Download Modification and Inactivation of Human Cu,Zn

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 protected]
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.