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Communications The Cyclopentenone Product of Lipid Peroxidation, 15-A2t-Isoprostane (8-Isoprostaglandin A2), Is Efficiently Conjugated with Glutathione by Human and Rat Glutathione Transferase A4-4 Ina Hubatsch,† Bengt Mannervik,† Ling Gao,‡ L. Jackson Roberts,‡ Yan Chen,‡ and Jason D. Morrow*,‡ Department of Biochemistry, Uppsala University, SE-75123 Uppsala, Sweden, and Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received April 8, 2002
Glutathione transferases (GSTs) are a large family of enzymes that can be divided into different classes based on structure. There has been considerable interest in the ability of GSTs to conjugate and inactivate endogenously derived reactive lipid peroxidation products that contain R,β-unsaturated carbonyl moieties such as 4-hydroxyalkenals. One enzyme with prominent activity toward these substrates is human GST A4-4. Recently, we described a novel series of compounds termed A2/J2-isoprostanes (IsoPs) that are formed endogenously in humans from the free radical-initiated peroxidation of arachidonic acid. These compounds contain R,βunsaturated carbonyl groups and have structures similar to cyclooxygenase-derived PGA2 and PGJ2. Because of their chemical reactivity, these compounds may mediate tissue injury associated with oxidant stress. Herein, we report that the A-ring IsoP 15-A2t-IsoP (8-iso-PGA2) is efficiently conjugated to glutathione (GSH) by human GST A4-4 with a kcat/Km value of >200 s-1 mM-1. The kcat/Km value for conjugation of 15-A2t-IsoP by the homologous rat GST A4-4 is >2000 s-1 mM-1. Similar high enzyme activities were observed when PGA2 was used as a substrate. In contrast, the human GSTs A1-1, M1-1, M2-2, P1-1, and T1-1 and rat GST T2-2 did not significantly metabolize 15-A2t-IsoP. These studies have therefore defined a potentially important route by which cyclopentenone IsoPs are metabolized that may serve as a mechanism for the inactivation of these highly reactive compounds.
Introduction (GSTs)1
Glutathione transferases comprise a large and widespread enzyme family whose soluble members in mammalian tissues are currently grouped into eight classes (alpha, kappa, mu, pi, sigma, theta, zeta, and omega) based on similarities in structure (1, 2). The substrate range of GSTs is broad and includes a number of xenobiotics and environmental pollutants such as crotonaldehyde, arene and alkyl epoxides, and various dihaloalkanes (1, 2). Generally, the GSTs are regarded as important components of cellular defense, catalyzing detoxication reactions through conjugation of electrophilic compounds with glutathione (GSH) (3). More recently, there has been considerable interest regarding the ability of GSTs to conjugate various en* Correspondence should be addressed to this author at the Departments of Pharmacology and Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6602. Phone: 615/343-1124, Fax: 615/322-3669, Email:
[email protected]. † Uppsala University. ‡ Vanderbilt University School of Medicine. 1Abbreviations: GST, glutathione transferase; GSH, glutathione; PG, prostaglandin; IsoP, isoprostane; LC, liquid chromatography; MS, mass spectrometry; ESI electrospray ionization.
dogenously derived products of intracellular metabolism that contain R,β-unsaturated carbonyl moieties including base propenals and 4-hydroxyalkenals (1, 4, 5). Hydroxyalkenals such as 4-hydroxynonenal are generated as a consequence of the peroxidation of polyunsaturated fatty acids (6). In particular, GST A4-4 displays high catalytic efficiency in the conjugation of 4-hydroxyalkenals to GSH (5, 7). It has also been reported that GSTs conjugate prostaglandins (PGs) that contain R,β-unsaturated carbonyls such as PGA2 and PGJ2 (8, 9), although it remains to be determined definitively whether these eicosanoids are formed in vivo (10, 11). Previously, we described a group of novel products resulting from the free radical-initiated peroxidation of arachidonic acid termed the isoprostanes (IsoPs) that are formed in vivo in humans (12). Different classes of IsoPs have been characterized. Those with an F-type prostane ring (F2-IsoPs) were the first reported, and since that time we have also determined that IsoPs with D-type and E-type prostane rings are formed in vivo (13). More recently, we have also found that D/E-IsoPs are rapidly metabolized with the loss of water to form J-ring and A-ring IsoPs (J2-IsoPs and A2-IsoPs, respectively) (11). One A2-IsoP generated in abundance in vivo is 15-A2t-
10.1021/tx020027r CCC: $22.00 © 2002 American Chemical Society Published on Web 08/28/2002
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IsoP (8-iso-PGA2) (14). We have shown this molecule to be highly reactive, and thus it may be responsible for adverse cellular effects associated with oxidative injury owing to its reactivity. 15-A2t-IsoP readily forms an adduct with GSH via Michael addition when incubated in vitro with a mixture of GSTs derived from bovine liver (Figure 1A) (11). However, the specific GST(s) responsible for the conjugation was (were) not determined in these studies. Because 15-A2t-IsoP is an R,β-unsaturated carbonyl compound, like the 4-hydroxyalkenals, we examined the catalytic activity of human GST A4-4 in comparison with other GSTs to conjugate this eicosanoid. We report that 15-A2t-IsoP is, in fact, an excellent substrate for human GSTA4-4.
Kinetic Measurements. Enzymatic activities were measured at 30 °C in 0.1 M sodium phosphate, pH 6.5, in the presence of 0.5 mM GSH and 5% ethanol (used as a solvent for the electrophiles). The reactions were monitored spectrophotometrically by following the disappearance of the electrophile used. Measurement of 15-A2t-IsoP was made at 220 nm, and reaction rates were calculated using the determined extinction coefficient 220 ) 12 mM-1 cm-1; for PGA2, the wavelength 221 nm and 221 ) 9.8 mM-1 cm-1 were used (11). In steady-state kinetic experiments, the electrophilic substrate concentration was varied across the range 1.5-100 µM for 15-A2t-IsoP and 1.5-150 µM for PGA2. Specific activities of the enzymes were determined with 0.1 mM electrophile. The steady-state kinetic parameters were obtained by nonlinear regression analysis using the GraphPadPrism program (GraphPad Software Inc., San Diego, CA). Preparation of GSH Conjugates of 15-A2t-IsoP and PGA2 for Mass Spectrometric Analysis. Two micrograms of either 15-A2t-IsoP or PGA2 was incubated with 20 µg of GSH in the presence of 10 units of mixed GSH transferases from bovine liver (Sigma, St. Louis, MO) for 1 h as described (11). The conjugate was purified by Sep-Pak extraction (Waters Associates, Milford, MA) and analyzed by liquid chromatography (LC)/ tandem mass spectrometry (MS). Briefly, LC was carried out utilizing a MAGIC 2002 LC system (Michrom BioResources, Auburn, CA) operating in an isocratic mode with the mobile phase of H2O/acetonitrile/acetic acid (77:22.9:0.1, v/v/v), and compounds were separated on an Eclipse XDB-C18 column (2.1 mm × 50 mm, 5 µm particle size; Aligent, Palo Alto, CA) at a flow rate of 75 µL/min. Following on-line chromatography, samples were characterized employing a Finnigan TSQ-7000 (San Jose, CA) triple quadrupole mass spectrometer operating in the positive-ion mode. An electrospray (ESI) source was fitted with a 100 µm internal diameter deactivated fused silica capillary and used nitrogen for both sheath and auxiliary gas, operating at 60 psi and 10 L/min, respectively. The ESI potential was maintained at 3.5 kV, the heated capillary at 20 V and 200 °C, and the tube lens at 70 V. For tandem MS, parent compounds were collisionally activated at an energy of -20 eV and under 2.5 mT argon. Data acquisition and analysis were performed using an Alpha workstation (Digital Equipment Corp., Maynard, MA) running Finnigan ICIS software, version 8.3.2.
Experimental Procedures
Results
Preparation of 15-A2t-IsoP. 15-A2t-IsoP was prepared as described by incubating 15-E2t-IsoP in the presence of 0.1 M HCl for 12 h at 37 °C (14). It was then purified by normal phase HPLC, and the structure was confirmed by NMR and mass spectrometry. PGA2 was purchased from Cayman Chemical Co. (Ann Arbor MI) or from ICN (Costa Mesa CA). Preparation of Human and Rat GSTs. Recombinant GST A4-4 was heterologously expressed and purified as reported (5) except that the elution of enzyme from the GSH-Sepharose gel was effected by 50 mM glycine buffer, pH 10. After adjustment of the pH to 7.8, the eluate was concentrated and subsequently run over a PD-10 gel filtration column equilibrated with 10 mM Tris/HCl, pH 7.8. Other recombinant GSTs (human A1-1, M11, M2-2, P1-1, and T1-1 and rat A4-4 and T2-2) used in this study were kindly provided by members of the Uppsala laboratory, and the enzymes were prepared according to published methods (15-22). A GST A1-1 mutant “GIMF helix” was prepared as described (23). Enzyme preparations were tested for activity by measurement with 1-chloro-2,4-dinitrobenzene as described except that GST T1-1 was tested using 1,2-epoxy3-(4-nitrophenoxy)propane (19, 22). Generally the enzymes showed 80-100% activitity with the standard substrate 1-chloro2,4-dinitrobenzene [or 1,2-epoxy-3-(4-nitrophenoxy)propane in the case of GST T1-1] with the exception of GST M1-1 (61% active) and GST T2-2 (55% active). Enzyme concentrations in studies were calculated as the amount of enzyme active with the standard substrates.
Figures 1A and 1B show the structures of the predicted GSH conjugation products for 15-A2t-IsoP and its cyclooxygenase-derived stereoisomer, PGA2. Figures 2A and 2B provide definitive evidence employing LC/MS/MS that 15-A2t-IsoP and PGA2 conjugate GSH in the presence of a mixture of GSH tansferases. Figures 2A and 2B represent the collision-induced dissociation analysis of GSH conjugates of 15-A2t-IsoP and PGA2, respectively. The parent ion for each conjugate is present at m/z 642. Relevant daughter ions include m/z 624 [M-H2O]+, m/z 317 [M-GSH-H2O] (the eicosanoid portion of each molecule), and m/z 308, which represents either [M-15A2t-IsoP] (Figure 2A) or [M-PGA2] (Figure 2B). 15-A2t-IsoP and its cycloxygenase-derived stereoisomer PGA2 were then tested as substrates for different soluble glutathione transferases belonging to classes alpha (A), mu (M), pi (P), and theta (T). Among the human GSTs, the highest conjugation rate for 15-A2t-IsoP was exhibited by GST A4-4; the rat homologue, rat GST A4-4, was even more efficient (Table 1). Similar results were obtained when PGA2 was used as a substrate. The rates of conjugation of 15-A2t-IsoP by these enzymes are extremely high although somewhat lower than those for 4-hydroxynonenal (5). 15-A2t-IsoP was not a substrate for
Figure 1. Structures of 15-A2t-IsoP (A) and PGA2 (B) and their respective GSH conjugation products.
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Table 1. Specific Activities of Recombinant GSTs with 15-A2t-IsoP and PGA2 as Electrophilic Substratesa specific activity (µmol min-1 mg-1) 15-A2t-IsoP human GST A4-4 rat GST A4-4 human GST A1-1 GST A1-1 mutant “GIMF helix” human GST M1-1 human GST M2-2 human GST P1-1 (Val105) human GST P1-1 (Ile105) human GST T1-1 rat GST T2-2
27 ( 3 112 ( 15
