J. Pineal Res. 2008; 45:524–531
2008 The Authors Journal compilation 2008 Blackwell Munksgaard
Doi:10.1111/j.1600-079X.2008.00631.x
Journal of Pineal Research
MT3/QR2 melatonin binding site does not use melatonin as a substrate or a co-substrate Abstract: Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 lm of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 lm).
Jean A. Boutin1, Estelle Marcheteau1, Philippe Hennig2, Natacha Moulharat1, Sylvie Berger1, Philippe Delagrange3, Jean-Paul Bouchet2 and Gilles Ferry1 1 Pharmacologie Mole´culaire et Cellulaire, Institut de Recherches Servier, Croissy-surSeine; 2Physico-Chimie Analytique, Institut de Recherches Servier, Suresnes; 3De´partement des Sciences Expe´rimentales, Institut de Recherches Servier, Suresnes, France
Key words: co-substrate, melatonin, MT3, nuclear magnetic resonance, quinone reductase 2, redox, substrate Address reprint requests to Jean A. Boutin, Pharmacologie mole´culaire et cellulaire, Institut de Recherches Servier, 125, chemin de Ronde, 78290, Croissy-sur-Seine, France. E-mail:
[email protected] Received July 3, 2008; accepted August 19, 2008.
Introduction Melatonin binding sites belong to two family of proteins [1]: the 7TM, G-coupled receptors, MT1 and MT2 that are well characterized at the molecular level in several species, including human [2–4], with the third receptor reportedly being the controversial MT3 binding site initially evidenced in hamster brain [5, 6]. This site has very peculiar characteristics, particularly regarding its fast kinetics of association/dissociation [7]. It also has a different pharmacology profile than MT1 and MT2, particularly in recognizing the distant melatonin analogue 5-methoxycarbonylamino-N-acetyltryptamine (MCA-NAT), N-acetylserotonin and prazosin. This have been reported from several laboratories [7, 8], validating the initial observations. None of these compounds have a strong affinity for MT1/MT2. Using an affinity chromatography based on an analogue of MCA-NAT, the purification of QR2 as MT3 [9, 10] was reported. Although surprising at first glance, a series of studies was conducted to better validate the hypothesis according to which MT3 was QR2. A large amount of pharmacological data for QR2/MT3 has been 524
described [11]. QR2)/) mice were constructed. These animals did not present any MT3-like binding site [12] further validating the original hypothesis according to which QR2 was indeed a low affinity binding site for melatonin. Several chemical programmes were then launched, leading to the discovery of several classes of potent inhibitors [13–17]. Bacterial recombinant human QR2 was purified and co-crystallized with melatonin [18], MCA-NAT and prasozin [S.D. Pegan, G. Ferry, J.A. Boutin, A.D. Mesacar, unpublished data], as well as with other inhibitors of QR2. Melatonin is a 1 lm inhibitor of quinone reductase 2 activity, but not of quinone reductase 1 [11, 18]. Several lines of evidences differentiated QR2 from the closely related protein QR1. Indeed, if QR1 is a detoxification protein of drug metabolism phase II [19], QR2 is less, contrarily to what has been generally admitted a detoxification enzyme, since its presence clearly enhances the toxicity of quinones, as reported by Long et al. [20] and confirmed in our laboratory [P. Delagrange and J.A. Boutin, unpublished data] in vivo, but also in cellulo as shown by Buryanoskyy et al. [21] and confirmed on another cell line by us [22]. Furthermore, several observations
Melatonin is not a co-substrate of QR2/MT3 linked the presence of an over-expressed QR2 to neurogenerative diseases [23–26]. It is obvious therefore, that more information is required on the relationship between potential enhanced reactive oxygen species production capacity of QR2, degenerative diseases and melatonin, considering the use of melatonin at high concentrations, often have been reported to delay such degenerative processes [27–29]. It came as an interesting hypothesis [30] that melatonin itself might serve as a substrate or a co-substrate of QR2. The present work aims at clarifying this particular subject. By use of direct observation of QR2 activity using nuclear magnetic reso-
nance (NMR), we show that melatonin remains unchanged during the course of the QR2 enzyme catalytic activity and that the substitution of either the substrate or the co-substrate of the enzyme by melatonin did not lead to any measurable catalytic activity.
Materials and methods Biological source of quinone reductase 2 The 6His-hQR2 cDNA was obtained by PCR amplification from pcDNA3.1(+)/hQR2 plasmid [9] using forward
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Fig. 1. Nuclear magnetic resonance spectra of the three main compounds: melatonin, AFMK and AMK. These spectra were obtained with the pure compounds in solution of deuterated dimethylsulfoxide in deuterated water.
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Boutin et al. primer (5¢-gattccaccatgcatcaccatcaccatcacgcaggttaagaaag tactc-3¢) and reverse primer (3¢-ggtgaccgtgaagcccgttattg tagac-5¢) to adduct six histidine residues between first and second amino acids of hQR2 and a BglII site in 3¢-end of cDNA. The amplified 6His-hQR2 cDNA was then cloned into EcoRI and BglII restriction endonuclease sites of pVL1393 vector (BD Biosciences-Pharmingen, San Diego, CA, USA). The construct was controlled by sequencing. The Sf9 Spodoptera frugiperda ovarian cell line (ATCC CRL-1711) was maintained on Grace medium (Invitrogen, Carlsbad, NM, USA) supplemented with 10% foetal calf serum, 50 lg/mL streptomycin sulphate and 50 units/mL penicillin. pVL1393/6His-hQR2 plasmid (2 lg) was co-transfected with 0.5 lg of BaculoGold AcNPV DNA into 2 106 Sf9 insect cells following the recommendations of the supplier (BD Biosciences-Pharmingen). Eight days after transfection, the medium was centrifuged and the recombinant baculovirus contained in the supernatant was measured by real-time PCR. Recombinant virus in the supernatant was then amplified by infecting Sf9 cells to obtain a large stock of virus with a titre of 3 · 108 virus/ mL. For the 6His-hQR2 production, 7.5 · 108 Sf9 cells in grace medium were infected in spinner flasks at a multiplicity of infection (MOI) of two and cells culture were harvested 72 hr after the initial infection by centrifuging at 1000 · g for 5 min. The pelleted cells were stored at )80C.
The pellet were suspended in 150 mL of a buffer (50 mm Tris-HCl, pH 8.5, with 1 mm of n-octyl-b-d-glucopyranoside) in the presence of anti-protease, EDTA-free, cocktail (Roche, Basel, Switzerland), vigorously mixed at 4C and centrifuged at 100,000 · g, 1 hr, at 4C. The process was repeated twice. The supernatants were combined and applied at 1 mL/min onto a 1.5 mL Ni-NTA chromatography column, previously equilibrated in the extraction buffer supplemented with 10 mm imidazole. The column was washed with 10 volumes of the same buffer. The protein was eluted with stepwise with increasing concentrations of imidazole in the same buffer, 50, 100, 200, 300 and 500 mm. The fractions with QR2 activity were pooled and dialysed overnight against the activity buffer (50 mm Tris-HCl, pH 8.5, with 1 mm of n-octyl-b-d-glucopyranoside). The enzyme preparation was then aliquoted and snap frozen at )80C until further use. NMR studies Nuclear magnetic resonance was used to follow QR2 activity. All kinetic studies followed by NMR spectroscopy were performed on a Bruker Avance 500 MHz spectrometer (Bruker Instruments, Wissembourg, France) equipped with a triple axes gradient 5 mm BBI probe. The experiments were conducted in a medium containing,
Fig. 2. Nuclear magnetic resonance spectroscopy analysis of the quinone reductase 2 reaction in the presence of a complete system as a function of incubation time. The experiments were conducted as described in experimental procedures. The enzymatic reaction, in deuterated buffer, comprised the co-substrate, BNAH, the enzyme and the substrate, menadione. The exemplified spectra were recorded 5 min after the beginning of the reaction (bottom) and 45 min (top).
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Melatonin is not a co-substrate of QR2/MT3 appearance of new signals, concomitant with, for instance, the cleavage of the indole moiety of melatonin. This particular catabolism route has previously been studied in our laboratory catalysed by indoleamine 2,3-dioxygenase and myeloperoxidase [31]. NMR is a noninvasive technique that permits the catalytic activity of an enzyme at the proton level to be observed. This approach was successfully applied in studying the characteristics of arylalkylamine N-methyl transferase activity using new substrates of this enzyme involved in melatonin synthesis [32]. First, the various spectra from melatonin, AFMK and AMK were acquired (Fig. 1A–C respectively). These spectra clearly show the various protons that could be followed in order to see the appearance of AFMK/AMK in the incubation media. Second, two sets of experiments were run, in which the co-substrates of the reactions were either BNAH or NMH, as depicted from Figs 2 and 3. The spectra taken at the very beginning of the experiments and after 45 min incubation clearly show the changes in the depicted protons (see figures for details). Intermediary spectra were acquired every 3 min, leading to the possibility to draw the kinetic progress of the reaction. BNAH disappearance and BNA appearance were clearly seen (Fig. 4A,B). A strange feature, though, was the constancy of the amount of menadione during the experiment (Fig. 4C). Although repeated under several experimental conditions, this confirmed the former
substrate and co-substrate, [100 lm in 5% (v/v) d6dimethylsulphoxide final volume, in 50 mm Tris-HCl, pH = 8.5 with 1 mm of n-octyl-b-d-glucopyranoside (and 8 ng of enzyme)] as described in the Results and discussion section. Monitoring of the enzymatic reaction was carried out by recording a series of 1D 1H NMR spectra (the temperature of the sample was continuously set to 300 K, 32 scans per spectra), using 32 K data points. The data were then Fourier-transformed, using a line broadening exponential treatment (lb = 1.5 Hz). All spectra were phased and the baseline was corrected before integration of the signals of interest. For the integration a specific signal for each species has been selected [5.75 ppm for N-benzylnicotinamide (BNA); 5.80 ppm for N-benzyldihydronicotinamide (BNAH); 7.05 ppm for melatonin; 7.70 ppm for menadione; 5.70 for N-methyldihydronicotinamide (NMH); 8.75 ppm for N-methylnicotinamide].
Results and discussion Using a highly purified source of recombinant human QR2, the hypothesis according to which QR2 might cleave off melatonin into N1-acetyl-N2-formyl-5-methoxykynurenine/ N1-acetyl-5-methoxykynurenine (AMFK/AMK) [30] was experimentally tested. In order to do so, NMR spectroscopy was preferred as this methodology can easily detect the
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Fig. 3. Nuclear magnetic resonance spectroscopy analysis of the quinone reductase 2 reaction in the presence of a complete system as a function of incubation time. The experiments were conducted as described in Materials and methods. The enzymatic reaction, in deuterated buffer, comprised a co-substrate, N-methyldihydronicotinamide, the enzyme and the substrate, menadione. The exemplified spectra were recorded 5 min after the beginning of the reaction (bottom) and 45 min (top).
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Boutin et al. results [33], where no menadione consumption were measured during the course of the experiment, as detected by high performance liquid chromatography (HPLC) and absorbance detection. Nevertheless, controls experiments were run then as now, clearly showing that in the absence of menadione (i.e. substrate), no enzymatic activity was recorded, either by fluorescence detection, by HPLC or by NMR, strongly suggesting, as seen in more complex experimental contexts [34], a re-cycling of menadiol, back to menadione, in a time-frame less than a millisecond. This demonstrated that, under the incubation conditions used, the system regenerates the quinone. The same observation was done when NMH, a suspected natural substrate of quinone reductase 2 was used instead of BNAH. In a next step, the full system was incubated with melatonin, as substrate (with BNAH or NMH as
co-substrates) or as a co-substrate (with menadione as substrate). Figs 5–7 show the results obtained. In all conditions, no changes were observed after 20 min incubation. Indeed, not only there were no quantitative changes in the spectra recorded for melatonin, but also there were no proton signals fitting the AFMK spectrum, as recorded in Fig. 1. These experiments demonstrated that melatonin does not serve as a substrate or a co-substrate. These features have been anticipated very early in our discovery of the oneness of the melatonin binding site MT3 and the enzyme quinone reductase 2 [9, 11, 33]. Furthermore, the position of melatonin into the QR2 crystals clearly showed the relationship of the hormone with the enzyme [18]. It was, nevertheless, a fascinating hypothesis, as the relationship between melatonin and QR2 raised two main questions: (a) Is melatonin a natural modifier (inhibitor, IC50
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Fig. 4. Kinetic representation of the nuclear magnetic resonance spectroscopy analyses. The proton b¢ peak height was measured and plotted versus time, during the complete course of the experiment, corresponding to about 20 spectra, i.e. one spectrum every 150 s.
Melatonin is not a co-substrate of QR2/MT3
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Fig. 5. Nuclear magnetic resonance spectroscopy analysis of the quinone reductase 2 reaction in the presence of a partial system as a function of incubation time. The experiments were conducted as described in Materials and methods. The enzymatic reaction, in deuterated buffer, comprised a co-substrate, N-benzyldihydronicotinamide, the enzyme and the putative substrate, melatonin. The exemplified spectra were recorded 5 min after the beginning of the reaction (bottom) and 45 min (top).
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Fig. 6. Nuclear magnetic resonance spectroscopy analysis of the quinone reductase 2 reaction in the presence of a partial system as a function of incubation time. The experiments were conducted as described in Materials and methods. The enzymatic reaction, in deuterated buffer, comprised another co-substrate, N-methyldihydronicotinamide, the enzyme and the putative substrate, melatonin. The exemplified spectra were recorded 5 min after the beginning of the reaction (bottom) and 45 min (top).
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Fig. 7. Nuclear magnetic resonance spectroscopy analysis of the quinone reductase 2 reaction in the presence of a partial system as a function of incubation time. The experiments were conducted as described in Materials and methods. The enzymatic reaction, in deuterated buffer, comprised a substrate, menadione, the enzyme and the putative co-substrate, melatonin. The exemplified spectra were recorded 5 min after the beginning of the reaction (bottom) and 45 min (top).
1 lm depending on the nature of the substrate/ co-substrate pair used in the catalytic activity measurement [11, 18]) or a pharmacological modifier of this activity? Indeed, the natural level of circulating hormone, reaches in the best case (night-time) 50–150 pg/mL, a feature that does not seem to fit the capacity of melatonin to inhibit the enzyme activity in an acellular assay. During ingestion of melatonin, though, such a level could be reached in vivo, as melatonin freely travels through cellular membranes and its ingestion seems to lead to urine excretion of the unchanged compound [35, 36]. This strongly suggests that at least part of melatonin remains unchanged in mammalian body. (b) If melatonin has some effects in vivo, through this particular enzyme, how does this inhibition translate in terms of cell biology? We have hypothesized, based on our own data and on data from others [20, 37] that QR2, contrary to common belief, is an activating enzyme. Indeed, its deletion from living systems (animals or cells) leads to a reduced toxicity of quinones [20–22], as exemplified by menadione, strongly suggesting that the reduction of quinone produces radical oxygen species with high deleterious effects. Further exemplification and analyses of this phenomenon is currently studied in vitro in our laboratory (K. Reybier, G. Ferry, J.A. Boutin, F. Nepveu, unpublished). Another feature remains to be understood. QR2 does not accept classical hydrure donors as co-substrates such as NAD(P)H [38]. It is believed, so far, that its natural co-substrates are N-ribosyldihydronicotinamide (NRH) 530
and NMH. These compounds can be produced during the catabolism of NADH [39] or during the biosynthesis of NADH, through a step catalysed by nicotinamide phosphoribosyltransferase [40]. Nevertheless, at the moment, it is not known how much of NMH or NRH are present in a cell under basal (i.e. nonstressed) conditions. In conclusion, the present work provides evidence according to which melatonin is not a substrate or a co-substrate of QR2. It also shows that under these experimental conditions, the quinone is recycled, generating constantly a fresh substrate for the enzyme until the consumption of the co-substrate is complete.
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