Lysophosphatidylserine-induced functional switch of human cytochrome P450 1A2 and 2E1 from monooxygenase to phospholipase D

Biochemical and Biophysical Research Communications 376 (2008) 584–589

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Lysophosphatidylserine-induced functional switch of human cytochrome P450 1A2 and 2E1 from monooxygenase to phospholipase D Eun Yi Cho a, Chul-Ho Yun a, Ho-Zoon Chae a, Han-Jung Chae b, Taeho Ahn c,* a

School of Biological Sciences and Technology and Hormone Research Center, Chonnam National University Gwangju 500-757, Republic of Korea Department of Pharmacology and Institute of Cardiovascular Research, School of Medicine, Chonbuk National University, Jeonju, Chonbuk 561-181, Republic of Korea c College of Veterinary Medicine, Department of Biochemistry, Chonnam National University, Buk-ku, Yong-bong 300, Gwangju 500-757, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 27 August 2008 Available online 16 September 2008

Keywords: Cytochrome P450 Lysophosphatidylserine Phospholipase D

a b s t r a c t Interaction of human cytochrome P450 1A2 (CYP1A2) and 2E1 (CYP2E1) with phospholipid, lysophosphatidylserine (LysoPS) in the context of a PC matrix specifically stimulated the PLD activity of both enzymes in a LysoPS concentration-dependent manner. However, other anionic lysophospholipids as well as the neutral lysophospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine, had no effect. LysoPS also accompanied conformational changes in both CYPs when assayed by circular dichroism. Although the PLD activity was decreased in the presence of components required for the monooxygenase (MMO) activity, including 100% PC membranes, NADPH-cytochrome P450 reductase and NADPH, as compared to the activity in the absence of the reducing system, LysoPS recovered the PLD activity in a concentration-dependent manner. Considering that LysoPS induced a decrease in the MMO activities of both CYPs, the results suggest that the functional roles of CYP1A2 and 2E1 can be switched by interaction with a specific anionic lysophospholipid in vivo. Ó 2008 Elsevier Inc. All rights reserved.

Phospholipids regulate the enzymatic activities of cytochrome P450s (CYPs) and provide a matrix for the incorporation of the proteins into the membrane bilayer [1]. For example, phospholipids in the immediate vicinity of CYPs in liver microsomes have been reported to be highly organized [2] and specific phospholipids such as PC are essential components of a reconstituted CYP monooxygenase (MMO) system measuring fatty acid and drug hydroxylation [3]. Similarly, it has also been proposed that the interaction of CYPs with phospholipids might be necessary for maintaining an active conformation and for efficient electron transfer [4]. Lysophosphatidylserine (LysoPS), generated by a serine phospholipid-selective phospholipase in activated platelets [5], has been known to regulate many biological functions, for example, by inducing a transient increase in intracellular calcium in cancer cell lines [6] and stimulating the production of interleukin-2 in Jurkat T cells [7]. It has also been suggested that LysoPS as well as other lysophospholipids regulates the calcium transport in the sarcoplasmic reticulum of skeletal and cardiac muscle cells [8]. However, since there have been a limited number of studies investigating the LysoPS-induced modulation of biological func-

tion, its precise role in various cellular processes has remained unclear. Cytochrome P450 1A2 (CYP1A2), located predominantly in the liver, participates in the metabolism of a variety of compounds, including the activation of potentially carcinogenic aryl and heterocyclic amines [9]. Cytochrome P450 2E1 (CYP2E1) is a classical ethanol-inducible enzyme that metabolizes various endogenous compounds such as lipid hydroperoxides, ketone bodies, and acetone [10] in addition to xenobiotics such as ethanol and chlorzoxazone [11]. It has been shown that both CYPs are primarily localized to the endoplasmic reticulum of cells. It has been previously reported that CYP1A2 and 2E1 each have PC-specific phospholipase D (PLD) activity, hydrolyzing PC but not other phospholipids, and they act as the major source of PLD activity in human liver microsomes [12]. However, the molecular characteristics and functional regulation of the PLD activities of these CYPs are unknown. Here, we expand the previous results in more detail and show that LysoPS incorporated into membranes specifically stimulates the PLD activities of the CYPs.

Materials and methods Abbreviations: E. coli, Escherichia coli; PLD, phospholipase D; CYP1A2, cytochrome P450 1A2; CYP2E1, cytochrome P450 2E1; NPR, NADPH-dependent cytochrome P450 reductase; LysoPS, lysophosphatidylserine * Corresponding author. Fax: +82 62 530 2809. E-mail address: [email protected] (T. Ahn). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.023

Materials. All phospholipids and lysophospholipids were obtained from Avanti Polar Lipids (Alabaster, AL). The radioactive reagent, 1-palmitoyl-[2-palmitoyl-9,10-3H]phosphatidylcholine, was

E.Y. Cho et al. / Biochemical and Biophysical Research Communications 376 (2008) 584–589

purchased from DuPont NEN (Boston, MA). Silica Gel 60 plates were purchased from Merck (Darmstadt, Germany). Preparation of lipid vesicles. Phospholipid membranes as large unilamellar vesicles (LUV) with a diameter of 100 nm were prepared as described [13]. To prepare liposomes containing a binary mixture of PC/lysophospholipids, LysoPS, lysophosphatidic acid (LysoPA), lysophosphatidylinositol (LysoPI), lysophosphatidylcholine (LysoPC) or lysophosphatidylethanolamine (LysoPE) was included at concentrations of up to 3 mol.% at the expense of PC. The concentrations of non-radioactive phospholipids were determined by a phosphorus assay [14]. Expression and purification of recombinant human CYP1A2, CYP2E1, and rat NPR. Recombinant human CYP1A2 containing C-terminal (His)5 and CYP2E1 were expressed in Escherichia coli (E. coli) and purified using Ni2+-affinity and ion-exchange chromatography, respectively, as described in an earlier report [15,16]. Recombinant rat NADPH-cytochrome P450 reductase (NPR) was expressed in E. coli and purified as described previously [17,18]. The catalytic MMO activities of CYP1A2 and CYP2E1 were determined by monitoring the 7-ethoxyresorufin O-deethylation and chlorzoxazone 6b-hydroxylation, respectively [15,16]. The concentrations of CYPs were determined by Fe2+-CO vs. Fe2+ difference spectroscopy [19]. Assay of PLD activity. PLD activity was measured as described previously with slight modifications. Briefly, purified recombinant CYPs (50 pmol) were added to lipid substrate (100% PC or a binary mixture of PC/anionic phospholipids, with a lipid/protein ratio of 800) in the form of LUV containing radioisotope-labeled phospholipids (1-palmitoyl-[2-palmitoyl-9,10-3H]PC, 0.25 lCi). The assay was carried out in a reaction volume of 0.4 ml for 20 min at 37 °C and stopped by adding 2 volumes of CHCl3/MeOH (2:1). After brief centrifugation, the PLD reaction products were separated on a reverse-phase silica TLC plate and identified by autoradiography as described [12]. The radioactive spots were then scraped, and radioactivity was measured by scintillation counting (Beckman counter) in 5 ml of Ecolume (ICN). The assay was linear over time for up to 1 h. In order to examine the effect of lysophospholipids on the PLD activities of the CYPs, 100% 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) was used as a standard matrix phospholipid. The reaction was also repeated under the same conditions

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for the measurement of CYP catalytic MMO activity. Reactions mixtures consisted of 0.4 lM CYP, 0.8 lM NPR, and 320 lM POPC (or a binary mixture of PC/lysophospholipids) in the presence of each specific substrate (100 lM) in 100 mM potassium phosphate buffer (pH 7.4). Reactions were initiated at 37 °C by the addition of an NADPH-generating system (0.5 mM NADP+, 10 mM glucose 6phosphate, and 1.0 IU glucose 6-phosphate dehydrogenase ml 1) as described previously [20]. Circular dichroism (CD) spectroscopy and fluorescence measurements. CD spectra in the far-ultraviolet range were monitored at 30 °C with a Jasco J-715 spectropolarimeter (Japan Spectroscopic, Tokyo). The optical path length was 0.1 cm and measurements were conducted in 100 mM potassium phosphate (pH 7.4) containing 1 lM CYP and 800 lM liposomes on the basis of phosphate concentration. Blanks (buffer with or without phospholipid) were routinely recorded and subtracted from the original spectra. On average, data from 20 scans were accumulated. Statistical analysis. Data were analyzed by analysis of variance (ANOVA) in the dose-response experiments, as well as by twotailed Student’s t-tests. A p value < 0.05 was considered significant. In each case, the statistical test used is indicated, and the number of experiments is stated individually in the legend of each figure. Results and discussion Effect of lysophospholipids on the PLD activity of CYPs In this work, the previous findings regarding the PLD activity of cytochrome P450 enzymes were expanded using recombinant human CYP1A2 and 2E1, which were shown to have the most significant PLD activities of those previously tested [12], by changing the phospholipid composition of the PC substrate membranes. In order to investigate the effect of lysophospholipids, which have been shown to regulate many biological functions in cells, on the PLD activities of CYP1A2 and CYP2E1 in membranes, a fraction of PC substrate was replaced with each of the anionic lysophospholipids, LysoPA, LysoPI, or LysoPS, and the resulting PLD activities were measured. Fig. 1 shows that LysoPS stimulated PLD activity in a concentration-dependent manner and approxi-

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Fig. 1. Effect of lysophospholipids on the PLD activities of CYP1A2 and 2E1. PLD activities were measured with increasing concentrations of lysophospholipids at the expense of PC in the membrane. The activity in the presence of 100 mol.% PC membranes was set to 100% in the y-axis as a control. Each number represents the concentrations of lysophospholipids in lipid substrate. Values are means ± SD of three experiments.

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mately fivefold and eightfold increases in CYP1A2 and CYP2E1 activities, respectively, were shown in the presence of 2 mol.% of the LysoPS when compared to a 100% PC matrix. In contrast, further incorporation of LysoPS (3 mol.%) into the membranes led to a slight decrease in the PLD activities of both CYPs. This decrease in the activity may be explained by a reduction in the concentration of PC as a substrate for the CYPs. In contrast, other anionic lysophospholipids such as LysoPA, and LysoPI as well as LysoPC and LysoPE, neutral lysophospholipids, did not enhance CYP1A2 and 2E1 PLD activities. Therefore, these results suggest that the specific lysophospholipid, LysoPS, but not negative charge in the anionic lipid per se or membrane properties induced by lysophospholipids such as non-bilayer formation [21] stimulates the PLD activities of the CYPs. The result also implies that specific interactions between LysoPS and the CYPs are present

and important for the activity increase. The PLD activities were also measured with sphingosine 1-phosphate and anionic phospholipids such as PG, PS, and PI incorporated into the membrane matrix at concentration of up to 3 mol.% instead of lysophospholipid type. However, they did not confer any enhancement of PLD activity and rather, there was a decrease in the activity levels of CYP1A2 and 2E1 with increasing concentrations of these lipids (results not shown), which may have resulted from decreasing the PC concentration as a PLD substrate. This result, therefore, implies a specific role of LysoPS in the regulation of the PLD activity. Effect of experimental conditions for MMO on the PLD activity In order to mimic the in vivo states, PLD activity levels were measured under the conditions for determining the catalytic

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Fig. 2. Effect of reducing system on the relative PLD activities of CYP1A2 and 2E1. The PLD assay was carried out under the conditions for the measurement of the MMO catalytic activities as described in ‘Materials and methods’ at a NPR/CYP ratio of 2 (A). Each number represents the mol.% of the indicated anionic lysophospholipid incorporated into the membranes at the expense of PC. In figure (B), the increase in the PLD activity of both CYPs was plotted against decreasing ratios of NPR/CYP in the presence of 2 mol.% LysoPS. Each number represents the ratio of NPR/CYP. In both figures, the control represents the PLD activity of CYP in the absence of the reducing system.

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MMO activities of CYPs. The reaction samples contained 100% PC membranes, NPR (the ratio of NPR/CYP was two), a NADPH-generating system and a substrate, 7-ethoxyresorufin or chlorzoxazone. As shown in Fig. 2A, the PLD activities of both CYPs were significantly reduced by approximately 70–80% compared to the activity levels in the absence of the reducing system. This result suggests that the reducing system and/or the experimental conditions for measuring MMO activity in the absence of LysoPS inhibit the PLD activity although the molecular mechanisms are currently unknown. However, the incorporation of LysoPS at the expense of PC recovered the PLD activity in a concentration-dependent manner. In order to confirm the inhibitory effect of the reducing conditions, when the assays were performed using decreased ratios of

NPR/CYP (actually, the concentration of CYP was fixed) in the presence of 2 mol.% LysoPS in the lipid substrate, the level of PLD activity increased (Fig. 2B). In cells CYP and NPR interact through lateral diffusion to form a functional complex for electron transfer [22]. Also, CYP is present in membranes in high molar excess relative to the reductase, the limiting component in microsomes, with molar ratios ranging from 10:1 to 25:1 depending on treatment with inducers [23,24]. Therefore, taken together, these results suggest that LysoPS can induce a molecular functional switch of both CYPs from MMO to PLD. As a control experiment, the assay was performed in the presence of MMO substrate only without the reducing system and under this condition the PLD activity was not influenced, which implies that

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[ANF or 4-MP], μM Fig. 3. The catalytic MMO activities of CYPs in the presence of LysoPS (A) and effects of specific inhibitors of CYP1A2 and 2E1 on their MMO and PLD activities(B). 7Ethoxyresorufin O-deethylation by CYP1A2 and chlorzoxazone 6b-hydroxylation by CYP2E1 were measured as described in ‘Materials and methods’ with increasing concentrations of LysoPS. Values are means ± SD of three experiments. In figure (B), activity levels were measured with increasing concentrations of ANF or 4-MP. The y-axis represents normalized activities as relative percentages of the activity in the absence of the inhibitor, which was set to 100%. The inhibitors were preincubated with each enzyme for 10 min and then MMO or PLD substrates were added to the appropriate reaction samples.

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Wavelength (nm) Fig. 4. Effect of LysoPS on the CD spectra of CYPs. Spectra of 1 lM CYP1A2 (A) and CYP2E1 (B) were recorded in the presence of 800 lM liposomes containing 0, 1, or 2 mol.% LysoPS. The protein was incubated with liposomes for 20 min before measurement. Molar ellipticity, [h]R, is expressed on the basis of their molar concentrations.

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the substrate per se had no effect on the PLD activities of the CYPs (result not shown). LysoPS-induced decrease in MMO acitivities and PLD activity of CYPs in the presence of specific inhibitors Regarding the comparison between MMO and PLD activities of the CYPs, it should also be noticed that the actual MMO activities are much higher than the PLD activities for both CYPs: for example, the product formation rate of CYP1A2 was 1.8 nmol/min/nmol protein as a MMO and the value was 0.1 nmol/min/nmol protein as a PLD when the enzyme was incorporated into POPC membranes. To investigate the effect of lysophospholipids on the MMO activities of CYP1A2 and 2E1 and support the possibility of the functional switch of CYPs, a 7-ethoxyresorufin O-deethylation and clorzoxazone 6-hydroxylation were measured as described [25] with increasing concentrations of anionic lysophospholipids in liposomes. As a result, the incorporation of LysoPS up to 3 mol.% induced decreases of about 50–60% in the MMO catalytic activities of both CYPs when the activities without LysoPS were considered to be 100% (Fig. 3A). As expected, other anionic lysophospholipids including LysoPC and LysoPE in this work did not induce changes in the catalytic activities (result not shown). Considering the results that LysoPS causes a decrease in the catalytic activity of CYPs, it can be postulated that the PLD and MMO activities of the CYPs are catalyzed by different mechanisms and that the involvement of a reducing system exerts a decrease in PLD activity in the absence of LysoPS. In addition, these results imply that CYP1A2 and 2E1 can change their molecular functions from MMOs to PLDs by interaction with a specific lysophospholipid in vivo, and that this molecular switch may also be regulated by a reducing system(s). However, the present work does not provide the exact molecular mechanisms inducing the functional switch of the CYPs. In order to speculate on the active site(s) for the PLD activity in both CYPs, PLD assays with 100% PC membranes were carried out in the presence of a-naphthoflavone (ANF) or 4-Methylpyrazole (4MP), inhibitors specific for the MMO activities of CYP1A2 [26] and 2E1 [27], respectively. As shown in Fig. 3B, the PLD activities were gradually decreased with increasing concentrations of ANF or 4MP. However, in the presence of excess amounts of the inhibitors (100 lM), both CYPs exhibited considerable PLD activities, which is in contrast to the MMO activities that showed almost complete inhibition at equivalent concentrations of ANF or 4-MP. Therefore, this result suggests that the active site(s) for the PLD activities in both CYPs are different from those of their MMO activities although more information should be provided to prove the direct binding of these inhibitors to the MMO active sites in both CYPs. This result also parallels our previous results that potent CYP inhibitors (ketoconazole and 7,8-benzoflavone against CYP1A2, and diethyldithiocarbamate against CYP2E1) at 50 lM did not inhibit their PLD activities [12]. LysoPS-induced conformational changes in CYPs The potential for lysophospholipids to induce conformational changes in the CYPs was studied by CD spectroscopy. CD spectra of both CYP1A2 and 2E1 upon interaction with PC vesicles in the absence or presence of LysoPS (1% or 2%) were examined. LysoPS induced secondary structural changes in both CYPs in a concentration-dependent manner (Fig. 4). However, the exact secondary structure of the spectra did not fit well to the reference data derived from five proteins (myoglobin, lysozyme, ribonuclease A, papain and lactate dehydrogenase) [28] using the least-squares method or other curve-fit methods. Instead, when the a-helical contents were estimated from the mean residue ellipticity ([h]R)

at 222 nm, its value increased from 32% for CYP2E1 bound to 100% PC to 48% in the presence of 2 mol.% LysoPS. Therefore, upon interaction with LysoPS, it seems that a rather drastic change in the a-helical content of CYP2E1 occurs and this conformational change is important for the PLD activity. Although the role of the PLD activity in the in vivo function of the CYPs is currently unknown, CYP-catalyzed PLD activity and the regulation of this activity by LysoPS could exert its biological effects through several mechanisms including signaling and changes in membrane properties. First, PA, the product of the PLD reaction, serves as a precursor of diacylglycerol and lysoPA, which are both agonists in signal transduction cascades [29–31]. Therefore, the regulated PLD activity of CYP may alter the levels of PA (also thereby altering the properties of ER membrane), leading to substantial changes in physiological properties of the membrane. In a view of other CYP types as well as both CYPs currently studied, it has been shown that the catalytic activity, conformation, and membrane topology of CYP are changed depending upon the type of phospholipids [1]. In conclusion, our investigations suggest that CYP1A2 and 2E1 interact specifically with LysoPS in membranes and this interaction induces a functional change from a MMO to a PLD in both CYPs, which is also regulated by the reducing system including NPR. Although the molecular mechanisms and biological significances of the molecular switch are not clear at present, the current results may show another example of the functional regulation of CYP enzymes by the phospholipid compositions of cellular membranes. Acknowledgment This work was supported by a Korea Research Foundation Grant (KRF-2005-070-C00081). References [1] T. Ahn, M. Kim, C-H. Yun, H-J. Chae, Functional Regulation of Hepatic Cytochrome P450 Enzymes by Physicochemical Properties of Phospholipids in Biological Membranes, Curr. Protein Pep. Sci. 8 (2007) 496–505. [2] A. Stier, E. Sackmann, Spin labels as enzyme substrates. Heterogeneous lipid distribution in liver microsomal membranes, Biochim. Biophys. Acta 311 (1973) 400–408. [3] H.W. Strobel, A.Y. Lu, J. Heidema, M.J. Coon, Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation, J. Biol. Chem. 245 (1970) 4851–4854. [4] J.S. French, F.P. Guengerich, M.J. Coon, Interactions of cytochrome P-450 NADPH-cytochrome P-450, reductase phospholipid and substrate in the reconstituted liver microsomal enzyme system, J. Biol. Chem. 255 (1980) 4112–4119. [5] T. Sato, J. Aoki, Y. Nagai, N. Dohmae, K. Takio, T. Doi, H. Arai, K. Inoue, Serine Phospholipid-specific phospholipase A that is secreted from activated platelets, A new member of the lipase family. J. Biol. Chem. 272 (1997) 2192–2198. [6] Y. Xu, X.J. Fang, G. Casey, G.B. Mills, Lysophospholipids activate ovarian and breast cancer cells, Biochem. J. 309 (1995) 933–940. [7] Y. Xu, G. Casey, G.B. Mills, Effect of lysophospholipids on signaling in the human Jurkat T cell line, J. Cell. Physiol. 163 (1995) 441–450. [8] I.S. Ambudkar, E.S. Abdallah, A.E. Shamoo, Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum, Mol. Cell Biochem. 79 (1988) 81–89. [9] T. Shimada, M.V. Martin, D. Pruess-Schwartz, L.J. Marnett, F.P. Guengerich, Roles of individual human cytochrome P-450 enzymes in the bioactivation of benzo(a)pyrene, 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons, Cancer Res. 49 (1989) 6304–6312. [10] C.S. Yang, J.S. Yoo, H. Ishizaki, J.Y. Hong, Cytochrome P450IIE1: roles in nitrosamine metabolism and mechanism of regulation, Drug Meta. Rev. 22 (1990) 147–159. [11] F.J. Gonzalez, H.V. Gelboin, Role of human cytochromes P450 in the metabolic activation of chemical carcinogens and toxins, Drug Metab. Rev. 26 (1994) 165–183. [12] C-H. Yun, T. Ahn, F.P. Guengerich, H. Yamazaki, T. Shimada, Phospholipase D activity of cytochrome P450 in human liver endoplasmic reticulum, Arch. Biochem. Biophys. 367 (1999) 81–89. [13] T. Ahn, F.P. Guengerich, C-H. Yun, Membrane insertion of cytochrome P450 1A2 promoted by anionic phospholipids, Biochemistry 37 (1998) 12860– 12866.

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