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Cu-catalyzed oxidation of cholesterol in 1,2-dipalmitoyl phosphatidylcholine liposome membrane
Journal of Bioscience and Bioengineering VOL. 109 No. 2, 145 – 148, 2010 www.elsevier.com/locate/jbiosc
Aβ/Cu-catalyzed oxidation of cholesterol in 1,2-dipalmitoyl phosphatidylcholine liposome membrane Toshinori Shimanouchi,1 Makoto Tasaki,1 Huong Thi Vu,1 Haruyuki Ishii,1 Noriko Yoshimoto,2 Hiroshi Umakoshi,1 and Ryoichi Kuboi1,⁎ Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan 1 and Department of Applied Molecular Bioscience, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan 2 Received 10 June 2009; accepted 3 August 2009 Available online 9 September 2009
The amyloid β protein with 42 amino acid residues (Aβ), which is a causative protein of Alzheimer's disease (AD), forms the complex with copper (II) to induce the cholesterol oxidase-like activity by the proton transfer from the cholesterol. In this study, the oxidation of cholesterol by Aβ/Cu complex was investigated on the surface of the zwitterionic phospholipid liposome including the bound water advantageous for the enhancement of the proton transfer. The bound water was pooled by the formation of cholesterol-rich domain within liposomes. The resulting reactivity was enhanced by the proton transfer mediated by the bound water. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Alzheimer's disease; Amyloid β/Cu Complex; Cholesterol; Domain; Liposome]
Amyloid β protein (Aβ) is considered to be the pathological factor for Alzheimer's disease (AD) and has the amyloid fibrilogenesis to induce the cytotoxicity (1). Aβ with 42 amino acid residues (Aβ(142)) is a main component enriched in amyloid plaques in AD (2). Copper ion is also enriched there at relatively high concentration level (Cu ∼ 400 μM) (3). Aβ(1–42) binds to copper ion to form the complex Aβ1Cu2 with very high affinity (4). The bi-nuclear copper complex of Aβ (Aβ/Cu) has been shown to induce the enzyme-like activity (5–7). The cholesterol oxidase-like activity by Aβ/Cu complex has been observed not only in bulk phase (6) but also on the lipid membrane surface (7). Oxidized cholesterol is found to be released from the lipid membrane at its higher rate constant in comparison with cholesterol molecule (8). Considering that the cholesterol content in neuronal cell membrane is linked to the AD pathology (9), a cholesterol oxidation could also be related to the AD. Recent studies on liposome indicate that the cholesterol shows a variety of phase states on the lipid bilayer membrane, such as liquiddisordered, liquid-ordered phases, and so on (10, 11). The cholesterol within the liposome membrane could also affect the activities of membrane-related enzymes, such as phospholipase D (12), cholesterol oxidase (13), gene expression system (14), and Aβ/Cu complex (7). In our previous study, it has been reported that the Aβ/Cu complex-catalyzed oxidation of cholesterol on the liposome membrane was controlled by the membrane structure affected by cholesterol (7). Meanwhile, the effect of a cholesterol-rich domain on the liposome membrane has not been investigated yet. The ⁎ Corresponding author. Tel./fax: +81 6 850 6271. E-mail address: [email protected] (R. Kuboi).
investigation of the Aβ/Cu-catalyzed oxidation of cholesterol on the liposome membrane would contribute to the understanding of the pathological aspect of AD. In this study, we investigated the cholesterol oxidation by the Aβ/ Cu complex on liposome membrane to study the effect of the cholesterol-rich domain on its oxidation. MATERIALS AND METHODS Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Avanti Polar lipids (Alabaster, AL). Cholesterol (Ch) and deuterated water were obtained from Sigma-Aldrich (St. Louis, MO, USA). Amyloid β with 42 amino acid residues (Aβ(1–42)) was purchased from Peptide Institute (Osaka, Japan). Dichlorofluorescein diacetate (DCF) was obtained from Dojindo (Kumamoto, Japan). Other chemicals were in analytical grade. Vesicle preparation Large unilamellar vesicles (LUVs) were prepared by the previous method (7, 15). In brief, a desired quantity of DPPC and Ch was dissolved into chloroform in a rounded bottom flask. The solvent was removed under vacuum using a rotary evaporator. This process was performed twice using chloroform and was pursued once more using chloroform in order to form a homogeneous thin layer of phospholipid on the wall of the flask. For the preparation of the liposome solution, an appropriate quantity of PBS (phosphatebuffered saline) buffer was added into the round bottom flask containing the lipid film and, thereafter, five freezing–thawing cycles were applied before forcing the solution 15 times through a polycarbonate filter (100 nm) using an extruder device (Avestin, LiposoFast) (15). Hydrogen peroxide assay Since the oxidative activity of Aβ/Cu is proportional to the production of H2O2 (5), the H2O2 was measured according to the previous reports (5–7). DCF was dissolved (5 mM) in 100% dimetyl sulfoxide (bubbled with N2 gas for 30 min to remove oxygen), deacetylated with 0.25 M NaOH for 30 min, and then neutralized, pH 7.4, to a final concentration of 1 mM. Horseradish peroxidase (HRP) stock solution was prepared to 1 μM in PBS, pH 7.4. The oxidative reactions for cholesterol in DPPC liposome by Aβ/Cu complex were carried out in PBS buffer, pH 7.4, under ambient conditions. H2O2 concentrations were measured by fluorescence
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spectrometer (FP-6500 SpectroFluoroMeter) (Ex: 485 nm, Em: 520 nm). Aβ was used at 500 nM. During the reaction, no fibril formation was confirmed with SEM observation and thioflavin T fluorescence measurement. FTIR measurement Samples for infrared spectroscopy analysis were prepared in 50-μm-thick cells with CaF2 windows. The infrared spectra were measured with a FT/IR-4100ST (Nihon Bunko Co. Ltd.). The temperature of sample was controlled by means of a block assembly equipped with a circulating water jacket and monitored by a thermosensor placed at the edge of the cell window. The resolution was 4 cm-1. The subtraction of spectra in buffer was carried out to remove the contribution from water bands. The accuracy of the frequency reading was better than ± 0.1 cm- 1.
RESULTS AND DISCUSSION Analysis of cholesterol oxidation on liposome membrane The oxidation of substrate catalyzed by the Aβ/Cu complex has been reported to be mediated by the proton-coupled electron transfer (PCET) mechanism (16). Based on this mechanism, Cu bound to Aβ gets two protons from the cholesterol molecule and brings them to O2 to form H2O2, followed by the cholesterol oxidation accompanied with the transfer of two electrons. The cholesterol oxidation and the reduction of O2 to H2O2 occur by 1:1 stochiometry (5). From the previous reports (5–7), the oxidation of cholesterol catalyzed by the Aβ/Cu complex (500 nM/1 μM) could be estimated as the H2O2 production rate, V. Although the neutral phospholipid liposomes could decompose H2O2 (17), no decomposition of H2O2 by the DPPC liposome membrane was observed for 1 h or so. The 1/V value was plotted against the reciprocal of cholesterol concentration in DPPC/ Ch, 1/S (Fig. 1). In the concentration range of cholesterol within DPPC liposome membrane between 10 and 50 mol%, a linear relationship was observed although some scattering in 33 or 50 mol% was thought to possibly result from the phase transition by cholesterol or the accuracy of the H2O2 measurement in the low substrate concentration. This implies that a cholesterol oxidation by the Aβ/Cu complex on liposome membranes is one step and that no furthermore oxidation occurred within DPPC liposome membrane. The possible oxidized product of this reaction system should show no further oxidation. The 4-cholesten-3-one is one of the products by Aβ/Cucatalyzed oxidation of cholesterol (6). The oxidation of 4-cholesten-3one within DPPC liposome was then examined. A significant reduction in H2O2 production rate was observed (data not shown), implying that the Aβ/Cu complex-catalyzed oxidation of cholesterol produced 4-cholesten-3-one, as a product, by a one-step reaction. This implication was supported by the confirmation with a mass spec-
FIG. 1. Lineweaber–Burk plot on the production rate of hydrogen peroxide associated with Aβ/Cu catalyzed oxidation of cholesterol incorporating into DPPC liposomes (10 to 50 mol%). The diameter of liposomes was 100 nm. All the experiments were performed at 37 °C.
J. BIOSCI. BIOENG., troscopy (data not shown), according to the previous paper (6). In the study with a density functional theory (DFT) calculation, the orientation of a C-3 hydroxyl group of cholesterol or its analogue to binuclear copper ions is indispensable for the cholesterol oxidation (16). Thus, the 4-cholesten-3-one might be a compound disadvantageous for oxidation catalyzed by the Aβ/Cu complex and one of the possible oxidants in DPPC/Ch liposome system. In order to investigate the reactivity of cholesterol oxidation on the liposome membrane, the second-order rate constant, kcat/Km, was estimated from Fig. 1. The stepwise change in the kcat/Km value was observed from 2.2 × 10- 5 nM/min for DPPC/Ch (10–30 mol%) to 5.1 × 10- 5 nM/min for DPPC/Ch (33–50 mol%) as shown in Fig. 2. In the case of DPPC/Ch (10–30 mol%), the kcat/Km value was similar to the value for cholesterol dispersed by sonication in the bulk phase (2.6 × 10- 5 nM/min) (6). In order to investigate the relationship between the reactivity of the Aβ/Cu complex and its orientation on the liposome membrane, the fluidity of the membrane surface was measured by using the fluorescence probe, 1-(4-trimethylamoniumphenyl)- 6-phenyl-1,3,5-hexatriene (TMA-DPH). No significant change in membrane fluidity of the surface of liposome was observed in spite of the addition of the Aβ/Cu complex (data not shown), supporting its peripherally binding on liposome membrane. This result is consistent with the previous finding that the cholesterol inhibited the insertion of Aβ/Cu complex (18). Its orientation on the liposome membrane is advantageous to utilize the bound water for the enhancement of a proton transfer in the PCET process of cholesterol oxidation. Relationship between the bound water and the reactivity of cholesterol oxidation The bound water per a phosphatidylcholine (PC) molecule estimated by Bach and his co-workers (11) was replotted in Fig. 2. This value is an index independent of temperature because this value was obtained from the plot of the enthalpy of melting of water as a function of relative number of water molecules per single phospholipid. The DPPC liposome was considered to hold about 6 to 7 bound waters (non-freezable) per a PC molecule (Fig. 2). The hydration of liposome membrane was also investigated with a FTIR. The peak at around 1230 cm- 1 indicates the asymmetric stretching vibration for PO-2 of phospholipid headgroup (12). The peak shift to lower frequency range was observed in the cholesterolcontaining DPPC liposome (Fig. 3), suggesting that the cholesterol pooled water in the liposome membrane. The variation of the water
FIG. 2. Concentration dependency of cholesterol in DPPC liposome against the secondorder rate constant, the bound water per PC and the kD2O/kH2O value. The diameter of liposomes was 100 nm. The concentrations of Aβ(1–42) and Cu were 200 and 400 nM, respectively. The data on the bound water to PC were referred to the previous report (11). All the experiments were performed at 37 °C, and the data with a standard error were obtained from four independent experiments. so: solid-ordered phase, lo: liquidordered phase.
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FIG. 3. FTIR spectra for DPPC/Ch liposomes. Xch was (a) 0, (b) 10, (c) 33, (d) 40 mol%, respectively. Arrows mean the peak corresponding to the asymmetric stretching vibration for PO-2 of phospholipid headgroup.
content in liposome was corresponding with that of the reactivity of the Aβ/Cu complex on the liposome surface (Fig. 2), indicating the contribution of bound water to liposome membranes. In order to clarify the role of the bound water, the exchange effect of water by D2O on the reactivity was investigated to show the significance of the bound water. The apparent ratio of the rate constant for H2O production k in D2O and H2O (kD2O and kH2O) was measured. The kD2O/kH2O value in bulk solution was larger than that in the presence of liposome. It is strongly suggested that the proton transfer was mediated by the bound water molecules to liposome membranes. It is, therefore, concluded that the bound water to liposome membrane could promote the cholesterol oxidation by the PCET mechanism. The effect of cholesterol-rich domain on liposome membrane The substrate specificity (Km) was estimated from Fig. 1. In a lower
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cholesterol concentration range (10 to 30 mol%), the Km value was found to be approximately 1.0 μM. Above 33 mol% of cholesterol, the value decreased to 0.5 μM. The circular dichroism analysis shows the similarity of the secondary structure of the Aβ/Cu complex, in the presence of DPPC/Ch (up to 30 mol%) with that in bulk solution (data not shown). The liposome with higher membrane fluidity like a 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Ch liposome could strongly interact with the Aβ/Cu complex to accelerate the cholesterol oxidation (the decrease in Km) (7). Meanwhile, in the case of liposome with low membrane fluidity like a DPPC/Ch liposome, it is considered that the change in Km value might result from the association of the Aβ/Cu complex with cholesterol-rich domain on DPPC liposome (Fig. 4). Considering the early work in relation to the phase state of DPPC/Ch liposome (10, 11), it is likely that the Aβ/Cu complex partitioned into the liquid-ordered (lo) phase rather than into the solid-ordered (so) and the so + lo phase of DPPC/Ch liposome. Perspective in AD The present study indicates that the conversion of cholesterol into neurotoxic 4-cholesten-3-one could be promoted on the lipid membrane with cholesterol-rich domain although the possibility of the production of other oxidants including 7-hydroxy-cholesterol (19) could not be excluded. In general, the oxidized cholesterol is rapidly released from the lipid bilayer membrane (8). Thus, the long-termed oxidation of cholesterol would lead to the change in the structure of liposome membrane or its phase state, by means of the release of its oxidant. The cholesterol content for normal brain tissues is 33 mol% (20), and the tissues in AD patients showed the abnormal cholesterol content (21). The increase in cholesterol in lipid membrane (and cholesterol-rich domain as a raft) enhances the production of Aβ from its precursor (22) and inhibits the amyloid fibril formation of Aβ (23). Therefore, we speculated that the Aβ/Cu complex might oxidize the excess cholesterol in membrane to release its oxidant from the neuronal membrane, leading to the suppression of unnecessary Aβ production. It has also been reported that the 4-cholesten-3-one is related to the apoptosis of neuronal cell and the level of 4-cholesten-3one was
FIG. 4. Schematic illustration on Aβ/Cu-catalyzed oxidation of cholesterol within a lipid membrane. Cholesterol content in brain tissue has been reported to be 33 mol% (20).
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higher in AD patients than in normal (6). It is, therefore, likely that the Aβ/Cu complex on the lipid membrane would contribute to the pathological process of AD in a certain manner although it is difficult to exclude the contribution of 4-cholesten-3-one produced by cholesterol oxidase. In conclusion, the bound water molecules, which could enhance the proton transfer, are considered to be pooled in the microdomain formed by cholesterol on liposome membrane. The liposome membrane with the cholesterol-rich domain could regulate the reactivity of the Aβ/Cu-catalyzed oxidation of cholesterol based on the PCET mechanism. ACKNOWLEDGMENTS The authors would thank Mr. Dane Cohen for his experimental contribution. The authors also thank the group of Engineering Science of Liposome and International Seminar between Japan Society for the Promotion of Science (JSPS)-Swiss National Scientific Foundation (SNSF) for the helpful discussion. The author would also thank the group of Membrane Stress Biotechnology. This work was partly supported by Grants-in-Aid for Scientist Research (no.21246121, 20760539, 20360350, 19656220, 19656203) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also partly supported by the Core University Program between JSPS and Vietnamese Academy of Science and Technology (VAST). It was partly supported by the Global COE program “Bio-Environmental Chemistry” of JSPS. The authors also thank the Gas Hydrate Analyzing System of Osaka University. H. Ishii also thanks the JSPS-Fellowship. References 1. Mattson, M. P.: Pathways towards and away from Alzheimer's disease, Nature, 430, 631–639 (2004). 2. Masters, C. L., Multhaup, G., Smith, G., Pottgiesser, J., Martins, R. N., and Beyreuther, K.: Neuronal origin of cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels, EMBO J., 4, 2757–2763 (1985). 3. Smith, M. A., Harris, P. L. R., Sayre, L. M., and Perry, G.: Iron accumulation in Alzheimer disease is a source of redox-generated free radicals, Proc. Natl. Acad. Soc. USA, 94, 9866–9868 (1997). 4. Atwood, C. S., Scarpa, R. C., Huang, X., Moir, R. D., Jones, W. D., Fairlie, D. P., Tanzi, R. E., and Bush, A. I.: Characterization of copper interactions with Alzheimer's amyloid β peptides: identification of an attomolar-affinity copper binding site on amyloid β1–42, J. Neurochemi., 75, 1219–1233 (2000). 5. Opazo, C., Huang, X., Cherny, R. A., Moir, R. D., Roher, A. E., White, A. R., Cappai, R., Masters, C. L., Tanzi, R. E., Inestrosa, N. C., and Bush, A. I.: Metalloenzyme-like activity of Alzheimer's disease β-amyloid, Cu dependent catalytic conversion of
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dopamine, cholesterol, and biological reducing agents to neurotoxic H2O2, J. Biol. Chem., 277, (43) 40302–40308 (2002). Puglielli, L., Friedlich, A. L., Setchell, K. D. R., Nagano, S., Opazo, C., Cherny, R. A., Barnham, K. J., Wade, J. D., Melov, S., Kovacs, D. R. M., and Bush, A. I.: Alzheimer disease β-amyloid activity mimics cholesterol oxidase, J. Clin. Invest., 115, (9) 2556–2663 (2005). Yoshimoto, N., Tasaki, M., Shiamnouchi, T., Umakoshi, H., and Kuboi, R.: Oxidation of cholesterol catalyzed by amyloid β-peptide (Aβ)-Cu complex on lipid membrane, J. Biosci. Bioeng., 100, (4) 455–459 (2005). McLean, L. R. and Phillips, M. C.: Mechanism of cholesterol and phosphatidylcholine exchange or transfer between unilamellar vesicles, Biochemistry, 20, 2893–2900 (1981). Ji, S. R., Wu, Y., and Sui, S. F.: Cholesterol is an important factor affecting the membrane insertion of amyloid peptide (Aβ1–40), which may potentially inhibit the fibril formation, J. Biol. Chem., 277, 6273–6279 (2001). Sankaram, M. B. and Thompson, T. E.: Cholesterol-induced fluid-phase immiscibility in membranes, Proc. Natl. Acad. Sci. USA, 88, 8686–8690 (1991). Bach, D. and Miller, I. R.: Hydoration of phospholipid bilayers in the presence and absence of cholesterol, Chem. Phys. Lipids, 136, 67–72 (2005). Yamamoto, I., Konto, A., Handa, T., and Miyajima, K.: Regulation of phospholipase D activity by neutral lipids in egg-yolk phosphatidylcholine small unilamellar vesicles and by calcium ion in aqueous medium, Biochim. Biophys. Acta, 1233, 21–26 (1995). Ahn, K. W. and Sampson, N. S.: Cholesterol oxidase sense subtle changes in lipid bilayer structure, Biochemistry, 43, 827–836 (2004). Bui, T. H., Umakoshi, H., Ngo, K. X., Nishida, M., Shimanouchi, T., and Kuboi, R.: Liposome membrane itself can affect gene expression in the Escherichia coli cellfree translation system, Langmuir, 24, 10537–10542 (2008). MacDonald, R. C., MacDonald, R. I., Menco, B. Ph M., Takeshita, K., Subbarao, N. K., and Hu, L.: Small-volume extrusion apparatus for preparation of large unilammelar vesicles, Biochim. Biophys. Acta, 1061, 297–303 (1991). Haeffner, F., Smith, D. G., Barnham, K. J., and Bush, A. I.: Model studies of cholesterol and ascorbate oxidation by copper complexes: relevance to Alzheimer's disease β-amyloid metallochemistry, J. Inorg. Biochem., 99, 2403–2442 (2005). Yoshimoto, M., Miyazaki, Y., Umemoto, A., Walde, P., Kuboi, R., and Nakao, K.: Phospholipid vesicle-mediated decomposition of hydrogen peroxide, Langmuir, 23, (18) 9416–9422 (2007). Curtain, C. C., Ali, F. E., Smith, D. G., Bush, A. I., Masters, C. L., and Barnham, K. J.: Metal ions, pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-β peptide with membrane lipid, J. Biol. Chem., 278, 2977–2982 (2003). Nelson, T. J. and Alkon, D. L.: Oxidation of cholesterol by amyloid precursor protein and beta-amyloid, J. Biol. Chem., 280, 7377–7387 (2005). Edidin, M.: The state of lipid rafts: from model membranes to cells, Ann. Rev. Biophys. Biomol. Struct., 32, 257–283 (2003). Chochina, S. V., Avdulov, N. A., Igbavboa, U., Cleary, J. P., O'Hare, E. O., and Wood, W. G.: Amyloid β-peptide1–40 increases neuronal membrane fluidity: role of cholesterol and brain region, J. Lipid Res., 42, 1292–1297 (2001). Abad-Rodriguez, J., Ledesma, M. D., Craessaerts, K., Perga, S., Medina, M., Delacourte, A., Dingwall, C., De Strooper, B., and Gotti, C. G.: Neuronal membrane cholesterol loss enhances amyloid peptide generation, J. Cell Biol., 167, (5) 953–960 (2004). Yip, C. M., Elton, E. A., Darabie, A. A., Morrison, M. R., and McLaurin, J.: Cholesterol, a modulator of membrane-associated Aβ-fibrillogenesis and neurotoxicity, J. Mol. Biol., 311, 723–734 (2001).
Aβ/Cu-catalyzed oxidation of cholesterol in 1,2-dipalmitoyl phosphatidylcholine liposome membrane Toshinori Shimanouchi,1 Makoto Tasaki,1 Huong Thi Vu,1 Haruyuki Ishii,1 Noriko Yoshimoto,2 Hiroshi Umakoshi,1 and Ryoichi Kuboi1,⁎ Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan 1 and Department of Applied Molecular Bioscience, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan 2 Received 10 June 2009; accepted 3 August 2009 Available online 9 September 2009
The amyloid β protein with 42 amino acid residues (Aβ), which is a causative protein of Alzheimer's disease (AD), forms the complex with copper (II) to induce the cholesterol oxidase-like activity by the proton transfer from the cholesterol. In this study, the oxidation of cholesterol by Aβ/Cu complex was investigated on the surface of the zwitterionic phospholipid liposome including the bound water advantageous for the enhancement of the proton transfer. The bound water was pooled by the formation of cholesterol-rich domain within liposomes. The resulting reactivity was enhanced by the proton transfer mediated by the bound water. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Alzheimer's disease; Amyloid β/Cu Complex; Cholesterol; Domain; Liposome]
Amyloid β protein (Aβ) is considered to be the pathological factor for Alzheimer's disease (AD) and has the amyloid fibrilogenesis to induce the cytotoxicity (1). Aβ with 42 amino acid residues (Aβ(142)) is a main component enriched in amyloid plaques in AD (2). Copper ion is also enriched there at relatively high concentration level (Cu ∼ 400 μM) (3). Aβ(1–42) binds to copper ion to form the complex Aβ1Cu2 with very high affinity (4). The bi-nuclear copper complex of Aβ (Aβ/Cu) has been shown to induce the enzyme-like activity (5–7). The cholesterol oxidase-like activity by Aβ/Cu complex has been observed not only in bulk phase (6) but also on the lipid membrane surface (7). Oxidized cholesterol is found to be released from the lipid membrane at its higher rate constant in comparison with cholesterol molecule (8). Considering that the cholesterol content in neuronal cell membrane is linked to the AD pathology (9), a cholesterol oxidation could also be related to the AD. Recent studies on liposome indicate that the cholesterol shows a variety of phase states on the lipid bilayer membrane, such as liquiddisordered, liquid-ordered phases, and so on (10, 11). The cholesterol within the liposome membrane could also affect the activities of membrane-related enzymes, such as phospholipase D (12), cholesterol oxidase (13), gene expression system (14), and Aβ/Cu complex (7). In our previous study, it has been reported that the Aβ/Cu complex-catalyzed oxidation of cholesterol on the liposome membrane was controlled by the membrane structure affected by cholesterol (7). Meanwhile, the effect of a cholesterol-rich domain on the liposome membrane has not been investigated yet. The ⁎ Corresponding author. Tel./fax: +81 6 850 6271. E-mail address: [email protected] (R. Kuboi).
investigation of the Aβ/Cu-catalyzed oxidation of cholesterol on the liposome membrane would contribute to the understanding of the pathological aspect of AD. In this study, we investigated the cholesterol oxidation by the Aβ/ Cu complex on liposome membrane to study the effect of the cholesterol-rich domain on its oxidation. MATERIALS AND METHODS Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Avanti Polar lipids (Alabaster, AL). Cholesterol (Ch) and deuterated water were obtained from Sigma-Aldrich (St. Louis, MO, USA). Amyloid β with 42 amino acid residues (Aβ(1–42)) was purchased from Peptide Institute (Osaka, Japan). Dichlorofluorescein diacetate (DCF) was obtained from Dojindo (Kumamoto, Japan). Other chemicals were in analytical grade. Vesicle preparation Large unilamellar vesicles (LUVs) were prepared by the previous method (7, 15). In brief, a desired quantity of DPPC and Ch was dissolved into chloroform in a rounded bottom flask. The solvent was removed under vacuum using a rotary evaporator. This process was performed twice using chloroform and was pursued once more using chloroform in order to form a homogeneous thin layer of phospholipid on the wall of the flask. For the preparation of the liposome solution, an appropriate quantity of PBS (phosphatebuffered saline) buffer was added into the round bottom flask containing the lipid film and, thereafter, five freezing–thawing cycles were applied before forcing the solution 15 times through a polycarbonate filter (100 nm) using an extruder device (Avestin, LiposoFast) (15). Hydrogen peroxide assay Since the oxidative activity of Aβ/Cu is proportional to the production of H2O2 (5), the H2O2 was measured according to the previous reports (5–7). DCF was dissolved (5 mM) in 100% dimetyl sulfoxide (bubbled with N2 gas for 30 min to remove oxygen), deacetylated with 0.25 M NaOH for 30 min, and then neutralized, pH 7.4, to a final concentration of 1 mM. Horseradish peroxidase (HRP) stock solution was prepared to 1 μM in PBS, pH 7.4. The oxidative reactions for cholesterol in DPPC liposome by Aβ/Cu complex were carried out in PBS buffer, pH 7.4, under ambient conditions. H2O2 concentrations were measured by fluorescence
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.08.003
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spectrometer (FP-6500 SpectroFluoroMeter) (Ex: 485 nm, Em: 520 nm). Aβ was used at 500 nM. During the reaction, no fibril formation was confirmed with SEM observation and thioflavin T fluorescence measurement. FTIR measurement Samples for infrared spectroscopy analysis were prepared in 50-μm-thick cells with CaF2 windows. The infrared spectra were measured with a FT/IR-4100ST (Nihon Bunko Co. Ltd.). The temperature of sample was controlled by means of a block assembly equipped with a circulating water jacket and monitored by a thermosensor placed at the edge of the cell window. The resolution was 4 cm-1. The subtraction of spectra in buffer was carried out to remove the contribution from water bands. The accuracy of the frequency reading was better than ± 0.1 cm- 1.
RESULTS AND DISCUSSION Analysis of cholesterol oxidation on liposome membrane The oxidation of substrate catalyzed by the Aβ/Cu complex has been reported to be mediated by the proton-coupled electron transfer (PCET) mechanism (16). Based on this mechanism, Cu bound to Aβ gets two protons from the cholesterol molecule and brings them to O2 to form H2O2, followed by the cholesterol oxidation accompanied with the transfer of two electrons. The cholesterol oxidation and the reduction of O2 to H2O2 occur by 1:1 stochiometry (5). From the previous reports (5–7), the oxidation of cholesterol catalyzed by the Aβ/Cu complex (500 nM/1 μM) could be estimated as the H2O2 production rate, V. Although the neutral phospholipid liposomes could decompose H2O2 (17), no decomposition of H2O2 by the DPPC liposome membrane was observed for 1 h or so. The 1/V value was plotted against the reciprocal of cholesterol concentration in DPPC/ Ch, 1/S (Fig. 1). In the concentration range of cholesterol within DPPC liposome membrane between 10 and 50 mol%, a linear relationship was observed although some scattering in 33 or 50 mol% was thought to possibly result from the phase transition by cholesterol or the accuracy of the H2O2 measurement in the low substrate concentration. This implies that a cholesterol oxidation by the Aβ/Cu complex on liposome membranes is one step and that no furthermore oxidation occurred within DPPC liposome membrane. The possible oxidized product of this reaction system should show no further oxidation. The 4-cholesten-3-one is one of the products by Aβ/Cucatalyzed oxidation of cholesterol (6). The oxidation of 4-cholesten-3one within DPPC liposome was then examined. A significant reduction in H2O2 production rate was observed (data not shown), implying that the Aβ/Cu complex-catalyzed oxidation of cholesterol produced 4-cholesten-3-one, as a product, by a one-step reaction. This implication was supported by the confirmation with a mass spec-
FIG. 1. Lineweaber–Burk plot on the production rate of hydrogen peroxide associated with Aβ/Cu catalyzed oxidation of cholesterol incorporating into DPPC liposomes (10 to 50 mol%). The diameter of liposomes was 100 nm. All the experiments were performed at 37 °C.
J. BIOSCI. BIOENG., troscopy (data not shown), according to the previous paper (6). In the study with a density functional theory (DFT) calculation, the orientation of a C-3 hydroxyl group of cholesterol or its analogue to binuclear copper ions is indispensable for the cholesterol oxidation (16). Thus, the 4-cholesten-3-one might be a compound disadvantageous for oxidation catalyzed by the Aβ/Cu complex and one of the possible oxidants in DPPC/Ch liposome system. In order to investigate the reactivity of cholesterol oxidation on the liposome membrane, the second-order rate constant, kcat/Km, was estimated from Fig. 1. The stepwise change in the kcat/Km value was observed from 2.2 × 10- 5 nM/min for DPPC/Ch (10–30 mol%) to 5.1 × 10- 5 nM/min for DPPC/Ch (33–50 mol%) as shown in Fig. 2. In the case of DPPC/Ch (10–30 mol%), the kcat/Km value was similar to the value for cholesterol dispersed by sonication in the bulk phase (2.6 × 10- 5 nM/min) (6). In order to investigate the relationship between the reactivity of the Aβ/Cu complex and its orientation on the liposome membrane, the fluidity of the membrane surface was measured by using the fluorescence probe, 1-(4-trimethylamoniumphenyl)- 6-phenyl-1,3,5-hexatriene (TMA-DPH). No significant change in membrane fluidity of the surface of liposome was observed in spite of the addition of the Aβ/Cu complex (data not shown), supporting its peripherally binding on liposome membrane. This result is consistent with the previous finding that the cholesterol inhibited the insertion of Aβ/Cu complex (18). Its orientation on the liposome membrane is advantageous to utilize the bound water for the enhancement of a proton transfer in the PCET process of cholesterol oxidation. Relationship between the bound water and the reactivity of cholesterol oxidation The bound water per a phosphatidylcholine (PC) molecule estimated by Bach and his co-workers (11) was replotted in Fig. 2. This value is an index independent of temperature because this value was obtained from the plot of the enthalpy of melting of water as a function of relative number of water molecules per single phospholipid. The DPPC liposome was considered to hold about 6 to 7 bound waters (non-freezable) per a PC molecule (Fig. 2). The hydration of liposome membrane was also investigated with a FTIR. The peak at around 1230 cm- 1 indicates the asymmetric stretching vibration for PO-2 of phospholipid headgroup (12). The peak shift to lower frequency range was observed in the cholesterolcontaining DPPC liposome (Fig. 3), suggesting that the cholesterol pooled water in the liposome membrane. The variation of the water
FIG. 2. Concentration dependency of cholesterol in DPPC liposome against the secondorder rate constant, the bound water per PC and the kD2O/kH2O value. The diameter of liposomes was 100 nm. The concentrations of Aβ(1–42) and Cu were 200 and 400 nM, respectively. The data on the bound water to PC were referred to the previous report (11). All the experiments were performed at 37 °C, and the data with a standard error were obtained from four independent experiments. so: solid-ordered phase, lo: liquidordered phase.
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FIG. 3. FTIR spectra for DPPC/Ch liposomes. Xch was (a) 0, (b) 10, (c) 33, (d) 40 mol%, respectively. Arrows mean the peak corresponding to the asymmetric stretching vibration for PO-2 of phospholipid headgroup.
content in liposome was corresponding with that of the reactivity of the Aβ/Cu complex on the liposome surface (Fig. 2), indicating the contribution of bound water to liposome membranes. In order to clarify the role of the bound water, the exchange effect of water by D2O on the reactivity was investigated to show the significance of the bound water. The apparent ratio of the rate constant for H2O production k in D2O and H2O (kD2O and kH2O) was measured. The kD2O/kH2O value in bulk solution was larger than that in the presence of liposome. It is strongly suggested that the proton transfer was mediated by the bound water molecules to liposome membranes. It is, therefore, concluded that the bound water to liposome membrane could promote the cholesterol oxidation by the PCET mechanism. The effect of cholesterol-rich domain on liposome membrane The substrate specificity (Km) was estimated from Fig. 1. In a lower
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cholesterol concentration range (10 to 30 mol%), the Km value was found to be approximately 1.0 μM. Above 33 mol% of cholesterol, the value decreased to 0.5 μM. The circular dichroism analysis shows the similarity of the secondary structure of the Aβ/Cu complex, in the presence of DPPC/Ch (up to 30 mol%) with that in bulk solution (data not shown). The liposome with higher membrane fluidity like a 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Ch liposome could strongly interact with the Aβ/Cu complex to accelerate the cholesterol oxidation (the decrease in Km) (7). Meanwhile, in the case of liposome with low membrane fluidity like a DPPC/Ch liposome, it is considered that the change in Km value might result from the association of the Aβ/Cu complex with cholesterol-rich domain on DPPC liposome (Fig. 4). Considering the early work in relation to the phase state of DPPC/Ch liposome (10, 11), it is likely that the Aβ/Cu complex partitioned into the liquid-ordered (lo) phase rather than into the solid-ordered (so) and the so + lo phase of DPPC/Ch liposome. Perspective in AD The present study indicates that the conversion of cholesterol into neurotoxic 4-cholesten-3-one could be promoted on the lipid membrane with cholesterol-rich domain although the possibility of the production of other oxidants including 7-hydroxy-cholesterol (19) could not be excluded. In general, the oxidized cholesterol is rapidly released from the lipid bilayer membrane (8). Thus, the long-termed oxidation of cholesterol would lead to the change in the structure of liposome membrane or its phase state, by means of the release of its oxidant. The cholesterol content for normal brain tissues is 33 mol% (20), and the tissues in AD patients showed the abnormal cholesterol content (21). The increase in cholesterol in lipid membrane (and cholesterol-rich domain as a raft) enhances the production of Aβ from its precursor (22) and inhibits the amyloid fibril formation of Aβ (23). Therefore, we speculated that the Aβ/Cu complex might oxidize the excess cholesterol in membrane to release its oxidant from the neuronal membrane, leading to the suppression of unnecessary Aβ production. It has also been reported that the 4-cholesten-3-one is related to the apoptosis of neuronal cell and the level of 4-cholesten-3one was
FIG. 4. Schematic illustration on Aβ/Cu-catalyzed oxidation of cholesterol within a lipid membrane. Cholesterol content in brain tissue has been reported to be 33 mol% (20).
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higher in AD patients than in normal (6). It is, therefore, likely that the Aβ/Cu complex on the lipid membrane would contribute to the pathological process of AD in a certain manner although it is difficult to exclude the contribution of 4-cholesten-3-one produced by cholesterol oxidase. In conclusion, the bound water molecules, which could enhance the proton transfer, are considered to be pooled in the microdomain formed by cholesterol on liposome membrane. The liposome membrane with the cholesterol-rich domain could regulate the reactivity of the Aβ/Cu-catalyzed oxidation of cholesterol based on the PCET mechanism. ACKNOWLEDGMENTS The authors would thank Mr. Dane Cohen for his experimental contribution. The authors also thank the group of Engineering Science of Liposome and International Seminar between Japan Society for the Promotion of Science (JSPS)-Swiss National Scientific Foundation (SNSF) for the helpful discussion. The author would also thank the group of Membrane Stress Biotechnology. This work was partly supported by Grants-in-Aid for Scientist Research (no.21246121, 20760539, 20360350, 19656220, 19656203) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also partly supported by the Core University Program between JSPS and Vietnamese Academy of Science and Technology (VAST). It was partly supported by the Global COE program “Bio-Environmental Chemistry” of JSPS. The authors also thank the Gas Hydrate Analyzing System of Osaka University. H. Ishii also thanks the JSPS-Fellowship. References 1. Mattson, M. P.: Pathways towards and away from Alzheimer's disease, Nature, 430, 631–639 (2004). 2. Masters, C. L., Multhaup, G., Smith, G., Pottgiesser, J., Martins, R. N., and Beyreuther, K.: Neuronal origin of cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels, EMBO J., 4, 2757–2763 (1985). 3. Smith, M. A., Harris, P. L. R., Sayre, L. M., and Perry, G.: Iron accumulation in Alzheimer disease is a source of redox-generated free radicals, Proc. Natl. Acad. Soc. USA, 94, 9866–9868 (1997). 4. Atwood, C. S., Scarpa, R. C., Huang, X., Moir, R. D., Jones, W. D., Fairlie, D. P., Tanzi, R. E., and Bush, A. I.: Characterization of copper interactions with Alzheimer's amyloid β peptides: identification of an attomolar-affinity copper binding site on amyloid β1–42, J. Neurochemi., 75, 1219–1233 (2000). 5. Opazo, C., Huang, X., Cherny, R. A., Moir, R. D., Roher, A. E., White, A. R., Cappai, R., Masters, C. L., Tanzi, R. E., Inestrosa, N. C., and Bush, A. I.: Metalloenzyme-like activity of Alzheimer's disease β-amyloid, Cu dependent catalytic conversion of
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