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Adsorption of a phospholipid-hydroperoxide glutathione peroxidase into phospholipid monolayers at the air–water interface
Colloids and Surfaces B: Biointerfaces 35 (2004) 99–105
Adsorption of a phospholipid-hydroperoxide glutathione peroxidase into phospholipid monolayers at the air–water interface Sandrine Morandat a,∗ , Muriel Bortolato a , Fergus Nicol b , John R. Arthur b , Jean-Paul Chauvet c , Bernard Roux a a
c
Laboratoire de Physico-Chimie Biologique, UMR-CNRS 5013, Bˆat. E. Chevreul 4ième étage, Université Claude Bernard Lyon I, 43 Bd du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France b The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK Laboratoire d’Ingénierie et de Fonctionnalisation des Surfaces, UMR-CNRS 5621, Equipe Bioingénierie et Reconnaissance Génétique, Ecole Centrale de Lyon, 36 Av. G. de Collongue, F-69134 Ecully Cedex, France Accepted 25 February 2004
Abstract The interfacial behavior differences of two glutathione peroxidase isoforms have been investigated. The first isoform is the phospholipidhydroperoxide glutathione peroxidase (EC 1.11.1.12) (GPx-4) isolated from rat testes and the second one is the cytosolic glutathione peroxidase (EC 1.11.1.9) (GPx-1) from bovine erythrocytes. Injected in the subphase buffer of a Langmuir trough, GPx-4 was able to adsorb quickly at the air–water interface whereas the GPx-1 was not. Then, the protein interaction with phospholipid monolayers was explored. Indeed, a monolayer of phospholipids containing a different number of polyunsaturated fatty acyl chains was prepared at the air–water interface. Under each kind of monolayer, the protein solution was injected and its adsorption was visualized by the measurement of successive pressure–area isotherms. We have, then, determined the molecular area increase due to the protein adsorption. It was found that the GPx-4 is adsorbed in each kind of monolayer tested whereas no molecular area increase was detected with the GPx-1. This indicates that the GPx-4 has a higher affinity for the interface, recovered or not by lipids, than the GPx-1. Moreover, the GPx-4 presents a different affinity for the phospholipid monolayers depending on the number of polyunsaturated fatty acyl chains. © 2004 Elsevier B.V. All rights reserved. Keywords: Glutathione peroxidase; Protein adsorption; Protein–lipid interactions; Langmuir monolayer; Polyunsaturated phospholipids
1. Introduction It is now well established that free radicals act as pathological agents in a several human diseases and various biological phenomena. The polyunsaturated phospholipids, which are abundant within cytoplasmic membranes, are particularly sensitive targets for free radical attack. The radical oxidation mechanism involves the abstraction of a hydrogen atom in bisallylic position yielding a new Abbreviations: DLiPC, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine; PLiPC, 1-palmitoyl, 2-linoleoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; Ks , surface elasticity modulus; GPx, glutathione peroxidase ∗ Corresponding author. Present address: BIOTEC, University of Technology of Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany. Tel.: +49-351-210-2693; fax: +49-351-210-2020. E-mail address: [email protected] (S. Morandat). 0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.02.011
lipid-derived radical species, i.e., polyunsaturated peroxyl radicals which play a crucial role in the free radical chain reaction of lipid peroxidation. Although the mechanism of cell membrane peroxidation has been extensively studied, attention has also been focused on the role of free radicals in aging, atherosclerosis, and neurodegenerative diseases such as Alzheimer’s or Parkinson’s. These medical consequences of cellular damage have stimulated investigations of the mechanism of lipid peroxidation, and also in the protective role of antioxidants in membrane model systems [1–3]. The antioxidants can be either amphiphilic molecules such as plasmalogen, cholesterol or Vitamin E [1,3,4] inserted directly within the lipid membrane or soluble compounds such as Vitamin C [2] or enzymes. The major antioxidant enzymes are catalases [5], superoxide dismutases [6], and gluthatione peroxidases [7,8]. For all the known glutathione peroxidases (GPx), the active site contains a selenocysteine, which is directly in-
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volved in the catalytic mechanism. Six isoforms of GPx are now described [7,9] and each isoform has distinct tissue and cell distributions and substrates. Indeed, the GPx-1 isoform (EC 1.11.1.9), also called cytosolic glutathione peroxidase, is located within the cell cytosol and it catalyses the reduction of hydrogen peroxides and fatty acid hydroperoxides in the presence of glutathione [7,10]. This tetrameric protein of about 22–23 kDa/monomer is unable to catalyze the reduction of phospholipid hydroperoxides without the prior intervention of phospholipase A2 [11]. One GPx isoform able to reduce the phospholipid hydroperoxides directly within the lipid membrane is GPx-4 (EC 1.11.1.12). This enzyme was first identified in 1982 by Ursini et al. [12]. The GPx-4 is a monomeric protein of 20 kDa which is either located in the mitochondria or in the cytosol. Indeed, the GPx-4 can be synthesized in two forms: a short form, the cytosolic GPx-4 (s-GPx-4) of 20 kDa and 170 aminoacids; and a long form, the mitochondrial GPx-4 (l-GPx-4) of 23 kDa and 197 aminoacids [8,13]. The long form contains a signal peptide to transport the enzyme into the mitochondria [13]. This signal peptide is then removed and the mitochondrial GPx-4 molecular weight becomes the same than the nonmitochondrial GPx-4, 20 kDa. This leader sequence is composed of two main parts: the first part is composed of basic residues and the second part of 20 hydrophobic aminoacids. In this, it can form an amphiphilic helix [14]. The catalytic activity of the GPx-4 on phospholipid hydroperoxides is stimulated by the addition of detergents such as Triton X100. Thus, it seems that in vivo, the protein is associated to membranes but not integrated into them [15,16]. The monolayer technique is a powerful method for studying the protein affinity for the air–water or for the lipid–water interface [17–20]. The present work deals with the two GPx isoforms—i.e., GPx-1 and GPx-4-interaction with the air–water or lipid–water interface. In order to determine the interfacial affinity of each protein alone, the protein was injected in the subphase buffer of a Langmuir trough at zero surface pressure. The variation of the molecular area recorded by the measurement of successive pressure–area isotherms (π–A isotherms) indicated the protein adsorption at the air–water interface. In a second experiment, the lipid affinity of each protein was examined. Indeed, several phospholipids, each with a different number of polyunsaturated fatty acyl chains, were spread in monomolecular films. The protein affinity for such monolayers was then tested by the injection of the protein solution under the lipid film.
2. Materials and methods 2.1. Materials Rat GPx-4 was purified from rat testes by the method of Roveri et al. [16]. The purified protein was a single band of 20 kDa confirmed by SDS-PAGE electrophoresis.
Silver staining showed that the GPx-4 was over 99% pure. N-terminal sequencing confirmed it to be the short form of the protein. Bovine erythrocyte GPx-1 (EC 1.11.1.9), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLiPC), 1palmitoyl, 2-linoleoyl-sn-glycero-3-phosphocholine (PLiPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Sigma (St. Louis, MO, USA). Phospholipids were spread in hexane/ethanol (9:1) purchased from Merck (Darmstadt, Germany) and from Carlo Erba, respectively. All other chemicals and reagents were of the highest purity available from Merck (Darmstadt, Germany) and from Fluka Chemika. The DLiPC, PLiPC, and DMPC solutions were protected against light and stored in a freezer until spreading. The distilled water was purified with a Millipore MilliQ filtering system, yielding a water resistance of 18.2 MW·cm. 2.2. Monolayer technique All experiments were performed at a constant temperature of 21.0±0.1 ◦ C. The film balance was built by R&K (Riegler & Kirstein, Wiesbaden, Germany) and equipped with a Wilhelmy-type surface-pressure measuring system. The subphase buffer used was 150 mM KCl, 10 mM Tris–HCl pH 7.2 (TBS). In all experiments, the subphase was continuously stirred with a magnetic stirrer spinning at 100 rpm. In order to measure π–A isotherms of the self-penetrated protein, a known quantity of protein was injected in the subphase buffer and after 15 min, the surface was compressed at a velocity of 6 cm2 /min up to a maximum lateral pressure of 33 mN/m. Then, compression–decompression– recompression π–A isotherms of proteins were measured. The adsorption times indicated on the compression– decompression–recompression isotherms correspond to the cumulated adsorption time at zero surface pressure. Indeed, we consider that the protein is weakly adsorbed at the interface during the decompression. The experiments to measure the interaction of proteins with lipids by compression–decompression–recompression π–A isotherms have been previously described [18,20]. Phospholipids, dissolved in 9:1 hexane/ethanol (v/v), were spread with a Hamilton syringe at the air–water interface to reach a final quantity of about 5 nmol. After 20 min solvent evaporation, the monolayer was compressed at a velocity of 6 cm2 /min up to a lateral pressure of 33 mN/m to obtain a π–A isotherm of the lipid alone. After 15 min at π = 0, the protein solution of GPx-1 or GPx-4 was injected in the subphase buffer through the lipid monolayer with a calibrated microsyringe (Hamilton). Thereafter, the monolayer was compressed at various times to obtain mixed protein–lipid π–A isotherms.The surface elasticity moduli (Ks ) were calculated from the π–A data obtained from the monolayer compressions using the following equation: Ks = −A ×
∂π ∂A
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where A is the molecular area at the indicated surface pressure π. High Ks values correspond to low interfacial fluidity among packed lipids forming a monolayer. This suggests that the higher the Ks value of a monolayer is, the more rigid it is.
3. Results and discussion 3.1. π–A isotherms of GPx-4 and GPx-1 adsorptions versus time A solution of GPx-4 was injected in the trough subphase buffer to reach the final concentration of 0.15 mg/l. The surface of the trough was compressed after various delays to obtain π–A isotherms represented in Fig. 1A. Other adsorption delays were measured, but are not shown for clarity. As one can see, the molecular area increased at each surface pressure indicating that the protein was adsorbed at the air–water interface [18,20]. The protein adsorption was not measured over 150 min due to the subphase evaporation. The surface elasticity modulus (Ks ) of the last π–A isotherm was represented against the surface pressure in Fig. 1B. The Ks increased up to 8 mN/m surface pressure and decreased from 12 mN/m. One may consider that the plateau observed between 8 and 12 mN/m could be due to an equilibrium between the protein desorption and adsorption at the air–water interface. It seems that above 12 mN/m, there was either a protein desorption or a reorganization of this protein at the air–water interface during the monolayer compression. Because of the possible desorption of a small amount of protein at a surface pressure above 12 mN/m, we have determined the molecular area at 5 mN/m surface pressure (A5 mN/m ). The A5 mN/m was plotted versus the adsorption delays and represented in Fig. 1C. This figure showed that GPx-4 needed only 15 min at zero surface pressure to induce a molecular area increase. This adsorption time can be compared with the adsorption time of an insect peptide: the defensin A. The defensin A was injected in the subphase buffer at the final concentration of 0.4 mg/l and the peptide adsorption was measured in terms of the surface pressure increase [21,22]. For a concentration of defensin A 2.7 times greater than that of GPx-4, 70 min are required to induce an increase in the surface pressure whereas only 10 min are sufficient for the GPx-4. In our experiments, A5 mN/m increased up to 40 min after the protein injection in the subphase buffer. After 40 min, the A5 mN/m did not vary significantly, so the protein did not seem to be adsorbed further. It can be the direct consequence of the surface saturation by GPx-4. By knowing the molar volume of a globular protein, the radius of a protein with the same molecular weight was estimated at about 1.81 nm. With the same molecule number injected, the surface occupied by the globular protein monolayer at zero surface pressure would be about 35 cm2 . With the last isotherm in Fig. 1A, we have determined that the GPx-4 film
Fig. 1. GPx-4 adsorption at the air–water interface at 21 ◦ C. The protein was injected in the subphase buffer to reach the final concentration of 0.15 mg/l (285 pmol). The GPx-4 adsorption was realized at zero surface pressure. (A) The different π–A isotherms were measured after 20 min (black circles), 30 min (black squares), 40 min (black triangles), 70 min (white squares), and 150 min (white triangles) at zero surface pressure. The symbols indicated are only guide to the eyes. (B) From 130 min π–A isotherm, the monolayer Ks was calculated and represented as a function of surface pressure. (C) The molecular area values at 5 mN/m (A5 mN/m ) determined on the π–A isotherms were plotted against the adsorption time. During the experiment, the interface was not protected from oxygen and light. The subphase buffer was 10 mM Tris–HCl, 150 mM KCl pH 7.2, thermostated at 21 ◦ C.
occupied a 32 cm2 surface. These two values (the calculated and the determined ones) of surface occupied are very similar and this confirms that after 150 min adsorption delay, the GPx-4 was mostly located at the air–water interface. The same experiment was performed with GPx-1 under the same conditions of temperature, atmosphere, and protein concentration in the subphase but even after 150 min
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adsorption at zero surface pressure, no molecular area variations were recorded (data not shown). 3.2. π–A isotherms of GPx-4 and GPx-1 adsorptions into lipid monolayers versus time The interfacial affinity of the glutathione peroxidases for three phospholipids was evidenced. The three phospholipids tested were composed of a different number of polyunsaturated fatty acyl chains. Indeed, we chose DMPC, which contains saturated fatty acyl chains; PLiPC, which contains only one polyunsaturated fatty acyl chain; and DLiPC, which
contains two polyunsaturated fatty acyl chains. For each phospholipid, a monolayer was formed at the air–water interface and the protein was injected in the subphase buffer at the final concentration of 0.15 mg/l. The protein adsorption into the lipid monolayer was analyzed by the measurement of successive π–A isotherms. The lipid monolayer was not protected from light and from oxygen during the measurement. Experiments were performed without glutathione in the subphase. Fig. 2A represents the successive π–A isotherms recorded after the GPx-4 adsorption into a DLiPC monolayer. As one can see, the molecular area increased with time indicating
Fig. 2. GPx-4 adsorption into a DLiPC monolayer at 21 ◦ C. Five nanomoles of DLiPC were spread at the air–water interface and after 20 min solvent evaporation, a π–A isotherm was recorded (black squares). After 15 min at zero surface pressure, the GPx-4 was injected in the subphase buffer at the final concentration of 0.15 mg/l (285 pmol). (A) Then, after different time intervals of 10 min (black triangles), 20 min (white squares), 30 min (white triangles), and 150 min (black circles) at zero surface pressure, π–A isotherms were measured. All the π–A isotherms measured were not represented for clarity. (B) From each π–A isotherm, the Ks was calculated and represented versus the surface pressure. The symbols indicated are only guide to the eyes. During the experiment, the interface was not protected from oxygen and light. The subphase buffer was 10 mM Tris–HCl, 150 mM KCl pH 7.2, thermostated at 21 ◦ C.
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protein adsorption into the lipid monolayer. The Ks corresponding to the π–A isotherms were calculated for each adsorption time and were plotted versus surface pressure in Fig. 2B. The protein insertion into the lipid monolayer led to a Ks decrease traducing the membrane fluidization. Moreover, after 60 min protein adsorption, we observed a Ks decrease over 25 mN/m surface pressure and 150 min after the protein injection, this decrease occurred at a lower surface pressure (15 mN/m). These decreases might be due to the protein desorption during the film compression. Moreover, at high surface pressures, the isotherms were shifted to higher molecular areas, indicating a stabilization of the protein within the monolayer [18]. Up to 90 min, the protein desorption did not seem to lead to lipid desorption because over 30 mN/m surface pressure, the molecular area observed on the π–A isotherm (Fig. 2A) is greater than that of the lipid alone. For the last π–A isotherm, the molecular area, for a surface pressure higher than 30 mN/m, was lower than that observed for the lipid alone. This indicates that the GPx-4 desorption from the interface induced an expulsion of some lipids adsorbed to the protein molecules [23]. Thus, the interactions between the GPx-4 and the DLiPC would appear to be rather strong. The same experiments were performed with GPx-4 injected under PLiPC or DMPC monolayers and as for the DLiPC monolayer, the molecular area increased with adsorption time (data not shown). We conclude that the GPx-4 was adsorbed as well in DLiPC monolayers as in PLiPC or DMPC monolayers. The GPx-1 was injected in the subphase buffer of each lipid monolayer but no molecular area variations were recorded, whatever be the fatty acyl chain composition of
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the phospholipid (data not shown). This indicates that the GPx-1 did not interact with the lipid monolayer. 3.3. π–A isotherms of polyunsaturated lipid monolayers versus time Because the monolayer was not protected from light and oxygen, it should take into account that the lipids were able to oxidize. That is why the successive π–A isotherms of lipid monolayers were measured in order to realize a correction on the molecular area variation due to not only the protein adsorption but also the lipid oxidation. The successive π–A isotherms of DLiPC autoxidation are shown in Fig. 3. The oxidation of DLiPC molecules at the air–water interface led to a dramatic molecular area decrease. Moreover, by calculating the Ks , we can see that the DLiPC autoxidation led to a fluidization of the monolayer (data not shown). The molecular area and Ks decrease is consistent with the results of the DLiPC monolayer UV-oxidation at 30 mN/m [1]. Moreover, even if no UV-irradiation is used, the molecular area decrease reached 6 Å2 /molecule. So it seems that the UV-irradiation enhanced the DLiPC oxidation. Moreover, whatever be the surface pressure and the atmosphere composition, this polyunsaturated phospholipid was able to autoxidize. In our study, the molecular area variation at 30 mN/m after 150 min reached about 30 Å2 /molecule. This value is greater than that observed in another study where the DLiPC monolayer was protected against oxygen and light [1]. Indeed, at zero surface pressure, the polyunsaturated fatty acyl chains were more exposed to the oxygen and light inducing a greater oxidation. Moreover, according to Morandat et al. [1] and Viitala and Peltonen [24], the
Fig. 3. DLiPC monolayer autoxidation at 21 ◦ C. Five nanomoles of DLiPC were spread at the air–water interface and after 20 min solvent evaporation, a π–A isotherm was recorded (black squares). Then, after different time intervals of 30 min (black triangles), 60 min (white squares), 90 min (white triangles), and 150 min (black circles) at zero surface pressure, π–A isotherms were measured. The symbols indicated are only guide to the eyes. All the π–A isotherms measured were not represented for clarity. During the experiment, the interface was not protected from oxygen and light. The subphase buffer was 10 mM Tris–HCl, 150 mM KCl pH 7.2, thermostated at 21 ◦ C.
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Fig. 4. Dependence of the phospholipid unsaturation number on the GPx-4 adsorption. The molecular areas were determined at 5 mN/m on each π–A isotherm of the GPx-4 adsorption into a lipid monolayer. Each value was corrected by the molecular area at the same surface pressure of the lipid only. This molecular area variation obtained was then corrected by the molecular area variation due to the lipid autoxidation. In this, we obtain the corrected A5 mN/m which was plotted versus adsorption time into a DLiPC (circles), PLiPC (squares) or DMPC monolayer (triangles).
polyunsaturated phospholipid oxidation led to the apparition of new molecular species at the air–water interface. In the case of a PLiPC monolayer, the lipid autoxidation led to a weak increase of the molecular area and to a Ks decrease (data not shown), indicating no dramatic physical properties changes. We have shown that successive π–A isotherms did not induce a change of the DMPC monolayer physical properties. 3.4. Dependence of the phospholipid unsaturation number on the GPx-4 adsorption For each kind of phospholipid, the molecular area was measured at 5 mN/m surface pressure after GPx-4 adsorption and it was corrected by those obtained at the same surface pressure for the lipid monolayer before GPx-4 injection. This molecular area variation obtained was then corrected by the molecular area variation due to the lipid autoxidation. In this, we defined a new parameter which is called corrected A5 mN/m . The corrected A5 mN/m was then plotted versus the adsorption time at zero surface pressure and it was represented in Fig. 4. For each phospholipid tested, an increase in corrected A5 mN/m was measured. This corrected A5 mN/m increase was more important in the DLiPC monolayer as compared to other lipids and the less important corrected A5 mN/m increase was observed for the DMPC monolayer. So, we can conclude that the GPx-4 was adsorbed into the three phospholipid monolayers but not with the same affinity. Moreover, the molecular area variation was observed from 10 min after the protein injection, whereas it was longer in the case of the protein adsorption when the surface was not covered by a lipid monolayer (Figs. 1C and 4). This means that the hydrophobic parts of the protein were more stabilized when a lipid film was at the
interface [23]. For each monolayer, the corrected A5 mN/m increase presented several steps. Indeed a slow corrected A5 mN/m increase was observed up to 10 min adsorption, then between 10 and 30 min, the curves presented a sharp corrected A5 mN/m increase. After 30 min protein adsorption, the corrected A5 mN/m increase became slower, and moreover, there is no corrected A5 mN/m variation after 90 min for the PLiPC and DMPC monolayers. It should be remembered that without glutathione, the enzyme was inactive on the phospholipid hydroperoxides formed. In this work, we have used a pure short form of GPx-4, as confirmed by SDS-PAGE electrophoresis and by N-terminal sequencing. Arai et al. [13] showed that the mitochondrial import of GPx-4 23 kDa precursor requires the leader sequence that induces import into mitochondria. Then, this precursor is processed to the 20 kDa mature form within the mitochondria. Our results showed that the GPx-4 short form can interact with membrane lipids. So, it seems evident that the leader sequence of the GPx-4 is not necessary to maintain the interaction between protein and lipids after the maturation has occurred.
4. Conclusions The GPx-4 adsorption at the air–water interface was detected whereas under the same conditions, GPx-1 was not adsorbed. Moreover, the molecular area increased only 15 min after the GPx-4 injection and after an adsorption time of 40 min, all the GPx-4 injected in the subphase buffer seemed to localize at the air–buffer interface. In this report, we have also shown the direct interaction of GPx-4 with phospholipid monolayers. Roveri et al. [16] described that no lipid post-translational modifications such as
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glycosylation, isoprenylation or acylation were detected on GPx-4. This means that no lipid modifications are required to target the GPx-4 to the phospholipid membranes. Nevertheless, we showed that the GPx-4 presented more affinity for lipid membranes than GPx-1. Moreover, the GPx-4 exhibited a higher affinity for the monolayer when it was composed of a potential substrate. Based on the present work, the affinity of GPx-4 was in the order DLiPC > PLiPC > DMPC.
Acknowledgements We would like to thank Dr. Karim El Kirat for helpful discussions. This work was supported by the “Centre National de la Recherche Scientifique” (CNRS), the “Ministère de l’Enseignement Supérieur et de la Recherche” (MESR) and by a grant of the “Région Rhˆone Alpes”. JRA’s lab is funded by the Scottish Executive Environment and Rural Affairs Department.
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Adsorption of a phospholipid-hydroperoxide glutathione peroxidase into phospholipid monolayers at the air–water interface Sandrine Morandat a,∗ , Muriel Bortolato a , Fergus Nicol b , John R. Arthur b , Jean-Paul Chauvet c , Bernard Roux a a
c
Laboratoire de Physico-Chimie Biologique, UMR-CNRS 5013, Bˆat. E. Chevreul 4ième étage, Université Claude Bernard Lyon I, 43 Bd du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France b The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK Laboratoire d’Ingénierie et de Fonctionnalisation des Surfaces, UMR-CNRS 5621, Equipe Bioingénierie et Reconnaissance Génétique, Ecole Centrale de Lyon, 36 Av. G. de Collongue, F-69134 Ecully Cedex, France Accepted 25 February 2004
Abstract The interfacial behavior differences of two glutathione peroxidase isoforms have been investigated. The first isoform is the phospholipidhydroperoxide glutathione peroxidase (EC 1.11.1.12) (GPx-4) isolated from rat testes and the second one is the cytosolic glutathione peroxidase (EC 1.11.1.9) (GPx-1) from bovine erythrocytes. Injected in the subphase buffer of a Langmuir trough, GPx-4 was able to adsorb quickly at the air–water interface whereas the GPx-1 was not. Then, the protein interaction with phospholipid monolayers was explored. Indeed, a monolayer of phospholipids containing a different number of polyunsaturated fatty acyl chains was prepared at the air–water interface. Under each kind of monolayer, the protein solution was injected and its adsorption was visualized by the measurement of successive pressure–area isotherms. We have, then, determined the molecular area increase due to the protein adsorption. It was found that the GPx-4 is adsorbed in each kind of monolayer tested whereas no molecular area increase was detected with the GPx-1. This indicates that the GPx-4 has a higher affinity for the interface, recovered or not by lipids, than the GPx-1. Moreover, the GPx-4 presents a different affinity for the phospholipid monolayers depending on the number of polyunsaturated fatty acyl chains. © 2004 Elsevier B.V. All rights reserved. Keywords: Glutathione peroxidase; Protein adsorption; Protein–lipid interactions; Langmuir monolayer; Polyunsaturated phospholipids
1. Introduction It is now well established that free radicals act as pathological agents in a several human diseases and various biological phenomena. The polyunsaturated phospholipids, which are abundant within cytoplasmic membranes, are particularly sensitive targets for free radical attack. The radical oxidation mechanism involves the abstraction of a hydrogen atom in bisallylic position yielding a new Abbreviations: DLiPC, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine; PLiPC, 1-palmitoyl, 2-linoleoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; Ks , surface elasticity modulus; GPx, glutathione peroxidase ∗ Corresponding author. Present address: BIOTEC, University of Technology of Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany. Tel.: +49-351-210-2693; fax: +49-351-210-2020. E-mail address: [email protected] (S. Morandat). 0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.02.011
lipid-derived radical species, i.e., polyunsaturated peroxyl radicals which play a crucial role in the free radical chain reaction of lipid peroxidation. Although the mechanism of cell membrane peroxidation has been extensively studied, attention has also been focused on the role of free radicals in aging, atherosclerosis, and neurodegenerative diseases such as Alzheimer’s or Parkinson’s. These medical consequences of cellular damage have stimulated investigations of the mechanism of lipid peroxidation, and also in the protective role of antioxidants in membrane model systems [1–3]. The antioxidants can be either amphiphilic molecules such as plasmalogen, cholesterol or Vitamin E [1,3,4] inserted directly within the lipid membrane or soluble compounds such as Vitamin C [2] or enzymes. The major antioxidant enzymes are catalases [5], superoxide dismutases [6], and gluthatione peroxidases [7,8]. For all the known glutathione peroxidases (GPx), the active site contains a selenocysteine, which is directly in-
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volved in the catalytic mechanism. Six isoforms of GPx are now described [7,9] and each isoform has distinct tissue and cell distributions and substrates. Indeed, the GPx-1 isoform (EC 1.11.1.9), also called cytosolic glutathione peroxidase, is located within the cell cytosol and it catalyses the reduction of hydrogen peroxides and fatty acid hydroperoxides in the presence of glutathione [7,10]. This tetrameric protein of about 22–23 kDa/monomer is unable to catalyze the reduction of phospholipid hydroperoxides without the prior intervention of phospholipase A2 [11]. One GPx isoform able to reduce the phospholipid hydroperoxides directly within the lipid membrane is GPx-4 (EC 1.11.1.12). This enzyme was first identified in 1982 by Ursini et al. [12]. The GPx-4 is a monomeric protein of 20 kDa which is either located in the mitochondria or in the cytosol. Indeed, the GPx-4 can be synthesized in two forms: a short form, the cytosolic GPx-4 (s-GPx-4) of 20 kDa and 170 aminoacids; and a long form, the mitochondrial GPx-4 (l-GPx-4) of 23 kDa and 197 aminoacids [8,13]. The long form contains a signal peptide to transport the enzyme into the mitochondria [13]. This signal peptide is then removed and the mitochondrial GPx-4 molecular weight becomes the same than the nonmitochondrial GPx-4, 20 kDa. This leader sequence is composed of two main parts: the first part is composed of basic residues and the second part of 20 hydrophobic aminoacids. In this, it can form an amphiphilic helix [14]. The catalytic activity of the GPx-4 on phospholipid hydroperoxides is stimulated by the addition of detergents such as Triton X100. Thus, it seems that in vivo, the protein is associated to membranes but not integrated into them [15,16]. The monolayer technique is a powerful method for studying the protein affinity for the air–water or for the lipid–water interface [17–20]. The present work deals with the two GPx isoforms—i.e., GPx-1 and GPx-4-interaction with the air–water or lipid–water interface. In order to determine the interfacial affinity of each protein alone, the protein was injected in the subphase buffer of a Langmuir trough at zero surface pressure. The variation of the molecular area recorded by the measurement of successive pressure–area isotherms (π–A isotherms) indicated the protein adsorption at the air–water interface. In a second experiment, the lipid affinity of each protein was examined. Indeed, several phospholipids, each with a different number of polyunsaturated fatty acyl chains, were spread in monomolecular films. The protein affinity for such monolayers was then tested by the injection of the protein solution under the lipid film.
2. Materials and methods 2.1. Materials Rat GPx-4 was purified from rat testes by the method of Roveri et al. [16]. The purified protein was a single band of 20 kDa confirmed by SDS-PAGE electrophoresis.
Silver staining showed that the GPx-4 was over 99% pure. N-terminal sequencing confirmed it to be the short form of the protein. Bovine erythrocyte GPx-1 (EC 1.11.1.9), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLiPC), 1palmitoyl, 2-linoleoyl-sn-glycero-3-phosphocholine (PLiPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Sigma (St. Louis, MO, USA). Phospholipids were spread in hexane/ethanol (9:1) purchased from Merck (Darmstadt, Germany) and from Carlo Erba, respectively. All other chemicals and reagents were of the highest purity available from Merck (Darmstadt, Germany) and from Fluka Chemika. The DLiPC, PLiPC, and DMPC solutions were protected against light and stored in a freezer until spreading. The distilled water was purified with a Millipore MilliQ filtering system, yielding a water resistance of 18.2 MW·cm. 2.2. Monolayer technique All experiments were performed at a constant temperature of 21.0±0.1 ◦ C. The film balance was built by R&K (Riegler & Kirstein, Wiesbaden, Germany) and equipped with a Wilhelmy-type surface-pressure measuring system. The subphase buffer used was 150 mM KCl, 10 mM Tris–HCl pH 7.2 (TBS). In all experiments, the subphase was continuously stirred with a magnetic stirrer spinning at 100 rpm. In order to measure π–A isotherms of the self-penetrated protein, a known quantity of protein was injected in the subphase buffer and after 15 min, the surface was compressed at a velocity of 6 cm2 /min up to a maximum lateral pressure of 33 mN/m. Then, compression–decompression– recompression π–A isotherms of proteins were measured. The adsorption times indicated on the compression– decompression–recompression isotherms correspond to the cumulated adsorption time at zero surface pressure. Indeed, we consider that the protein is weakly adsorbed at the interface during the decompression. The experiments to measure the interaction of proteins with lipids by compression–decompression–recompression π–A isotherms have been previously described [18,20]. Phospholipids, dissolved in 9:1 hexane/ethanol (v/v), were spread with a Hamilton syringe at the air–water interface to reach a final quantity of about 5 nmol. After 20 min solvent evaporation, the monolayer was compressed at a velocity of 6 cm2 /min up to a lateral pressure of 33 mN/m to obtain a π–A isotherm of the lipid alone. After 15 min at π = 0, the protein solution of GPx-1 or GPx-4 was injected in the subphase buffer through the lipid monolayer with a calibrated microsyringe (Hamilton). Thereafter, the monolayer was compressed at various times to obtain mixed protein–lipid π–A isotherms.The surface elasticity moduli (Ks ) were calculated from the π–A data obtained from the monolayer compressions using the following equation: Ks = −A ×
∂π ∂A
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where A is the molecular area at the indicated surface pressure π. High Ks values correspond to low interfacial fluidity among packed lipids forming a monolayer. This suggests that the higher the Ks value of a monolayer is, the more rigid it is.
3. Results and discussion 3.1. π–A isotherms of GPx-4 and GPx-1 adsorptions versus time A solution of GPx-4 was injected in the trough subphase buffer to reach the final concentration of 0.15 mg/l. The surface of the trough was compressed after various delays to obtain π–A isotherms represented in Fig. 1A. Other adsorption delays were measured, but are not shown for clarity. As one can see, the molecular area increased at each surface pressure indicating that the protein was adsorbed at the air–water interface [18,20]. The protein adsorption was not measured over 150 min due to the subphase evaporation. The surface elasticity modulus (Ks ) of the last π–A isotherm was represented against the surface pressure in Fig. 1B. The Ks increased up to 8 mN/m surface pressure and decreased from 12 mN/m. One may consider that the plateau observed between 8 and 12 mN/m could be due to an equilibrium between the protein desorption and adsorption at the air–water interface. It seems that above 12 mN/m, there was either a protein desorption or a reorganization of this protein at the air–water interface during the monolayer compression. Because of the possible desorption of a small amount of protein at a surface pressure above 12 mN/m, we have determined the molecular area at 5 mN/m surface pressure (A5 mN/m ). The A5 mN/m was plotted versus the adsorption delays and represented in Fig. 1C. This figure showed that GPx-4 needed only 15 min at zero surface pressure to induce a molecular area increase. This adsorption time can be compared with the adsorption time of an insect peptide: the defensin A. The defensin A was injected in the subphase buffer at the final concentration of 0.4 mg/l and the peptide adsorption was measured in terms of the surface pressure increase [21,22]. For a concentration of defensin A 2.7 times greater than that of GPx-4, 70 min are required to induce an increase in the surface pressure whereas only 10 min are sufficient for the GPx-4. In our experiments, A5 mN/m increased up to 40 min after the protein injection in the subphase buffer. After 40 min, the A5 mN/m did not vary significantly, so the protein did not seem to be adsorbed further. It can be the direct consequence of the surface saturation by GPx-4. By knowing the molar volume of a globular protein, the radius of a protein with the same molecular weight was estimated at about 1.81 nm. With the same molecule number injected, the surface occupied by the globular protein monolayer at zero surface pressure would be about 35 cm2 . With the last isotherm in Fig. 1A, we have determined that the GPx-4 film
Fig. 1. GPx-4 adsorption at the air–water interface at 21 ◦ C. The protein was injected in the subphase buffer to reach the final concentration of 0.15 mg/l (285 pmol). The GPx-4 adsorption was realized at zero surface pressure. (A) The different π–A isotherms were measured after 20 min (black circles), 30 min (black squares), 40 min (black triangles), 70 min (white squares), and 150 min (white triangles) at zero surface pressure. The symbols indicated are only guide to the eyes. (B) From 130 min π–A isotherm, the monolayer Ks was calculated and represented as a function of surface pressure. (C) The molecular area values at 5 mN/m (A5 mN/m ) determined on the π–A isotherms were plotted against the adsorption time. During the experiment, the interface was not protected from oxygen and light. The subphase buffer was 10 mM Tris–HCl, 150 mM KCl pH 7.2, thermostated at 21 ◦ C.
occupied a 32 cm2 surface. These two values (the calculated and the determined ones) of surface occupied are very similar and this confirms that after 150 min adsorption delay, the GPx-4 was mostly located at the air–water interface. The same experiment was performed with GPx-1 under the same conditions of temperature, atmosphere, and protein concentration in the subphase but even after 150 min
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adsorption at zero surface pressure, no molecular area variations were recorded (data not shown). 3.2. π–A isotherms of GPx-4 and GPx-1 adsorptions into lipid monolayers versus time The interfacial affinity of the glutathione peroxidases for three phospholipids was evidenced. The three phospholipids tested were composed of a different number of polyunsaturated fatty acyl chains. Indeed, we chose DMPC, which contains saturated fatty acyl chains; PLiPC, which contains only one polyunsaturated fatty acyl chain; and DLiPC, which
contains two polyunsaturated fatty acyl chains. For each phospholipid, a monolayer was formed at the air–water interface and the protein was injected in the subphase buffer at the final concentration of 0.15 mg/l. The protein adsorption into the lipid monolayer was analyzed by the measurement of successive π–A isotherms. The lipid monolayer was not protected from light and from oxygen during the measurement. Experiments were performed without glutathione in the subphase. Fig. 2A represents the successive π–A isotherms recorded after the GPx-4 adsorption into a DLiPC monolayer. As one can see, the molecular area increased with time indicating
Fig. 2. GPx-4 adsorption into a DLiPC monolayer at 21 ◦ C. Five nanomoles of DLiPC were spread at the air–water interface and after 20 min solvent evaporation, a π–A isotherm was recorded (black squares). After 15 min at zero surface pressure, the GPx-4 was injected in the subphase buffer at the final concentration of 0.15 mg/l (285 pmol). (A) Then, after different time intervals of 10 min (black triangles), 20 min (white squares), 30 min (white triangles), and 150 min (black circles) at zero surface pressure, π–A isotherms were measured. All the π–A isotherms measured were not represented for clarity. (B) From each π–A isotherm, the Ks was calculated and represented versus the surface pressure. The symbols indicated are only guide to the eyes. During the experiment, the interface was not protected from oxygen and light. The subphase buffer was 10 mM Tris–HCl, 150 mM KCl pH 7.2, thermostated at 21 ◦ C.
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protein adsorption into the lipid monolayer. The Ks corresponding to the π–A isotherms were calculated for each adsorption time and were plotted versus surface pressure in Fig. 2B. The protein insertion into the lipid monolayer led to a Ks decrease traducing the membrane fluidization. Moreover, after 60 min protein adsorption, we observed a Ks decrease over 25 mN/m surface pressure and 150 min after the protein injection, this decrease occurred at a lower surface pressure (15 mN/m). These decreases might be due to the protein desorption during the film compression. Moreover, at high surface pressures, the isotherms were shifted to higher molecular areas, indicating a stabilization of the protein within the monolayer [18]. Up to 90 min, the protein desorption did not seem to lead to lipid desorption because over 30 mN/m surface pressure, the molecular area observed on the π–A isotherm (Fig. 2A) is greater than that of the lipid alone. For the last π–A isotherm, the molecular area, for a surface pressure higher than 30 mN/m, was lower than that observed for the lipid alone. This indicates that the GPx-4 desorption from the interface induced an expulsion of some lipids adsorbed to the protein molecules [23]. Thus, the interactions between the GPx-4 and the DLiPC would appear to be rather strong. The same experiments were performed with GPx-4 injected under PLiPC or DMPC monolayers and as for the DLiPC monolayer, the molecular area increased with adsorption time (data not shown). We conclude that the GPx-4 was adsorbed as well in DLiPC monolayers as in PLiPC or DMPC monolayers. The GPx-1 was injected in the subphase buffer of each lipid monolayer but no molecular area variations were recorded, whatever be the fatty acyl chain composition of
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the phospholipid (data not shown). This indicates that the GPx-1 did not interact with the lipid monolayer. 3.3. π–A isotherms of polyunsaturated lipid monolayers versus time Because the monolayer was not protected from light and oxygen, it should take into account that the lipids were able to oxidize. That is why the successive π–A isotherms of lipid monolayers were measured in order to realize a correction on the molecular area variation due to not only the protein adsorption but also the lipid oxidation. The successive π–A isotherms of DLiPC autoxidation are shown in Fig. 3. The oxidation of DLiPC molecules at the air–water interface led to a dramatic molecular area decrease. Moreover, by calculating the Ks , we can see that the DLiPC autoxidation led to a fluidization of the monolayer (data not shown). The molecular area and Ks decrease is consistent with the results of the DLiPC monolayer UV-oxidation at 30 mN/m [1]. Moreover, even if no UV-irradiation is used, the molecular area decrease reached 6 Å2 /molecule. So it seems that the UV-irradiation enhanced the DLiPC oxidation. Moreover, whatever be the surface pressure and the atmosphere composition, this polyunsaturated phospholipid was able to autoxidize. In our study, the molecular area variation at 30 mN/m after 150 min reached about 30 Å2 /molecule. This value is greater than that observed in another study where the DLiPC monolayer was protected against oxygen and light [1]. Indeed, at zero surface pressure, the polyunsaturated fatty acyl chains were more exposed to the oxygen and light inducing a greater oxidation. Moreover, according to Morandat et al. [1] and Viitala and Peltonen [24], the
Fig. 3. DLiPC monolayer autoxidation at 21 ◦ C. Five nanomoles of DLiPC were spread at the air–water interface and after 20 min solvent evaporation, a π–A isotherm was recorded (black squares). Then, after different time intervals of 30 min (black triangles), 60 min (white squares), 90 min (white triangles), and 150 min (black circles) at zero surface pressure, π–A isotherms were measured. The symbols indicated are only guide to the eyes. All the π–A isotherms measured were not represented for clarity. During the experiment, the interface was not protected from oxygen and light. The subphase buffer was 10 mM Tris–HCl, 150 mM KCl pH 7.2, thermostated at 21 ◦ C.
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Fig. 4. Dependence of the phospholipid unsaturation number on the GPx-4 adsorption. The molecular areas were determined at 5 mN/m on each π–A isotherm of the GPx-4 adsorption into a lipid monolayer. Each value was corrected by the molecular area at the same surface pressure of the lipid only. This molecular area variation obtained was then corrected by the molecular area variation due to the lipid autoxidation. In this, we obtain the corrected A5 mN/m which was plotted versus adsorption time into a DLiPC (circles), PLiPC (squares) or DMPC monolayer (triangles).
polyunsaturated phospholipid oxidation led to the apparition of new molecular species at the air–water interface. In the case of a PLiPC monolayer, the lipid autoxidation led to a weak increase of the molecular area and to a Ks decrease (data not shown), indicating no dramatic physical properties changes. We have shown that successive π–A isotherms did not induce a change of the DMPC monolayer physical properties. 3.4. Dependence of the phospholipid unsaturation number on the GPx-4 adsorption For each kind of phospholipid, the molecular area was measured at 5 mN/m surface pressure after GPx-4 adsorption and it was corrected by those obtained at the same surface pressure for the lipid monolayer before GPx-4 injection. This molecular area variation obtained was then corrected by the molecular area variation due to the lipid autoxidation. In this, we defined a new parameter which is called corrected A5 mN/m . The corrected A5 mN/m was then plotted versus the adsorption time at zero surface pressure and it was represented in Fig. 4. For each phospholipid tested, an increase in corrected A5 mN/m was measured. This corrected A5 mN/m increase was more important in the DLiPC monolayer as compared to other lipids and the less important corrected A5 mN/m increase was observed for the DMPC monolayer. So, we can conclude that the GPx-4 was adsorbed into the three phospholipid monolayers but not with the same affinity. Moreover, the molecular area variation was observed from 10 min after the protein injection, whereas it was longer in the case of the protein adsorption when the surface was not covered by a lipid monolayer (Figs. 1C and 4). This means that the hydrophobic parts of the protein were more stabilized when a lipid film was at the
interface [23]. For each monolayer, the corrected A5 mN/m increase presented several steps. Indeed a slow corrected A5 mN/m increase was observed up to 10 min adsorption, then between 10 and 30 min, the curves presented a sharp corrected A5 mN/m increase. After 30 min protein adsorption, the corrected A5 mN/m increase became slower, and moreover, there is no corrected A5 mN/m variation after 90 min for the PLiPC and DMPC monolayers. It should be remembered that without glutathione, the enzyme was inactive on the phospholipid hydroperoxides formed. In this work, we have used a pure short form of GPx-4, as confirmed by SDS-PAGE electrophoresis and by N-terminal sequencing. Arai et al. [13] showed that the mitochondrial import of GPx-4 23 kDa precursor requires the leader sequence that induces import into mitochondria. Then, this precursor is processed to the 20 kDa mature form within the mitochondria. Our results showed that the GPx-4 short form can interact with membrane lipids. So, it seems evident that the leader sequence of the GPx-4 is not necessary to maintain the interaction between protein and lipids after the maturation has occurred.
4. Conclusions The GPx-4 adsorption at the air–water interface was detected whereas under the same conditions, GPx-1 was not adsorbed. Moreover, the molecular area increased only 15 min after the GPx-4 injection and after an adsorption time of 40 min, all the GPx-4 injected in the subphase buffer seemed to localize at the air–buffer interface. In this report, we have also shown the direct interaction of GPx-4 with phospholipid monolayers. Roveri et al. [16] described that no lipid post-translational modifications such as
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glycosylation, isoprenylation or acylation were detected on GPx-4. This means that no lipid modifications are required to target the GPx-4 to the phospholipid membranes. Nevertheless, we showed that the GPx-4 presented more affinity for lipid membranes than GPx-1. Moreover, the GPx-4 exhibited a higher affinity for the monolayer when it was composed of a potential substrate. Based on the present work, the affinity of GPx-4 was in the order DLiPC > PLiPC > DMPC.
Acknowledgements We would like to thank Dr. Karim El Kirat for helpful discussions. This work was supported by the “Centre National de la Recherche Scientifique” (CNRS), the “Ministère de l’Enseignement Supérieur et de la Recherche” (MESR) and by a grant of the “Région Rhˆone Alpes”. JRA’s lab is funded by the Scottish Executive Environment and Rural Affairs Department.
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