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Aminoacetone Induces Oxidative Modification to Human Plasma Ceruloplasmin Fernando Dutra,*,†,‡ Maria R. Ciriolo,§ Lilia Calabrese,| and Etelvino J. H. Bechara†,‡ Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, CP 26077, 05513-970, Sa˜ o Paulo, SP, Brazil, and Centro de Cieˆ ncias Biolo´ gicas e da Sau´ de, Universidade Cruzeiro do Sul, 08060-070, Sa˜ o Paulo, SP, Brazil, Dipartimento di Biologia, Universita´ degli Studi di Roma Torvergata, Via della Ricerca Scientifica s.n.c. 00133, Rome, Italy, and Dipartimento di Scienze Biochimiche, University of Rome “La Sapienza”, P.le A. Moro, 5, 00185 Rome, Italy Received December 14, 2004
Aminoacetone (AA), a putative endogenous source of cytotoxic methylglyoxal, and ceruloplasmin (CP), the antioxidant plasma copper transporter, are known to increase in diabetes. AA was recently shown in vitro to act as a pro-oxidant toward ferritin and isolated mitochondria. We now report AA oxidative effects on CP mediated by AA-generated reactive oxygen species (ROS). Incubation of 1.5 µM human CP with 0.05-1 mM AA resulted in extensive protein aggregation. That ROS-driven thiol cross-linking underlies the CP aggregation was evidenced by the inhibitory effects of added superoxide dismutase, catalase, mannitol, and dithiothreitol. The addition of CP to AA (mM) solutions accelerated oxygen consumption by AA and caused CP copper ion release and loss of ferroxidase and aminoxidase activities. If operative in vivo, this reaction would impair the antioxidant role of CP and iron uptake by ferritin and hence contribute to intracellular iron-induced oxidative stress during AA accumulation in diabetes mellitus.
Introduction Ceruloplasmin (CP), a blue copper glycoprotein produced by hepatocytes and secreted into the plasma (1), is the major copper carrier, binding 70-95% of circulating copper in mammals (2). CP is a member of the multicopper oxidase family and contains three type I copper sites, responsible for its strong absorption at 600 nm, conferring to it an intense blue color (3). This absorption is attributed to the charge transfer between the cysteine ligand sulfur and the copper at the type I sites (3). While the physiological functions of CP are still unclear, several biochemical roles have been proposed for it: copper transport in plasma (4), catalytic activity toward aromatic amines (4), ferroxidase activity (5), and catalysis of biological S-nitrosation (6). CP also modulates K+ channel activity in neuroblastoma cells (7), affects cardiodynamics of isolated rat hearts (8), and induces aggregation of newly differentiated neurons in vitro (9). The role of CP as a regulator of iron metabolism in mammalian cells appears to be well-established (10-12). CP has been shown to control the oxidative state of iron ions by oxidizing the ferrous form (FeII) to the ferric form (FeIII) and then promoting FeIII loading onto transferrin (13) and ferritin (14, 15), proteins responsible for iron transport in plasma and for nontoxic iron storage in the cell, respectively. Reilly and co-workers (16) found that * To whom correspondence should be addressed. Tel: 55-11-30913869. Fax: 55-11-3815-5579. † Universidade de Sa ˜ o Paulo. ‡ Universidade Cruzeiro do Sul. § Universita ´ degli Studi di Roma Torvergata. | University of Rome “La Sapienza”.
ferritin and CP form a protein-protein complex during iron loading into apoferritin, and intact CP was required for the protein-protein interaction (17). The participation of CP in iron uptake by cells (18), besides its capacity to regulate iron levels in the central nervous system (19), suggests a possible role for CP as an antioxidant in various neurodegenerative diseases (e.g., Parkinson’s disease, Alzheimer’s disease) in which iron deposition is known to occur (20). In vitro exposure of CP to oxidizing agents (e.g., low-intensity UV irradiation, incubation with peroxyl radicals) resulted in a loss of CP-ferroxidase activity, release of copper ions, and modification of protein structure (21, 22). It has been well-documented that the CP level is increased in patients with insulin-dependent (23, 24) and noninsulin-dependent (25-27) diabetes mellitus, probably as a response to the increased level of circulating unbound iron observed in human and experimental animal diabetes. Another consequence of intracellular hyperglycemia is the accumulation of triose phosphates, glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate that leads to the formation of the highly reactive R-dicarbonyl compound methylglyoxal (MG) (28). MG has been implicated in diabetes complications such as retinopathy, neuropathy, and cataracts (29). We have recently found that aminoacetone (AA), a threonine and glycine metabolite, implicated as a contributing source of MG in diabetes (29), undergoes aerobic oxidation to MG, NH4+, and H2O2. This reaction has been reported to be catalyzed in vitro by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) (30) as well as by CuII ions (31). Recently, we demonstrated that AA undergoes fast enolization at physiological pH, fol-
10.1021/tx049655u CCC: $30.25 © 2005 American Chemical Society Published on Web 03/15/2005
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Chem. Res. Toxicol., Vol. 18, No. 4, 2005 Scheme 1. Aerobic Oxidation of AA to MG
lowed by oxidation yielding MG with the intermediacy of reactive oxygen species (ROS), specifically superoxide anion radical, hydroxyl radical, and AA enoyl radical (32) (Scheme 1). The AA-generated oxyradicals were shown to cause chemical damage to the subunits of horse spleen ferritin, with consequent decreases in its ferroxidase activity and iron incorporation ability (33), and to induce transition permeability in isolated rat liver mitochondria (34). These findings contributed to explain a possible intracellular iron-induced oxidative stress during AA accumulation in diabetes mellitus. We now examine structural and functional characteristics of CP after its in vitro exposure to AA. Herein, we report (i) CP aggregation induced by AA-generated ROS; (ii) the effect of AA on the ferroxidase activity of CP; and (iii) comparative studies of effects of AA and MG on CP.
Experimental Procedures Materials. o-Dianisidine, Chelex-100, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), p-phenylenediamine, mannitol, diethyldithiocarbamate, dithiothreitol (DTT), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), catalase, and Cu,Zn-superoxide dismutase (SOD) were purchased from Sigma Chemical Co. (St. Louis, MO). MG obtained from Sigma Chemical Co. was purified prior to use by fractional distillation as described by McLellan and Thornalley (35). The organic solvents used were of HPLC grade, while other reagents were of analytical grade. All of the buffers and solutions were prepared with Milli-Q deionized water and pretreated with Chelex-100. AA hydrochloride was prepared according to Hepworth (36) and recrystallized from ethanol:ether (2:8). Stock solutions of AA were prepared daily in deaerated Milli-Q purified water and kept under nitrogen. Isolation of Human CP. CP was purified from human plasma by single step passage on derivatized sepharose as described by Musci et al. (37). Briefly, citrated plasma obtained from healthy volunteers was supplemented with 20 mM -aminocaproic acid and applied to the resin for 15 min. After the plasma was filtered away, the resin was washed with 80 and 100 mM phosphate buffer (pH 7.0), both containing -aminocaproic acid, to remove nonspecifically bound proteins. A further washing with 120 mM phosphate buffer (pH 7.0), which caused a 20-40% loss of impure CP, was followed by elution of 50-60 µM pure (>95%) CP with 200 mM phosphate buffer (pH 7.0). The whole purification procedure was carried out at 4 °C. Oxidation and Native PAGE Analysis of CP. A standard oxidation procedure was carried out by 8 h of incubation of 1.5 µM (0.2 mg/mL) CP, both in the presence and in the absence of AA, in 100 mM phosphate buffer (pH 7.4) at 37 °C. After this treatment, aliquots (∼5 µg of protein) of the reaction mixture were diluted with a 0.25 mM Tris-HCl, pH 6.8, 40% glycerol, and 0.01% bromophenol blue solution. The resulting solutions were subjected to native-PAGE using a 10% acrylamide slab gel as described by Laemmli (38). Other additions and AA concentrations were described on the figure captions. The gels
Dutra et al. were stained with 0.15% Coomassie brilliant blue R-250 or o-dianisidine. Triplicates were run with each of three distinct CP preparations. Oxygen Consumption, Spectrophotometry, and Protein Analyses. Oxygen uptake was followed in a Hansatech Oxygraph equipped with a Clark type electrode and magnetic stirring (39). Unless otherwise stated, the experiments were run with different AA concentrations in 100 mM phosphate buffer, pH 7.4, at 37 °C, in the presence or absence of CP (3.8 µM). Observed rate values were expressed as means ( SD of at least three independent experiments. Spectrophotometric measurements of CP thiol content and activity were carried out in a thermostatically controlled Hitachi U2000 spectrophotometer. Protein was measured by the Bradford method (40). CP Activities Measurement. The amine oxidase activity of CP was calculated by coupled assay on the basis of the NADH consumption by the first oxidation product of p-phenylenediamine (41). Amine oxidase activity of native or oxidized CP (1.5 µM aliquots) was assayed in 100 mM phosphate buffer (pH 6.3) containing 0.25 mM DTPA (to prevent any increase of the activity caused by iron traces), 0.25 mM NADH, and 10 mM NaCl. After the addition of p-phenylenediamine (0.5 mM final concentration), the reaction was followed by monitoring the decay of absorbance at 340 nm. The ferroxidase activity of CP was determined as described by Reilly and Aust (14), using acetate buffer to avoid nonenzymatic, phosphate-assisted oxidation of ferric ions. After the standard oxidation procedure, aliquots of native or oxidized CP (0.3 µM) were incubated at 37 °C in 300 mM acetate buffer (pH 6.0) containing 2.5 mM ferrozine and increasing concentrations of FeII:histidine (1:5) (prepared in argon-purged 50 mM NaCl solution, pH 7.0). Reactions were initiated by addition of iron, and their initial rates were monitored by absorbance changes at 564 nm (564 ) 27.9 mM-1 cm-1) for at least 5 min, when the absorbance variations were linear. Determination of Free Copper Ion Concentration. CP samples (7.5 µM) were incubated with 1 mM AA for various intervals and then subjected to ultrafiltration using Millipore Ultrafree-MC filter with a molecular mass cutoff of 3 kDa. The concentration of copper ion in the filtrate was determined by atomic absorption spectrophotometry on a Perkin-Elmer apparatus model 3030 equipped with graphite furnace. Analysis of Protein Thiols. CP (7.5 µM) was submitted to the standard oxidation procedure in the presence and absence of 1 mM AA for 8 h at 37 °C in 100 mM phosphate buffer (pH 7.4). Protein thiols were measured spectrophotometrically after reaction with DTNB (42). Briefly, aliquots of 50 µg of protein were added to 1 mL of 0.5 mM EDTA and 0.5 M Tris (pH 8.3) containing 0.1 mM DTNB. After 15 min, the resulting thiol conjugate was determined by its absorption at 412 nm. The assay was calibrated using glutathione to calculate the concentration of total thiols. The values represent the average of three independent experiments. Statistical Analysis. The results were analyzed by two-way ANOVA and Tukey’s post-hoc test. A probability of p < 0.05 was used as the criterion for statistical significance.
Results Endogenous AA may act as an in vivo pro-oxidant metabolite during the onset of diabetes mellitus, considering that (i) AA behaves in vitro as a source of reactive intermediates (O2•-, H2O2, and AA-enoyl radical) when it undergoes metal-catalyzed aerobic oxidation (32) and (ii) AA production is enhanced under conditions in which acetyl-CoA accumulates (e.g., ketoses) (43). Our previous work has shown the ability of AA to promote ferritin protein modification with consequent iron release and losses of ferritin ferroxidase and iron uptake activities (33). Because intact CP is required for iron incorporation into apoferritin, and both AA and CP levels are elevated
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Figure 1. Aggregation of CP after incubation with AA. CP (1.5 µM) was incubated with the indicated concentrations of AA in 100 mM phosphate buffer (pH 7.4) at 37 °C for 8 h.
Figure 2. Effect of radical scavengers and copper ion chelators on the aggregation of CP by AA. CP (1.5 µM) was incubated with 0.5 mM AA in the absence or presence of radical scavengers and copper chelators at 37 °C for 8 h. All effectors were added prior to the AA treatment. Lanes: 1, CP control; 2, incubated with AA; 3, lane 2 + SOD (40 U/mL); 4, lane 2 + catalase (5 µM); 5, lane 2 + mannitol (200 mM); and 6, lane 2 + diethyldithiocarbamate (20 mM).
in diabetes mellitus, we decided to investigate whether ROS produced during nonenzymatic AA aerobic oxidation were able to promote CP protein modification. A proaggregative effect of AA against CP was accompanied by the formation of visually observed precipitates that did not enter the stacking gel. Incubation (8 h) of CP with AA promoted an increase in the frequency of protein aggregation in a dose-dependent manner (Figure 1). Concentrations as low as 50 µM AA promoted slight protein aggregation while concentrations higher than 250 µM lead to extensive CP aggregation indicated by total disappearance of CP in the gel. To clarify the role of ROS and copper in the CP aggregation induced by AA, the effects of SOD, catalase, mannitol, and diethyldithiocarbamate were investigated. Both antioxidant enzymes, SOD (40 U/mL) and catalase (5 µM) (Figure 2, lanes 3 and 4 respectively), as well as mannitol (200 mM; Figure 2, lane 5) and the copper chelator diethyldithiocarbamate (20 mM; Figure 2, lane 6), were able to protect CP against aggregation induced by 0.5 mM AA. These results confirm that O2•- and H2O2 may be associated with the structural protein modification. Involvement of catalyst adventitious iron in AA oxidation may be evoked here, although AA (0.5 mM) has been shown to not be able to reduce FeIII ions (0.25 mM) in nitrogen-purged phosphate solution, pH 7.4 (not shown); therefore, AA could not recycle iron required to sustain ROS production under aerobic conditions. Oxidative damage of copper-containing enzymes by ROS has been shown by the modification of copper’s ligand environment and by the release of copper ions from the enzyme (44-46). Accordingly, incubation of CP with 1 mM AA for 8 h produced a 52% decrease in thiol content (9.5 ( 1.1 vs 18.2 ( 0.9 nM/mg protein, control; p < 0.05). This effect was almost totally eliminated (thiol content ) 16.9 ( 0.6 nM/mg protein) by the addition of 1 mM DTT to the reaction mixture. Altogether, these results suggest oxidative modification of CP type I copper sites. Copper ion release accompanied CP aggregation during reaction with AA (Figure 3). After 8 h of incubation with 1 mM AA, about 5 µM copper ion was released from CP vs 1 µM in the control incubation in the absence of AA, which may reflect the content of loosely bound copper ions in the CP sample and explain the protective effect of diethyldithiocarbamate against AA-induced CP aggregation. Copper ions released to the medium by oxidativemodified CP may catalyze AA aerobic oxidation (31). Accordingly, the presence of CP was shown to triple the rate of oxygen consumption by AA (Figure 4). For
Figure 3. Release of copper ions from CP during incubation with AA. CP (7.5 µM) was incubated with (-b-) or without (-[-, control) 1 mM AA at 37 °C under standard reaction conditions for different incubation periods. An aliquot was taken, and free copper ions were determined as described under the Experimental Procedures. Data represent the means ( SD (n ) 5).
Figure 4. Oxygen uptake by AA. The time course of oxygen consumption by AA was monitored in 100 mM Chelex-treated phosphate buffer (pH 7.4), at 37 °C, with the indicated concentrations of AA in the presence (line A) or absence (control, line B) of 3.8 µM CP. Data represent the means ( SD (n ) 3). Table 1. Rate of Oxygen Uptake by AA in the Presence of Copper Complexesa metal complexes (µM)
oxygen uptake (µM O2/min)b
none CuII(aquo) (10) CuII(aquo) (50) CuII:EDTA (100) CuII:histidine (30) CP (3.5)
3.9 ( 0.4 7.3 ( 0.6 77.7 ( 11.0 4.9 ( 0.6 26.5 ( 4.2 13.5 ( 2.1
a Oxygen uptake by 5 mM AA in 100 mM phosphate buffer (pH 7.4) at 37 °C. Ratio chelator/metal ) 1.2, except for CuII:histidine ratio ) 2. b Observed rate values were expressed as means ( SD of at least three independent experiments.
comparison, 50 µM CuII(aquo) increased 20-fold the rate of oxygen uptake, whereas 100 µM CuII:EDTA had no effect (Table 1). AA concentration (mM)-dependent loss of ferroxidase activity of CP was observed (Figure 5) during protein incubation with AA. Regarding these apparently nonphysiological AA concentrations, it must be stated that AA concentrations have not yet been determined in the plasma and tissues of either normal or diabetic subjects. The only available in vivo estimation was made long ago by Urata and Granick (43), who found “about 0.4 mg of a compound with chromatographic properties of ami-
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of ROS, and not MG, in the observed AA-driven CP aggregation.
Discussion and Conclusion
Figure 5. Kinetics of iron oxidation by native and oxidized CP. Increased concentrations of iron:histidine complex were added to mixtures containing 0.3 µM CP native (A) or 0.3 µM CP previously incubated with 2.5 mM (B), 5 mM (C), or 10 mM (D) AA. Reactions were initiated by the addition of iron, and the initial rate was monitored by the changes at 564 nm for at least 5 min, during which the absorbance changes were linear. Iron oxidation was measured as described under the Experimental Procedures. Data represent the means ( SD (n ) 5).
Figure 6. Aggregation of CP after incubation with AA or with MG. CP (1.5 µM) was incubated with 0.5 mM AA or with 50 mM MG at 37 °C for 8 h. After native PAGE analysis, the gels were stained with 15% Coomassie brilliant blue (A) or with o-dianisidine (B). Lanes: 1, CP control; 2, incubated with AA; 3, incubated with MG; and 4, after 48 h of incubation with MG.
noacetone” in the urine collected daily from normal human adults, i.e., 2 µM AA, assuming excretion of 1.5 L of urine/day. In our model studies, the millimolar concentrations of AA might reflect pathological rather than normal conditions. Furthermore, CP aminoxidase activity was abolished even in the absence of protein aggregation (less than 250 µM AA; data not shown). These results suggest that AAinduced protein aggregation with consequent copper release is associated with the loss of CP enzymatic properties. As SSAO- and iron-catalyzed AA oxidation by oxygen may contribute to MG accumulation in diabetes and a causal link between MG and diabetes symptoms may involve MG binding to CP (47), we also examined here the contribution of MG to the observed protein aggregation. Upon CP incubation with 0.5 mM AA (37 °C, 8 h), a faint protein band was observed in the native PAGE gel when it was stained with Coomassie Blue (Figure 6A, lane 2), whereas no aminoxidase activity band was observed using o-dianisidine (Figure 6B, lane 2). Incubation of CP with 50 mM MG did not alter significantly the protein nor the CP oxidase activity (Figure 6A,B, lane 3). Increasing the incubation time up to 48 h led to augmented CP electrophoretic mobility (Figure 6A,B, lane 4) but no changes were observed in the CP activity (Figure 6B, lane 4). These results stress the involvement
SOD (48), catalase (49), glutathione peroxidase (50), and CP (51) are among the antioxidant enzymes reported to be inactivated by Fenton type reactions. Of interest regarding iron metabolism and diabetes is CP as it participates in the iron uptake by transferrin and ferritin (16). Recently, we have found that endogenous aminoketones (AA and 5-aminolevulinic acid, a heme precursor accumulated in porphyric disorders) undergo ferritininduced oxidation by oxygen coupled with loss of ferritin ferroxidase activity and iron incorporation ability (33, 52). Because AA overproduction is implicated as a contributing source of MG in diabetes (43), the CP tissue levels are increased in diabetes mellitus types I and II (2427), and because the ability of CP to oxidize iron in vitro has been shown here to be susceptible to AA-induced oxidative damage, we suggest that an interplay of CP and AA may be relevant when studying the molecular bases underlying the pathogenesis of diabetes. Accordingly, in the current in vitro studies, we observed structural and functional changes in the CP after its exposure to AA. Specifically, we demonstrated (i) changes in the CP electrophoretic behavior induced by AA-generated reactive species; (ii) copper release from CP after protein incubation with AA; (iii) changes in the protein thiol content; and (iv) loss of ferroxidase and aminoxidase activities of human CP. AA was shown not only to act as a CP copper-releasing agent but also to damage the protein structure, probably by inducing protein aggregation. Several authors (5355) have reported the disappearance of the native CP bands and no generation of fragments by electrophoretic analysis of the reaction products from CP treated with H2O2. This finding and the conservation of oxidizable amino acid residues on CP structure led Aouffen and coworkers (55) to postulate that H2O2 can induce the aggregation of CP molecules. We have shown here that incubation of CP with AA causes CP aggregation as well (Figures 1 and 6A), concomitantly with loss of ferroxidase (Figure 5) and aminoxidase activities, and that the AAinduced CP aggregation is accompanied by copper release from the enzyme. The AA-induced loss of ferroxidase activity resulted in an inability of CP to promote iron oxidation and therefore is expected to affect the cooperative CP/ferritin activity, as proposed by Reilly and co-workers (16). Aggregation of CP induced by AA could contribute to preventing the CP-ferritin interaction, leading to potentially dangerous iron overload and consequent oxidative stress observed in neurodegenerative disorders (56, 57) and diabetes (58). The protective effects of antioxidant enzymes (SOD and catalase), a radical scavenger (mannitol), and a copper chelator (diethyldithiocarbamate) against AA-induced oxidative damage to CP (Figure 2) suggest that superoxide anion and hydrogen peroxide may play a critical role in AA-mediated CP aggregation. In addition, diethyldithiocarbamate protection of CP damage revealed a catalytic effect of copper released from CP on the aerobic oxidation of AA. This result is not surprising, since we had already demonstrated that AA undergoes oxidation by dioxygen propagated by O2•- and is accompanied by HO• formation (32).
Aminoacetone-Induced Ceruloplasmin Oxidation
Our measurements of protein thiol content of CP indicated cysteine residue oxidation induced by AA. The observed loss of thiol (52% decrease) may reflect changes in CP type I copper binding sites (3), resulting in loss of activity and copper ions release. Moreover, the observed protective effect of DTT suggests that CP aggregate formation involves disulfide covalent cross-linking. It has been shown that MG reacts with lysine residue of bovine serum albumin yielding Schiff adducts with increased electrophoretic mobility (59). Likewise, changes in electrophoretic behavior of CP after reaction with MG were observed here (Figure 6A), but it was not sufficient to promote the loss of CP oxidase activity toward primary amines (Figure 6B). In our experimental conditions, formation of CP aggregates is safely attributed to AAgenerated ROS instead of MG-promoted conjugation with CP. To our knowledge, this is the first study showing that an endogenous metabolite induces CP protein modification with consequent loss of enzymatic activity. If operative in vivo, this reaction would impair the antioxidant role of CP and iron uptake by ferritin and hence contribute to intracellular iron-induced oxidative stress during AA accumulation in diabetes mellitus.
Acknowledgment. We thank the Fundac¸ a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), the Conselho de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), the Programa de Apoio a Nu´cleos de Exceleˆncia (PRONEX), and the University of Sa˜o Paulo for financial support. We are also indebted to Prof. Giuseppe Rotilio for kindly supporting one of the authors (F.D.) to run the main experiments on the CP/AA system in his laboratories, to Dr. Giuseppe Filomeni and Dr. Katia Aquilano for the technical support, and to Dr. Brian Bandy for reading the manuscript.
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