnier, V. M., Vishwanath, V., Frank, K. E., Elmets, C. A., Dauchot, P., and Kohn, R. R. (1986) Relation between complications of type I diabetes mellitus and collagen-linked fluorescence. New. Engl. J. Med. 314, 403−408. Monnier, V. M., Sell, D. R., Nagaraj, R. H., Miyata, S., Grandhee, S., Odetti, P., and Ibrahim, S. A. (1992) Maillard reaction-mediated molecular damage to extracellular matrix and other tissue proteins in diabetes, aging and uremia. Diabete 41, 36−41. Nagaraj, R., Shipanova, I. N., and Faust, F. M (1996) Protein cross-linking by the Maillard reaction. Isolation, characterization, and in vivo detection of a lysine-lysine cross-link de- Jung Hoon Kang rived from methylglyoxal. J. Biol. Chem. 271, 19338−19345. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F.-H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76−85. Szent-Györgyi, A. and McLaughlin, J. A. (1975) Interaction of glyoxal and methylglyoxal with biogenic amines. Proc. Natl. Acad. Sci. USA 72, 1610−1611. Tainer, J. A., Gertzoff, E. D., Richardson, J. S., and Richardson, D. C. (1983) Structure and mechanism of copper,zinc superoxide dismutase. Nature 306, 284−287. Thornalley, P. J., Langborg, A., and Minhas, H. S. (1999) Forma- 199 tion of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem J. 344, 109−116 Vander Jagt, D. L. and Hunsaker, L. A. (1993) Substrate specificity of reduced and oxidized forms of human aldose reductase. Adv. Exp. Med. Biol. 328, 279−288. Vlassara, H., Brownlee, M., and Cerami, A. (1981) Nonenzymatic glycosylation of peripheral nerve protein in diabetes mellitus. Proc. Natl. Acad. Sci. USA 78, 5190−5192. Yim, H.-Y., Kang, S.-O., Hah, Y.-C., Chock, P. B., and Yim, M. B. (1995) Free radicals generated during the glycation reaction of amino acids by methylglyoxal. J. Biol. Chem. 270, 28228−28233.