Glycation damage targets glutamate dehydrogenase in the rat liver mitochondrial matrix during aging Maud Hamelin, Jean Mary, Michal Vostry, Bertrand Friguet* and Hilaire Bakala* Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite ´ Paris 7-Denis Diderot, France Aging is characterized by gradual deterioration of cel- lular functions [1] associated with cumulative damage to intracellular macromolecules, particularly proteins [2]. Several lines of evidence suggest that mitochondria play a key role in aging, both by producing intracellu- lar reactive oxygen species (ROS) and by being the most adversely affected organelles during aging [3]. The electron transport chain located in the inner mito- chondrial membrane is implicated in production of ATP via oxidative phosphorylation, and is also known to be the major intracellular site of free radical genera- tion, such as that of superoxide anions and, sub- sequently, other potentially deleterious ROS [4]. Numerous data have indicated an age-related increase in the rate of mitochondrial free radical generation and in the extent of oxidative damage to mitochondrial macromolecules [5–9], especially enzymes involved in the respiratory chain, leading to impairment of respira- tory activity [7,9–12]. Although direct oxidation of proteins and other macromolecules is believed to be the main type of endogenous damage during aging [3,13,14], ROS can Keywords aging; glycation; liver mitochondria; proteomics; urea cycle enzymes Correspondence H. Bakala, Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, EA3106 ⁄ IFR117, Universite ´ Paris 7-Denis Diderot, 2 place Jussieu, 75251 Paris, Cedex 05, France Fax: +33 1 44 27 39 25 Tel: +33 1 44 27 82 27 E-mail: bakala@paris7.jussieu.fr *These authors contributed equally to this work (Received 22 May 2007, revised 21 Septem- ber 2007, accepted 25 September 2007) doi:10.1111/j.1742-4658.2007.06118.x Aging is accompanied by gradual cellular dysfunction associated with an accumulation of damaged proteins, particularly via oxidative processes. This cellular dysfunction has been attributed, at least in part, to impair- ment of mitochondrial function as this organelle is both a major source of oxidants and a target for their damaging effects, which can result in a reduction of energy production, thereby compromising cell function. In the present study, we observed a significant decrease in the respiratory activity of rat liver mitochondria with aging, and an increase in the advanced gly- cation endproduct-modified protein level in the mitochondrial matrix. Wes- tern blot analysis of the glycated protein pattern after 2D electrophoresis revealed that only a restricted set of proteins was modified. Within this set, we identified, by mass spectrometry, proteins connected with the urea cycle, and especially glutamate dehydrogenase, which is markedly modified in older animals. Moreover, mitochondrial matrix extracts exhibited a signifi- cant decrease in glutamate dehydrogenase activity and altered allosteric regulation with age. Therefore, the effect of the glycating agent methylgly- oxal on glutamate dehydrogenase activity and its allosteric regulation was analyzed. The treated enzyme showed inactivation with time by altering both catalytic properties and allosteric regulation. Altogether, these results showed that advanced glycation endproduct modifications selectively affect mitochondrial matrix proteins, particularly glutamate dehydrogenase, a crucial enzyme at the interface between tricarboxylic acid and urea cycles. Thus, it is proposed that glycated glutamate dehydrogenase could be used as a biomarker of cellular aging. Furthermore, these results suggest a role for such intracellular glycation in age-related dysfunction of mitochondria. Abbreviations AGE, advanced glycation endproduct; CEL, N-(carboxyethyl)lysine; CML, carboxymethyl-lysine; GDH, glutamate dehydrogenase; GO, glyoxal; MGO, methylglyoxal; OCT, ornithine carbamoyl transferase; RCR, respiratory control ratio; ROS, reactive oxygen species; TCA, tricarboxylic acid. FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS 5949 also affect protein function through either lipoperoxi- dative production of reactive aldehydes [15] or glycoxi- dation pathways [16]. Indeed, there is a causal relationship between hyperglycemia-induced ROS generation and intracellular advanced glycation end- product (AGE) formation [17]. This rise in AGE was shown to be primarily if not exclusively due to a rapid increase in AGE-forming methylglyoxal concentration [18]. Alpha-dicarbonyl compounds such as glyoxal (GO) and methylglyoxal (MGO) are physiological, highly reactive intermediates involved in the Maillard reaction [19]. Interestingly, MGO may originate from various biochemical pathways, including dephosphorylation of glycolytic intermediates, metabolites of the polyol pathway and metabolism of aminoacetones [20]. At physiological concentrations, MGO primarily targets the arginine residues of proteins, leading primarily to AGE-imidazolone [21,22] and -lysine residues to form AGE adducts: N-(carboxyethyl)lysine (CEL) and meth- ylglyoxal lysine dimer [23]. In a previous study, using an immunochemical method, we showed that glycated proteins accumulate in the rat liver mitochondrial matrix with aging [24]. In the present study, we show that glycation targets a limited set of proteins, the most severely affected being glutamate dehydrogenase, a crucial enzyme at the interface between the tricarboxylic acid (TCA) and urea cycles, indicating the interference of matrix enzymes in mitochondrial impairment function with aging. Results Age-related changes in the respiratory function of isolated mitochondria Mitochondria were isolated from the livers of young (3-month-old) and old (27-month-old) Wistar rats and their respiratory parameters were measured. As shown in Table 1, there was a significant decrease in the rates of mitochondrial oxygen consumption between 3-month-old and 27-month-old rats with gluta- mate ⁄ malate or succinate as substrates. The ADP ⁄ O ratios (P ⁄ O) obtained whatever the substrate used indi- cated no change in coupling efficiency. Although the respiratory control ratio (RCR) slightly decreased with glutamate ⁄ malate substrate during aging, no change was observed using succinate. In the presence of 2,4-dinitrophenol, oxygen consumption rates were the same in the presence of ADP (state 3) at each age. Together, these results indicated age-related impair- ment of mitochondrial function. Determination of 6D12-detectable AGE content in mitochondrial matrix proteins We next investigated the levels of AGE-modified pro- teins to determine whether dysfunction of the organelle parallels the accumulation of modified mitochondrial matrix proteins. For this purpose, we used monoclonal anti-AGE IgG (6D12) in a competitive ELISA using carboxymethyl-lysine (CML)-modified BSA as standard to evaluate AGE-modified protein content (Fig. 1). AGE content increased significantly, by 48% from 12.01 ± 0.71 AUÆlg protein )1 (n ¼ 8) in 3-month-old rats to 17.81 ± 1.83 AUÆlg protein )1 (n ¼ 9) in 27-month-old rats (P<0.01). These data indicated age-associated accumulation of AGE adducts in mitochondrial matrix proteins. Identification of glycated mitochondrial matrix proteins by LC-MS ⁄ MS after 2D gel electrophoresis and western blotting 2D gel electrophoresis and western blotting Standard 2D gel electrophoresis of liver mitochondrial matrix proteins from young and old rats was run in parallel (Fig. 2). Electrophoregrams showed a typical pattern of total protein, and more than a thousand Table 1. Biochemical respiratory parameters in rat liver mitochondria from 3-month-old and 27-month-old rats. Oxygen consumption rates were measured polarographically in the presence of either 10 m M succinate or 5 mM glutamate ⁄ 5mM malate. State 3 respiration was deter- mined after adding 310 nmol of ADP. State 4 respiration is the rate of O 2 consumption after depletion of ADP. Data represent the mean ± SEM. n, number of animals. *P < 0.05; **P < 0.01 versus 3-month-old rats. Succinate (n ¼ 7) Malate + glutamate (n ¼ 5) 3 months 27 months 3 months 27 months State 4 (ng atom O min )1 Æmg )1 ) 74 ± 7 47 ± 8* 37.1 ± 2.9 35.8 ± 4.6 State 3 (ng atom O min )1 Æmg )1 ) 281 ± 24 167 ± 14** 153.3 ± 6.6 130.6 ± 4.5* RCR 3.79 ± 0.36 3.55 ± 0.28 4.42 ± 0.2 3.81 ± 0.45 P ⁄ O 1.87 ± 0.09 1.91 ± 0.06 2.83 ± 0.06 2.84 ± 0.09 Glycation of glutamate dehydrogenase with aging M. Hamelin et al. 5950 FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS spots were detected using 2D Elite Master Software. Western blot analysis using anti-AGE IgG (Fig. 3) in samples from both young and old rats revealed that only a small number of proteins among the thousand protein spots observed on gel stained with colloidal Coomassie blue were targeted for glycation. Moreover, AGE-modified proteins were already present, although to a lesser extent, in samples from young rats. We noted individual variations both in the nature of modi- fied proteins and in their extent of labelling within Fig. 1. Determination of 6D12-detectable AGE protein content in liver mitochondrial matrix with aging. The AGE adduct content in mitochondrial matrix proteins from 3-month-old and 27-month-old rats was assayed by competitive ELISA using monoclonal anti-AGE IgG (clone 6D12). The results are expressed as AUÆlg protein )1 and represent the mean ± SEM. **P < 0.01 versus 3-month-old rats. Number of animals is given in parentheses above the bar graphs. A 250 - 150 - 100 - 75 - 50 - 37 - 25 - pI 310 B - - - - - - - Molecular Mass (kDa) 250 150 100 75 50 37 25 Molecular Mass (kDa) pI 310 Fig. 2. 2D gel electrophoresis profile of liver mitochondrial matrix proteins. Liver mito- chondrial matrix proteins (150 lg) were sub- jected to isoelectrofocusing and subsequent SDS ⁄ PAGE electrophoresis under reducing conditions. Gels containing samples from (A) 3-month-old and (B) 27-month-old rats were stained with colloidal Coomassie blue. A B Fig. 3. 2D gel electrophoresis and western blotting analysis of liver mitochondrial matrix proteins; identification of AGE-modified pro- teins with aging. Samples (150 lg) were subjected to isoelectrofo- cusing and subsequent SDS ⁄ PAGE under reducing conditions. Gels were subjected to western blotting using monoclonal anti-AGE IgG (clone 6D12) to detect AGE-modified proteins in samples from (A) 3-month-old and (B) 27-month-old rats. Matched proteins were identified by tandem LC-MS ⁄ MS mass spectrometry as: (1) gluta- mate dehydrogenase; (2) catalase; and (3) ornithine carbamoyl transferase. M. Hamelin et al. Glycation of glutamate dehydrogenase with aging FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS 5951 each age group. Furthermore, we observed that anio- nic isoforms appeared to be modified with age for trails 1 and 3 (Fig. 3B). Only spots from trails of iso- forms numbered 1–3 that exhibited a significantly increased yield of modifications with aging in a repro- ducible way were retained for further analysis (Fig. 3A,B) and were matched with colloidal Coomas- sie blue staining spots (Fig. 2B), which were subjected to LC-MS ⁄ MS protein identification. Protein identification Proteins from selected spots were identified automati- cally by a computer program. Three series of MS ⁄ MS spectra were obtained (data not shown). At least two spots for each trail of isoforms were identified with sufficient peptide coverage (13–32%), and analysis led to the characterization of three proteins in a narrow pI range (7.15–9.12), with a molecular mass in the range Table 2. Identification of proteins located in spots 1–3. Tryptic peptides from in-gel digestion were subjected to LC ⁄ MS ⁄ MS analysis as described in the Experimental procedures. Peptide identification was evaluated using Xcorr scores that measure similarities between mass- to-charge (m ⁄ z) ratios for fragment ions predicted from amino acid sequences and fragment ions observed in the MS ⁄ MS spectrum. Xcorr, cross correlation scores; z, charge of the precursor ion; M*, oxidized methionine, C@, alkylated cysteine. Spot No. Protein UniProt accession No. Mass (kDa) pI Peptides matches Covearge (%) 1 Glutamate dehydrogenase 1, mitochondrial precursor, P10860 61.4 8.05 12 22 Sequence Position MH + DM z Xcorr K.M*VEGFFDR.G 69–76 1016.45 0.44 2 2.02 R.RDDGSWEVIEGYR.A 124–136 1581.73 0.31 3 2.83 R.DDGSWEVIEGYR.A 125–137 1425.63 0.17 2 3.01 R.YSTDVSVDEVK.A 152–162 1241.59 0.27 2 2.55 K.ALASLM*TYK.C 163–171 1013.53 0.34 2 2.64 K.C@AVVDVPFGGAK.A 172–183 1219.61 0.29 2 2.65 K.KGFIGPGIDVPAPDM*STGER.E 212–231 2060.01 0.64 3 2.72 K.GFIGPGIDVPAPDM*STGER.E 213–231 1931.9 0.43 2 3.04 K.HGGTIPVVPTAEFQDR.I 481–496 1723.88 0.62 2 2.10 K.DIVHSGLAYTM*ER.S 504–516 1507.72 0.46 3 3.46 R.TAM*KYNLGLDLR.T 524–535 1410.74 0.53 3 3.29 K.YNLGLDLR.T 527–535 963.53 0.57 2 2.50 R.TAAYVNAIEK.V 536–545 1079.57 0.35 2 2.80 2 Catalase, P04762 59.6 7.15 6 13 Sequence Position MH + DM z Xcorr K.LNIM*TAGPR.G 38–46 988.52 0.33 2 2.03 K.LVNANGEAVYC@K.F 221–232 1337.65 0.37 2 2.52 R.LAQEDPDYGLR.D 252–262 1276.62 0.38 2 2.65 R.LGPNYLQIPVNC@PYR.A 365–379 1803.92 0.53 2 2.07 R.FNSANEDNVTQVR.T 431–443 1493.70 0.44 2 4.38 K.DAQLFIQR.K 468–475 990.54 0.22 2 2.44 3 Ornithine carbamoyltransferase, mitochondrial, P00481 39.9 9.12 10 32 Sequence Position MH + DM z Xcorr K.GRDLLTLK.N 39–46 915.56 0.51 2 2.17 K.GEYLPLLQGK.S 71–80 1117.63 0.36 2 2.42 R.VLSSM*TDAVLAR.V 130–141 1278.67 0.57 2 3.41 R.VYKQSDLDILAK.E 142–153 1392.77 0.53 2 2.01 K.FGM*HLQAATPK.G 211–221 1216.61 0.43 2 2.20 K.GYEPDPNIVK.L 222–231 1131.57 0.26 2 2.07 K.LSM*TNDPLEAAR.G 244–255 1333.64 0.20 2 3.23 R.LQAFQGYQVTM*K.T 278–289 1429.71 0.54 2 3.98 R.KPEEVDDEVFYSPR.S 307–320 1709.80 0.71 2 2.91 R.SLVFPEAENR.L 321–330 1161.60 0.62 2 2.87 Glycation of glutamate dehydrogenase with aging M. Hamelin et al. 5952 FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS 39.9–61.4 kDa (Table 2). The number of peptides lead- ing to the identification of each protein varied between six and 12. The proteins identified corresponded to glutamate dehydrogenase (GDH) for trail 1, catalase for trail 2 and ornithine carbamoyl transferase (OCT) for trail 3. By contrast to catalase, a potent enzyme in antioxidant defense, the other two enzymes (GDH and OCT) involved in the mitochondrial urea cycle exhib- ited a wide extent of modification at 27 months compared to 3 months. Further investigations were performed with GDH because this enzyme is relevant due to its essential role in between the urea and TCA cycles. Age-associated increased glycation and decreased activity of GDH Using anti-GDH serum, we first checked by western blotting that GDH content within the mitochondrial matrix was unchanged with age (data not shown). GDH was then immunoprecipitated from matrix sam- ples and western blotting was carried out to investigate the increased GDH glycation rate with aging (Fig. 4). Spots of GDH protein exhibited identical intensity in 3-month-old (49.86 ± 3.10 AU) and 23-month-old rats (54.98 ± 2.39 AU), whereas AGE-labelling inten- sity significantly increased by 45%, from 32.71 ± 2.65 AU (n ¼ 4) in 3-month-old rats to 47.29 ± 3.67 AU (n ¼ 3) in 23-month-old rats (P<0.01) (Fig. 4B). The rate of glycation expressed as the ratio of AGE ⁄ GDH intensity was significantly increased by 30%, from 0.66 ± 0.05 (n ¼ 4) in 3-month-old rats to 0.86 ± 0.03 (n ¼ 3) in 23-month-old rats (P<0.01) (Fig. 4B). These data indicate that GDH underwent increased glycation with aging. We next examined whether the glycation modifica- tion demonstrated above was associated with impair- ment of GDH function. For this purpose, we measured GDH activity in mitochondrial matrix extracts (50 lg of total protein) in the absence and presence of allosteric effectors (ADP and GTP) (Table 3). The results obtained revealed a significant decrease (23%) in GDH activity with aging, from 2.47 ± 0.19 UÆmg )1 in 3-month-old rats to 1.89 ± 0.07 UÆmg )1 in 23-month-old rats ( n ¼ 4) (P ¼ 0.02). In the presence of the ADP activator, this activity was enhanced by 1.20-fold in young samples, although slightly less (1.14-fold) in old samples. However, this GDH activity markedly collapsed with the GTP inhibi- tor, with residual activity barely representing 8% in young samples compared to 12% in old samples (P<0.01). These data suggest an inhibitory effect of glycation on GDH activity, in addition to a slightly altered response to allosteric regulation with aging. Effect of MGO on purified GDH activity and its allosteric regulation To ascertain whether the glycation impact upon GDH activity was due to modifications in lysine ⁄ arginine A B Fig. 4. Immunochemical identification of glycated GDH from mitochondrial matrix. GDH from matrix samples (500 lg) was immunoprecipitat- ed and samples resolved by SDS ⁄ PAGE electrophoresis. Two identical gels from each sample run in parallel were subjected to western blotting using either monoclonal anti-AGE IgG or GDH polyclonal antibody to detect AGE modifications or GDH antigen content, respectively, in samples from 3-month-old and 23-month-old rats (A). Blots were semiquantified by densitometry scanning and density expressed in arbitrary units (AU). Results are presented as the ratio of the AGE ⁄ GDH rate (B). Values are the means ± SEM. **P<0.01 versus 3-month- old rats. M. Hamelin et al. Glycation of glutamate dehydrogenase with aging FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS 5953 residues, we used MGO as a potent glycating agent generated intracellularly to investigate the effect on purified GDH. In vitro MGO modification of GDH A purified GDH sample (10 lg) was incubated with varying concentrations of MGO for up to 24 h and subjected to western blotting (Fig. 5) with monoclonal anti-AGE IgG or polyclonal anti-GDH serum. After 24 h of incubation, the GDH protein was MGO- derived AGE-modified and the extent of modification increased with the concentration of MGO (Fig. 5A), whereas the GDH immunolabelling intensity concur- rently decreased (Fig. 5B). These results clearly demon- strated that glycated enzyme partially lost its antigenicity, indicating that some antigenic sites of GDH could be masked by glycation adducts. Effect of MGO on GDH activity and kinetic parameters In vitro MGO treatment of purified GDH resulted in decreased activity with time of incubation and an increasing MGO concentration (Table 4). With 1 mm MGO, GDH activity significantly decreased at 30 min of incubation, and markedly dropped by 37% within 5 h compared to control. This MGO effect on GDH activity was compared with that induced by GO, an a- dicarbonyl metabolite presumed to possess noxious specificity like MGO. Treatment with GO led to a sig- nificant inhibition effect with as little as 50 lm (38%), whereas a high concentration (1 mm) led to strong inhibition (67%), providing evidence that GDH activ- ity is altered by glycation modification. Using amino acid analysis, we noted that both arginine (24 : 30) and lysine (22 : 32) residues were damaged in MGO- treated GDH, whereas 22 and eight residues, respectively, were damaged with GO, indicating that MGO was equally noxious to lysine and arginine residues, leading to the loss of part of its activity. In addition, analysis of enzymatic parameters (V max and K m ) of GDH modified by 1 mm MGO for 24 h using a-ketoglutarate as substrate (Fig. 6) showed that MGO treatment altered only the maximum velocity (V max ), whereas the apparent K m value did not change. Effect of MGO on GDH allosteric regulation We next analyzed the allosteric regulator effects on purified GDH activity before and after treatment with MGO. At indicated times, the activity of the enzyme incubated with 1 mm MGO was measured in the pres- ence of constant concentrations of allosteric effectors (activators: 250 lm ADP or 10 mm leucine; inhibitor: 30 lm GTP). GDH activity at t 0 in the absence of ef- fectors (28.6 UÆmg enzyme )1 ) was set at 100% activity. As shown in Fig. 7, GDH activity spontaneously decreased with time of incubation. This decline was accentuated when GDH was incubated with 1 mm MGO (Fig. 7A) and the activity ratio shifted from 0.85-fold to 0.71-fold at t 240 versus t 0 . In the presence of an ADP effector (Fig. 7B), native GDH activity exhibited a significant 1.66-fold increase (Fig. 7B, left panel), whereas this increase fell to 1.44-fold when GDH was preincubated with 1 mm MGO (Fig. 7B, right panel). With leucine as an effector, we observed a similar phenomenon (Fig. 7C); the activity increased by 1.55-fold at 240 min with native enzyme (Fig. 7B, left panel) and by 1.24-fold when the enzyme was preincubated with MGO (Fig. 7B, right panel). These Table 3. Age-related changes in GDH activity and allosteric effector sensitivity. Liver mitochondrial matrix extracts (50 lg total protein) from young (3-month-old) and old (23-month-old) rats were sub- jected to the GDH activity assay in the direction of a-ketoglutarate amination, in the absence (control) or presence of constant concen- trations of allosteric effectors (activator: 250 l M ADP; inhibitor: 10 l M GTP). Values are expressed as specific activity (UÆmg )1 ) (control) and activity determined in the presence of effectors is given as a percentage of control in the absence of effectors, at each age. Data represented the mean ± SEM (n ¼ 4). *P ¼ 0.02; **P < 0.01 versus 3-month-old rats. GDH activity Age 3 months (n ¼ 4) 23 months (n ¼ 4) GDH activity (no effector) 2.47 ± 0.19 1.89 ± 0.07* % of GDH activity (+ ADP) 120.88 ± 4.75 114.71 ± 16.71 % of GDH activity (+ GTP) 8.23 ± 0.75 11.57 ± 0.59** 4321 EGA A 4321 HDG B Fig. 5. Western blot detection of purified GDH modified by MGO. Purified GDH (Sigma) samples (10 lg) were incubated with or with- out varying MGO concentrations for 5 h and aliquots (1 lg) were subjected to SDS ⁄ PAGE under reducing conditions. Gels were sub- mitted to western blotting using either monoclonal anti-AGE IgG (clone 6D12) to detect MGO-derived AGE in GDH samples (A) or GDH polyclonal antibody to determine whether the GDH load was preserved (B). Lane 1, control, 0 l M MGO; lane 2, 50 lM MGO; lane 3, 200 l M MGO; lane 4, 1 mM MGO. Glycation of glutamate dehydrogenase with aging M. Hamelin et al. 5954 FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS results indicate that GDH stimulation induced by allo- steric effectors was partly abolished when the enzyme was previously treated with MGO. On the other hand, the GTP inhibitor (Fig. 7D) exhibited an efficient effect on GDH activity; the activity, which was barely 0.44-fold at 240 min (Fig. 7D, left panel), rose to 0.71- fold when GDH was pretreated with MGO (Fig. 7D, right panel), indicating the capacity of MGO to abro- gate the inhibitory effect of GTP on GDH activity. Together, these results clearly demonstrate the inhibi- tory effect of MGO on GDH activity, which rein- forced the decline of its activity with time. In addition, this MGO modification deeply altered the responsive- ness of GDH to its respective allosteric effectors, in agreement with data observed in vivo. Discussion In the present study, we have demonstrated a decline in mitochondrial respiratory chain activity upon aging concomitant with an accumulation of AGE-modified proteins in the mitochondrial matrix. These modifica- tions affect several proteins that are targets for glyca- tion damage and, although some of these proteins are already modified at a young age (3-month-old rats), the level of damage significantly increased with increasing age (27-month-old rats). Among the glycated proteins, trails of isoforms appeared, in agreement with modifi- cations targeting the basic amino acids arginine and lysine, subsequently turning off their ionic charge. In support of this assertion, recent studies have shown that glycation was associated with both loss of basic groups and shifts in pK of the acidic groups, consistent with a reduction in effective anionic charge [23,25]. The identification of these proteins, which are increasingly glycated with aging, revealed three enzymes, GDH, OCT and catalase. Catalase, a crucial antioxidant defense enzyme highly expressed in peroxisomes, and also constitutively present in the heart and liver mito- chondria [26–28], is maintained with GDH and OCT, which belong to the urea cycle. GDH, in particular, which markedly emerged as being modified at 27 months, plays a key role in connecting TCA to the urea cycles, and exhibited a loss of activity and altera- tions in its allosteric properties with aging. Interest- ingly, all these modified proteins are different from those preferentially found to be oxidized during aging in the mitochondrial matrix, as previously identified (i.e. aconitase [29] and adenine nucleotide translocase [30]). To determine whether the age-related inhibition of GDH activity demonstrated here was related to glycation modification, purified GDH was treated with the a-dicarbonyl metabolite MGO. Incubation of GDH with this compound resulted in time-dependent inacti- vation of the enzyme, consistent with the damaging Table 4. Effect of MGO and GO concentrations on GDH activity. Purified GDH samples (10 lg) were incubated in 100 mM TEA-HCl buffer pH 7.3 with varying concentrations (0.05, 0.200 and 1 m M) of either MGO or GO as glycating agents for variable times. At indicated times, enzyme activity was determined spectrophotometrically on aliquots (1 lg). Data are expressed as a percentage of GDH activity control at t 0 for each concentration and represent the mean ± SEM (n, number of animals). *P < 0.05; **P < 0.01; ***P < 0.001 traited versus control at t 0 . MGO [m M] GDH activity (n ¼ 4) Incubation time GO [m M] Incubation time 30 min 2 h 30 5 h 5 h 0 98.0 ± 2.2 91.6 ± 2.7 86.3 ± 6.0 0 82.4 ± 3.9 0.05 87.9 ± 5.2 94.2 ± 2.7 83.4 ± 6.1 0.05 51.2 ± 2.3** 0.2 94.0 ± 4.0 90.8 ± 3.4 73.7 ± 6.0 0.2 45.3 ± 1.9** 1 80.4 ± 5.7* 72.2 ± 5.4** 54.8 ± 7.2** 1 27.5 ± 1.5*** Fig. 6. Effect of MGO modification on GDH parameters V max and K m . Kinetic analyses were performed to determine the effect of MGO (1 m M) on GDH enzymatic parameters V max and K m . Purified GDH (Sigma) samples (10 lg) were incubated with or without MGO for 24 h and aliquots (1 lg) were taken for enzymatic assay. Lineweaver–Burk plots were used with a-ketoglutarate as substrate (S) in native (1) or MGO-treated (2) GDH. M. Hamelin et al. Glycation of glutamate dehydrogenase with aging FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS 5955 effect of glycation. Interestingly, kinetic analysis of modified GDH showed that treatment with MGO reduced only the maximum velocity without affecting K m , indicating that MGO-modified GDH is inacti- vated. In addition, amino acid analysis performed on MGO- and GO-treated GDH revealed that lysine resi- dues were more sensitive to MGO than to GO modifi- cations, whereas arginine was equally sensitive to both dicarbonyl compounds, suggesting that GDH inactiva- tion was at least partly due to MGO-lysine ⁄ arginine modifications, in accordance with the observed trails of isoforms in the 2D electrophoregram. Indeed, numer- ous data have claimed that MGO modifications of criti- cal arginine ⁄ lysine residues cause structural distortion, leading to enzyme inactivation [23,31]. In addition, recent data on GDH studies indicate that among 33 lysine residues constitutive of its primary sequence, the prominent lysine 126 directly interacts with the a-carbon constituent on the substrate [32], suggesting that MGO modification affects enzymatic activity. In support of this assertion, a lysine residue involved in inactivation of brain GDH isoproteins by O-phthal- aldehyde has been identified [33]. Moreover, loss of GDH activity was reported under multiple system atro- phy conditions in which GDH activity was decreased to a greater extent than other mitochondrial enzymes [34] indicating that GDH is more sensitive to insults. Interestingly, a recent in vitro study showed that incu- bation of mitochondria with MGO led to rapid inhibi- tion of mitochondrial respiratory rates through particular protein target modifications [35]. Both the TCA cycle and the electron respiratory chain were inhibited, indicating a link between mitochondrial MGO modified enzymes and altered function. Mamma- lian GDH is allosterically regulated by a number of small molecules [36] and its regulation is of particular biological importance, as exemplified by the observa- tion that some regulatory mutations of the gene for GDH are associated with severe clinical manifestation in children [32]. In addition, as shown in a recent study Control 0 20 40 60 80 100 120 140 160 180 01 MGO (m M) Activity (%) 0 15 120 240 ADP 250 µM 0 20 40 60 80 100 120 140 160 180 0 MGO (m M) Activity (%) Leu 10 mM 0 20 40 60 80 100 120 140 160 180 0 MGO (m M) Activity (%) GTP 30 µM 0 20 40 60 80 100 120 140 160 180 01 MGO (m M) Activity (%) B C D A 1 1 1 ** ** **** *** # # # # # # ** ** ** ** # # # # # # # **** **** **** **** # # # # # # # # # # # # # # # # # # # # # # Fig. 7. MGO effect on GDH activity and allosteric regulation. Puri- fied GDH samples (10 lg; Sigma) was incubated with 1 m M MGO for up to 240 min and aliquots (1 lg) were subjected to the GDH activity assay in the absence (A) or presence of constant concentra- tions of allosteric activators ADP (B) and leucine (C), or inhibitor GTP (D). Values expressed as specific activity (UÆmg )1 ) are given as percentage of control (activity determined in the absence or pres- ence of MGO at t 0 ; left and right panels, respectively) in each range. Data represented the mean ± SEM (n ¼ 6). *t-test: effector versus control at each time; #t-test: MGO 1 m M-treated versus nontreated MGO at each time. °Equivalent to * or #; °P < 0.05; °°P < 0.01; °°°P < 0.005; °°°°P < 0.001. Glycation of glutamate dehydrogenase with aging M. Hamelin et al. 5956 FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS [37], the arginine side chain at position 463 of GDH is thought to be involved in ADP allosteric activation because the R463A mutant form of this enzyme is insensitive to ADP stimulation. In the present study, MGO modifications altered the allosteric regulation properties of the enzyme, suggesting that these effects are not only due to a change in charge profile, but also in the conformation of the molecule resulting from gly- cation of the charged arginine ⁄ lysine side-chain resi- dues. The fact that arginine ⁄ lysine residues are targets of glycation [38,39] suggests that the modifications observed in the present study involve some of these res- idues, leading to an impairment of allosteric regulation and the catalytic properties of the enzyme. GDH is important in converting free ammonia and a-ketoglutatrate to glutamate; it utilizes nicotinamide nucleotide cofactor NAD + for nitrogen liberation and NADP + for nitrogen incorporation; however, it should be recognized that the reverse reaction is a key anapleurotic process linking amino acid metabolism with TCA cycle activity. In a reverse reaction, GDH provides an oxidizable carbon source for the produc- tion of energy, as well as the reduced electron carrier NADH. Thus, GDH is considered to be significant not only because it catalyzes a reaction directly connected to the TCA cycle, but also because of the pivotal posi- tion in metabolism occupied by both glutamate and a- ketoglutarate as a result of their ability to enter into many metabolic pathways [40]. Accordingly, the build- up of an inactive form of GDH demonstrated in the present study could contribute to a decreased produc- tion of a-ketoglutarate and a diminished flux through the TCA cycle, which might be at least partly be responsible for impairment of mitochondrial function with advanced age, as demonstrated by the decrease in respiration driven by the glutamate–malate substrate. In summary, the results obtained in the present study demonstrate that age-related impairment of mitochon- drial respiration runs parallel to an accumulation of AGE-modified matrix proteins. Identification of selec- tively glycated proteins revealed that two of these are key urea cycle enzymes, among which GDH was the main target protein and showed a loss of both activity and sensitivity to allosteric effectors with aging. In vitro alterations in both allosteric regulation and catalytic properties of this enzyme by the glycating agent MGO during short-term incubation support the notion of the dysfunctional power of intracellular glycation. In line with the central role played by this enzyme in cellular metabolism and energy homeostasis, we hypothesize that AGE modifications of GDH may contribute, at least in part, to a defect in mitochondria with aging and could be used as a biomarker of cellular aging. Experimental procedures Animals Experiments were performed on male Wistar rats (WAG ⁄ Rij) born and raised in the animal care facilities of the Commissar- iat a ` l’Energie Atomique (CEA, Gif-sur-Yvette, France). This strain remains lean even when fed ad libitum and does not suffer from age-associated nephropathy, hypertension or diabetes [41]. Cohorts were constituted of young adult (3-month-old) and senescent (27-month-old) animals. All stu- dies were conducted in accordance with the animal care policy of national and European regulations. Chemicals GDH (bovine liver; EC 1.4.1.3) a nd horseradish peroxidase- conjugated anti-(mouse IgG) or anti-(rabbit IgG) sera were purchased from Sigma Chemicals (Saint Quentin Fallavier, France). GDH was dissolved in 100 mm of triethanolamine, pH 7.3. Monoclonal antibody to AGE (clone no. 6D12) from Trans Genic Inc. (Kumamoto, Japan) shows cross-reaction both to CEL and CML [ 42]. Polyclonal antibody against GDH was obtained from Interchim (Montluc¸ on, France) and pro- tein G-agarose bed (ImmunoPure immobilized proteinG Plus) from Pierce (Perbio Science Company, Brebie ` res, Franc e). Isolation of mitochondria A 10% (w ⁄ v) tissue homogenate was prepared using a Pot- ter apparatus in an ice-cold medium containing 220 mm mannitol, 70 mm sucrose, 0.1 mm EDTA and 2 mm Hepes, pH 7.4, supplemented with 0.5% BSA (w ⁄ v). Nuclei and cellular debris were pelleted by centrifugation for 10 min at 800 g and 4 °C. Supernatant was centrifuged at 8000 g for 10 min at 4 °C. The mitochondrial pellet was then washed three times with the homogenization medium and used for polarographic measurements. To prepare mitochondrial matrix extract, mitochondria were suspended in 50 mm Tris ⁄ HCl, pH 7.9, then dis- rupted by sonication (four times for 10 s). The resulting suspension was centrifuged at 15 000 g for 10 min and then at 100 000 g for 45 min at 4 °C. The supernatant (containing matrix proteins) was stored at )80 °C for fur- ther analysis of AGE-modified proteins. The protein con- centration was assessed using a Bradford protein assay (Biorad, Mu ¨ nchen, Germany). To estimate contamination of mitochondrial preparation with lysosomes, we used acid phosphatase activity as a marker. Measurements of mitochondrial respiration Oxygen consumption was measured polarographically with a Clark electrode in the sample, as described by Aprille M. Hamelin et al. Glycation of glutamate dehydrogenase with aging FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS 5957 and Asimakis [43], in a thermostatically controlled closed 2 mL chamber (30 °C). The rate of oxygen consumption was measured in the presence of 310 nmol of ADP and 10 mm of succinate or 5 mm glutamate ⁄ 5mm malate (state 3) and after all ADP had been consumed (state 4 or resting state). Oxygen consumption rates are expressed as ng atoms of oxygen consumedÆmin )1 Æmg protein )1 . The rate of oxygen consumption in state 3 and in state 4, RCR (the ratio of state 3 to state 4 respiration), an index of electron transport chain activity, and the ADP ⁄ O 2 ratio were calculated. Oxygen consumption in the presence of 40 lm of dinitrophenol (uncoupled state) was also checked. Determination of 6D12-detectable AGE content in mitochondrial matrix proteins by competitive ELISA The ELISA assay was conducted as previously described [24]. Briefly a 96-well microtiter Nunc-immuno plate (Nunc, Roskilde Denmark) was coated with 100 lLof CML-BSA (6.4 nmol CMLÆmL )1 ) by incubation overnight at 4 °C. Wells were washed with NaCl ⁄ P i )0.05% Tween 20 (v ⁄ v) (buffer A) and free binding sites were blocked by incubation for 1 h at room temperature with 100 lL of NaCl ⁄ P i )6% skimmed milk or NaCl ⁄ P i )1% BSA (w ⁄ v). After washing with buffer A, 50 lL of com- peting antigen (test samples at 0.1 lgÆlL )1 or serial dilu- tions of standard CML-BSA from 0.64 mm to 128 mm) was added, followed by 50 lL of monoclonal anti-AGE IgG (clone 6D12) (dilution 1 : 3000). The plate was incu- bated for 2 h at room temperature, washed and then incubated with 50 lL horseradish peroxidase-conjugated anti-(mouse IgG) (dilution 1 : 10 000) for 2 h at room temperature. The wells were washed, then 100 lL of sub- strate solution (40 mm ABTS and 200 lL of 30% hydro- gen peroxide in 20 mL sodium acetate-phosphate buffer, pH 7.2) were added per well and incubated. Absorbance (A) was measured at 405 nm on a micro-ELISA plate reader (Spectra Rainbow, SLT. Labinstruments, Salzburg, Austria). Results are expressed as the ratio B ⁄ Bo (bound ⁄ total), calculated as: experimental A ) background A (no antibody) ⁄ total A (no competitor) ) background A, versus CML added, as pmol CMLÆlg protein )1 . Finally, data were expressed as arbitrary unitsÆlg protein )1 (AUÆlg pro- tein )1 ) because anti-AGE IgG recognizes both CML and CEL. The use of 6% skimmed milk (w ⁄ v) as an alternative to 1% BSA (w ⁄ v) as a blocking agent in the immunochemical assay introduced an increment of less than 10% at the CML level, with GO-modified BSA used as standard (data not shown). We took advantage of these results and used skimmed milk in all further immunochemical assays (ELISA and western blotting). 1D and 2D gel electrophoresis of mitochondrial matrix proteins and western blotting Mitochondrial matrix protein samples (150 lg) from 3- month-old and 27-month-old rats were mixed with 200 l L of 2D sample buffer (7 m urea, 2 m thiourea, 4% Chaps, 1% dithithreitol, 2% Pharmalytes, Amersham Biosciences, Saclay, France; pH 3.0–10.0). The strips were allowed to rehydrate overnight. 1D isoelectric focusing was performed on Immobiline Drystrips (Amersham Biosciences; pH 3.0– 10.0, 13 cm) in a Multiphor II device (Amersham Bio- sciences) for 49 325 Vh. After electrofocusing, immobilines were prepared for SDS ⁄ PAGE and 2D SDS ⁄ PAGE was run vertically on a 12% polyacrylamide gel using the cool- ing Protean II system (Bio-Rad, Marne La-Coquette, France). The gels were either fixed and stained with colloi- dal Coomassie blue for total protein pattern and LC- MS ⁄ MS analysis, or western blotted onto a nitrocellulose membrane (Bio-Rad) overnight at 30 V. The membrane was saturated with NaCl ⁄ P i , pH 7.4, 0.1% Tween 20 (v ⁄ v), 5% skimmed milk (w ⁄ v) overnight at 4 °C, followed by four washes (10 min each) with NaCl ⁄ P i , pH 7.4, 0.2% Tween 20 (washing buffer). The membrane was then incu- bated for 2 h at room temperature with monoclonal anti- AGE IgG clone 6D12 (dilution 1 : 3000) in NaCl ⁄ P i , 0.1% Tween 20 (v ⁄ v), washed four times, incubated for 1 h with anti-(mouse IgG) coupled to horseradish peroxidase (dilu- tion 1 : 3000) and given a final wash. The proteins were revealed with a SuperSignal West Pico chemiluminescent reagent (Perbio Science Company, Brebie ` res, France). Protein identification by LC-MS ⁄ MS Colloidal Coomassie blue-stained spots matching with bands immunolabelled by monoclonal anti-AGE IgG were excised from gel, cut into 1 mm pieces and then treated for LC-MS ⁄ MS analysis. Gel pieces were washed twice in 100 mm ammonium bicarbonate buffer pH 8.8 and then dehydrated with acetonitrile. The gel pieces were rehydrated in 10 mm dithithreitol ⁄ ammonium bicarbonate solution and proteins alkylated with 50 mm iodoacetamide. After dehy- dratation with acetonitrile, gel pieces were rehydrated on ice for 10 min in 20 lLof20mm ammonium bicarbonate containing 50 ngÆlL )1 of sequence-grade modified porcine trypsin (Promega, Madison, WI, USA); then supernatants were replaced by 20 lLof20mm ammonium bicarbonate, and in-gel digestion was performed for 15 h at 37 °C. The resulting peptides were extracted twice with 20 l Lof 20 mm ammonium bicarbonate and then three times in 20 lL of 0.5% trifluoroacetic acid in 50% acetonitrile. The peptide extracts were concentrated to 20 lL using an RC 10.22 evaporator concentrator (Jouan, Saint Herblain, France). Samples were then subjected to mass spectrometry analysis. Glycation of glutamate dehydrogenase with aging M. Hamelin et al. 5958 FEBS Journal 274 (2007) 5949–5961 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... 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(unitsÆmL enzyme)1) and then reported as specific activity (unitsÆmg protein)1) One unit will reduce 1.0 lmol of a-ketoglutarate to l -glutamate per min at pH 7.4 and 25 °C in the presence of ammonium ions All assays were performed in triplicate Kinetic analyses were performed to determine the effect of MGO on purified GDH kinetic parameters Vmax and Km GDH samples (10 lg) were incubated in the absence or presence... contained 90 mm TEA-HCl, pH 7.4, 53 mm ammonium acetate, 60 lm NADH, 250 lm EDTA, at 25 °C according to the Sigma-Aldrich procedure The enzyme reaction was initiated by adding a-ketoglutarate to a final concentration of 13 mm in the absence or presence of allosteric effectors (activators: ADP, leucine; inhibitor: GTP) The activity of the reaction mixture devoid of ammonium ions was taken as blank The. .. 43 nase complexes elucidate the mechanism of purine regulation J Mol Biol 307, 707–720 Ahn JY, Choi S & Cho SW (1999) Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by O-phthalaldehyde Biochimie 81, 1123–1129 Sorbi S, Piacentini S, Fani C, Tonini S, Marini P & Amaducci L (1989) Abnormalities of mitochondrial enzymes in hereditary ataxias Acta Neurol... N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins Biochem J 324, 565–570 Broca C, Brennan L, Petit P, Newsholme P & Maechler P (2003) Mitochondria-derived glutamate at the interplay between branched-chain amino acid and glucose-induced insulin secretion FEBS Lett 545, 167– 172 Corman B & Michel JB (1987) Glomerular filtration, . Glycation damage targets glutamate dehydrogenase in the rat liver mitochondrial matrix during aging Maud Hamelin, Jean Mary, Michal. with aging. Interest- ingly, all these modified proteins are different from those preferentially found to be oxidized during aging in the mitochondrial matrix,