Tài liệu Báo cáo khoa học: Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane pdf

Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane Olga Nikolaeva, Yusuke Takahashi, Gennadiy Moiseyev and Jian-xing Ma Departments of Cell Biology and Medicine Endocrinology, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, OK, USA Keywords isomerohydrolase; liposome; retina; retinyl ester; RPE65 Correspondence G Moiseyev, Departments of Cell Biology and Medicine Endocrinology, Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, 941 Stanton L Young blvd, BSEB 302, Oklahoma City, OK 73104, USA Fax: +1 405 271 3973 Tel: +1 405 2718001 (ext 48443) E-mail: gennadiy-moiseyev@ouhsc.edu (Received February 2009, revised 23 March 2009, accepted 24 March 2009) Generation of 11-cis-retinol from all-trans-retinyl ester in the retinal pigment epithelium is a critical step in the visual cycle and is essential for perception of light Recent findings from cell culture models suggest that protein RPE65 is the retinoid isomerohydrolase that catalyzes the reaction However, previous attempts to detect the enzymatic activity of purified RPE65 were unsuccessful, and thus its enzymatic function remains controversial Here, we developed a novel liposome-based assay for isomerohydrolase activity The results showed that purified recombinant chicken RPE65 had a high affinity for all-trans-retinyl palmitate-containing liposomes and demonstrated a robust isomerohydrolase activity Furthermore, we found that all-trans-retinyl ester must be incorporated into the phospholipid membrane to serve as a substrate for isomerohydrolase This assay system using purified RPE65 enabled us to measure kinetic parameters for the enzymatic reaction catalyzed by RPE65 These results provide conclusive evidence that RPE65 is the isomerohydrolase of the visual cycle doi:10.1111/j.1742-4658.2009.07021.x In vertebrates, both rod and cone visual pigments require 11-cis-retinal as a chromophore [1] Upon absorption of photon, 11-cis-retinal is photoisomerized to all-trans-retinal, which triggers the conformational change of opsin and subsequently activates the G-protein transducin and initiates vision [2,3] The process of recycling 11-cis-retinal, termed the visual cycle (Fig 1), is essential for the regeneration of visual pigments [4,5] All-trans-retinal generated by photoactivation is dissociated from opsin and converted to all-trans-retinol by retinol dehydrogenase [6] The all-trans-retinol is then exported from photoreceptors to the retinal pigment epithelium (RPE), and all-transretinol is esterified by lecithin:retinol acyltransferase (LRAT) to all-trans-retinyl esters [7] The key enzyme of the visual cycle, isomerohydrolase (EC 5.2.1.7), processes all-trans-retinyl esters into 11-cis-retinol [8] It has been proposed that the free energy generated from ester hydrolysis is probably used by the enzyme to drive a thermodynamically uphill trans–cis isomerization of the retinoid double bond [9] The chemical nature of the isomerohydrolase has been undetermined thus far RPE65 is a membrane-associated protein expressed predominantly in the RPE [10] The molecular mass of bovine RPE65 as determined by MS is 61 961 Da; this is higher than its calculated value (60 944 Da) based on the derived amino acid sequence [11], indicating post-translational modifications [12] Hydropathy analysis of the RPE65 amino acid sequence revealed no obvious hydrophobic transmembrane domains [10] In Rpe65) ⁄ ) mice, it has been shown that 11-cis-retinoids are absent in the retina, and rhodopsin regeneration is thus impaired, suggesting that RPE65 is essential for Abbreviations Ad-RPE65, adenovirus expressing RPE65; LRAT, lecithin:retinol acyltransferase; MOI, multiplicity of infection; PC, phosphatidylcholine; RPE, retinal pigment epithelium 3020 FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS O Nikolaeva et al Isomerohydrolase activity of purified RPE65 Fig Scheme of retinoid visual cycle visual pigment regeneration in vivo [13] Mutations in the RPE65 gene have been linked to Leber’s congenital amaurosis, which is an inherited disease characterized by blindness at birth [14,15] Recently, we and two other groups reported that isomerohydrolase activity was detected in cultured cells that coexpress both RPE65 and LRAT, suggesting that RPE65 is the isomerohydrolase [16–18] However, as isomerohydrolase activity has never been shown using purified RPE65, there is skepticism about whether RPE65 is indeed the isomerohydrolase [19] Two groups have reported independently that purified RPE65 is a retinyl ester-binding protein [20,21] These studies led to speculation that RPE65 is not the isomerohydrolase itself, but rather that it is required to present the insoluble retinyl ester to some ubiquitous isomerohydrolase [20] In this study, we purified recombinant chicken RPE65 to apparent homogeneity and demonstrated its isomerohydrolase activity, exploiting a novel enzymatic assay system that utilizes all-trans-retinyl palmitate incorporated into liposomes Chicken RPE65 was selected as the ideal homolog for this study, because we have shown previously that chicken RPE65 has higher expression levels than the human homolog and higher isomerohydrolase activity than both the human and the bovine homologs [22] Using this system, we have performed a kinetic analysis of the enzymatic activity of purified RPE65 Results Expression and solubilization of recombinant RPE65 with isomerohydrolase activity To optimize the expression of chicken RPE65, 293ALRAT cells were infected with different titers of adenovirus expressing RPE65 (Ad-RPE65) [multiplicity of infection (MOI) 5–500] and harvested 24 h after infection The cells were disrupted by sonication, and RPE65 was solubilized using Chaps RPE65 expression levels were evaluated by western blot analysis, using the same amount (20 lg) of either total cellular protein (Fig 2A) or the Chaps-soluble fraction (Fig 2B) As shown by western blot analysis, RPE65 expression levels increased with MOI, and reached a plateau at MOI 150–500 both in total cell homogenates and in the Chaps-soluble fractions The cells expressing RPE65 were treated with different concentrations of Chaps to determine the optimal amount for solubilizing RPE65 As shown by western blot analysis, Chaps at concentrations of 0.1–0.5% solubilized significant amounts of recombinant RPE65 in the cells, whereas lower concentrations of the detergent did not adequately solubilize RPE65 from the membrane (Fig 2C) We also determined the effect of increasing Chaps concentrations on the enzymatic activity of RPE65 For these measurements, a novel enzymatic activity FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS 3021 Isomerohydrolase activity of purified RPE65 O Nikolaeva et al A D 80 60 RPE65 50 β-actin 50 10 15 20 30 50 BM F 25 40 A320 (1 × 10–2 AU) Total cell lysate kDa 3.0 0% CHAPS 2.0 1.0 0 10 MOI 15 20 25 Time (min) E CHAPS soluble fraction 80 60 RPE65 50 β-actin 15 20 30 50 3.0 0.5% CHAPS 2.0 1.0 10 50 25 40 A320 (1 × 10–2 AU) B kDa MOI 10 15 20 25 Time (min) F 80 RPE65 60 50 β-actin 01 0 00 40 11-cis retinol (pmol) C kDa 600 500 400 300 200 100 0 CHAPS (%) 0.1 0.2 0.3 0.4 0.5 0.6 CHAPS (%) Fig Optimization of expression and solubilization of recombinant RPE65 (A, B) The 293A-LRAT cells were infected with Ad-RPE65 with increasing MOI, and harvested at 24 h after infection Equal amounts (20 lg) of proteins from total cell lysates (A) and the Chaps (0.1%)-solubilized supernatant after ultracentrifugation (B) were analyzed by western blot analysis using an antibody specific for RPE65, and normalized by b-actin levels Proteins of the bovine RPE microsomal fraction (1 lg) were included as a control (C) To determine the effects of Chaps concentration on RPE65 solubility, the cells were infected with Ad-RPE65 at MOI 100, and harvested 24 h after infection The cell lysates were incubated with increasing concentrations of Chaps for h at °C, and then centrifuged at 200 000 g for 30 Equal amounts (2 lg) of total proteins from the supernatant fractions were blotted with antibody against RPE65 (D, E) The effect of Chaps concentration on the isomerohydrolase activity of RPE65 was evaluated using in vitro isomerohydrolase assays Liposomes preloaded with the all-trans-retinyl ester (50 lM lipids, 0.66 lM all-trans-retinyl palmitate) were incubated with 500 lg of total proteins from the cells expressing RPE65 in the presence of 0% (D) or 0.5% (E) Chaps for h The generated retinoids were analyzed by HPLC, and peaks were identified as follows: 1, retinyl esters; 2, all-trans-retinal; and 3, 11-cis-retinol (F) Dependence of the isomerohydrolase activity on Chaps concentration was measured for total cell lysates (4) and Chaps-soluble fractions ( ) and plotted The activity was calculated from the peak areas of the generated 11-cis-retinol in HPLC profiles (mean ± standard deviation, n = 3) assay with liposomes containing all-trans-retinyl ester was developed (see Experimental procedures) In the absence of Chaps, incubation of the total cell homogenate expressing RPE65 with all-trans-retinyl palmitate incorporated into liposomes generated a significant amount of 11-cis-retinol (Fig 2D) The addition of 0.5% Chaps to the assay system almost completely abolished the 11-cis-retinol formation (Fig 2E) To define the Chaps concentration that sufficiently solubilizes RPE65 while preserving its enzymatic activity, we measured the dependence of isomerohydrolase activity on Chaps concentration, both for total 293A- 3022 LRAT cell homogenates expressing RPE65 and for Chaps-solubilized fractions (Fig 2F) For total cell lysates, the production of 11-cis-retinol gradually decreased with increasing Chaps concentrations When the Chaps-soluble fractions were used for the isomerohydrolase assay, an initial plateau of enzymatic activity was observed up to 0.1% of Chaps In line with previous data [20], 11-cis-retinol generation was drastically decreased by 0.3% Chaps (Fig 2F) Taken together, these results suggest that 0.1% Chaps is optimal for solubilizing RPE65 while preserving its enzymatic activity, and this concentration was therefore employed FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS O Nikolaeva et al for the following RPE65 purification and enzymatic assays Isomerohydrolase activity of purified RPE65 A kDa Purification of recombinant RPE65 To facilitate the purification of recombinant chicken RPE65, a histidine-hexamer tag (6 · His) was fused to the N-terminus of RPE65 and expressed using Ad-RPE65 at MOI 500 The recombinant RPE65 was solubilized with 0.1% Chaps and purified through an Ni2+–nitrilotriacetic acid column The purified RPE65 appeared to be homogeneous, as shown by SDS ⁄ PAGE followed by Coomassie Brilliant Blue staining (Fig 3A) The identity of the purified RPE65 was confirmed by western blot analysis, using antibodies against RPE65 (Fig 3B) and the His-tag (Fig 3C) Purified RPE65 showed isomerohydrolase activity that was dependent upon association with liposomes Although all-trans-retinyl ester has been established as the substrate of the isomerohydrolase [24], the poor solubility of hydrophobic all-trans-retinyl ester has historically hindered its use as a substrate for assays of isomerohydrolase activity In this study, a novel isomerohydrolase activity assay was developed in which alltrans-retinyl ester was incorporated into liposomes, and all-trans-retinyl ester-containing liposomes were then used as the substrate for measuring the isomero- RPE65 50 37 25 20 B kDa 5 220 120 100 80 60 50 Reassociation of RPE65 with a phospholipid membrane To investigate the interaction of RPE65 with the lipid membrane, we performed a liposome flotation assay Using this technique, others have shown that centrifugal force causes liposomes to float to the top of the sucrose gradient, owing to inherent buoyancy, separating liposomes from unbound protein, which remains in the bottom fractions [23] All-trans-retinyl palmitate was incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine ⁄ 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes at a lipid ⁄ retinyl palmitate ratio of 75 : After centrifugation, liposomes were predominantly present in the fractions from the top of the gradient, regardless of the presence of RPE65 protein (Fig 4A) In the absence of liposomes, RPE65 was located only in the bottom fractions of the gradient (Fig 4D,E) However, in the presence of liposomes, significant amounts of RPE65 floated to the top of the gradient (Fig 4B,C), demonstrating that RPE65 efficiently binds to liposomes containing all-trans-retinyl palmitate 250 150 100 75 40 30 C kDa 220 120 100 80 60 50 40 30 20 Fig Purification of recombinant RPE65 The 293A-LRAT cells were infected with Ad-RPE65 expressing chicken RPE65 at an MOI of 500 RPE65 was solubilized by 0.1% Chaps and purified by Ni2+–nitrilotriacetic acid affinity chromatography (A) SDS ⁄ PAGE with Coomassie Brilliant Blue staining (B) Western blot analysis with antibody specific for RPE65 (C) Western blot analysis with antibody specific for the His-tag Lane 1: total cell lysate Lane 2: Chaps-solubilized supernatant after centrifugation at 200 000 g for h Lane 3: flow-through fraction not bound to the Ni2+–nitrilotriacetic acid column Lane 4: purified recombinant chicken RPE65 Lane 5: bovine RPE microsomal proteins The amounts of protein used for SDS ⁄ PAGE were 20 lg for lanes 1, 2, 3, and 5, and lg for lane For western blot analysis, the amount of protein was 500 ng for each lane hydrolase activity of purified RPE65 As shown in Fig 5A, incubation of purified RPE65 with the liposomes containing all-trans-retinyl palmitate generated a FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS 3023 Isomerohydrolase activity of purified RPE65 O Nikolaeva et al A D B kDa P kDa 80 P 80 60 60 E C D 2.5 A320 (1 × 10–2 AU) A320 (1 × 10–2 AU) A 2.0 1.5 1.0 0.5 0 10 15 20 1.2 1.0 0.8 0.6 0.4 0.2 0 25 10 Time (min) A320 (1 × 10–2 AU) E Absorbance (1 × 10–3 AU) 2.0 1.5 1.0 0.5 280 300 320 340 360 1.2 25 0.8 0.6 0.4 0.2 0 380 10 15 20 25 20 25 Time (min) F 1.2 A320 (1 × 10–2 AU) A320 (1 × 10–2 AU) 20 1.0 Wavelength (nm) 1.0 0.8 0.6 0.4 0.2 0 10 15 Time (min) 3024 15 Time (min) B C Fig Interaction of purified RPE65 with liposomes Purified RPE65 protein (25 lg) was incubated with the 14C-labeled liposomes (100 lM lipids, 1.3 lM all-trans-retinyl palmitate) for h at 37 °C The mixture was placed at the bottom of a sucrose gradient and centrifuged Six 500 lL fractions were collected from the top of the gradient (A) The lipid amount in each flotation fraction was quantified by scintillation counting of [14C]PC and expressed as a percentage of the total amount of [14C]PC in the gradient (means ± standard deviation, n = 3) (B–E) Purified RPE65 was incubated with (B, C) and without (D, E) 14C-labeled liposomes, and centrifuged in the gradient as described above The same volumes from each fraction (30 lL) and pellets (6 lL) were examined by western blot analysis using antibody against RPE65 RPE65 levels in each of the flotation fractions with liposomes (C) and without liposomes (E) were quantified by densitometry and averaged from three independent experiments (mean ± standard deviation, n = 3) 20 25 12 10 2 0 10 15 Time (min) Fig Isomerohydrolase activity of purified RPE65 reconstituted in liposomes Purified RPE65 (25 lg) was incubated with the following substrates in the presence of 25 lM cellular retinaldehyde-binding protein and 0.5% BSA for h at 37 °C The generated retinoids were analyzed by HPLC (A) Ten microliters of liposomes (250 lM lipids, 3.3 lM all-trans-retinyl palmitate); (B) UV absorbance spectrum recorded for the indicated 11-cis-retinol peak from (A); (C) no substrate was added; (D) 3.3 lM all-transretinol; (E) 3.3 lM all-trans-retinyl palmitate added in lL of N,N-dimethylformamide without liposomes; (F) 10 lL of liposomes (250 lM lipids, 3.3 lM all-trans-retinyl palmitate) in the absence of RPE65 protein Peaks were identified as follows: 1, retinyl esters; 2, all-trans-retinal; 3, 11-cis-retinol; 4, 13-cis-retinol; 5, all-trans-retinol FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS O Nikolaeva et al Kinetics of the isomerohydrolase activity of purified RPE65 To determine the steady-state kinetics of RPE65 activity, the assay conditions were optimized to ensure that measurements were taken within the linear range First, we plotted the time course of 11-cis-retinol generation after incubation of the liposomes containing all-transretinyl palmitate with 25 lg of purified RPE65 for various time intervals The time course of 11-cis-retinol production was linear in its initial period (Fig 6A), and all of the further experiments in this study were therefore conducted within this range Second, to establish the dependence of 11-cis-retinol production on the concentration of purified RPE65, the liposomes containing all-trans-retinyl palmitate were incubated with increasing amounts of purified RPE65 The production of 11-cis-retinol was found to be a linear function of RPE65 concentration within a range of 20–250 lgỈmL)1 RPE65 (Fig 6B) Finally, to analyze the substrate dependence of the RPE65 isomerohydrolase, we measured the initial reaction velocity using different concentrations of retinyl ester incorporated into the liposomes Lineweaver–Burk analysis of these data yielded the 11-cis retinol (pmol) A 600 500 400 300 200 100 0 20 40 60 80 100 120 140 Time (min) 11-cis retinol production rate (pmol·h–1) B 300 250 200 150 100 50 0 50 100 150 200 250 300 RPE65 (µg·mL–1) C 0.35 1/V (pmol–1·min·mg) significant amount of 11-cis-retinol (Fig 5A) The identity of the 11-cis-retinol peak was validated by recording the UV spectrum during chromatography (kmax = 319 nm) (Fig 5B) and also confirmed by coelution with the 11-cis-retinol standard (data not shown) As a control, no 11-cis-retinol was generated when the purified RPE65 was incubated alone in the absence of the added liposomes (Fig 5C), suggesting that the purified recombinant protein did not contain endogenous all-trans-retinyl ester To exclude the possibility that trace amounts of LRAT were copurified with RPE65, all-trans-retinol was examined as a substrate Neither retinyl ester nor 11-cis-retinol was produced after incubation of all-trans-retinol with the purified RPE65 (Fig 5D), confirming that LRAT activity was absent from the system This result also provides further evidence confirming that all-trans-retinol is not an intrinsic substrate for RPE65 When the liposomes containing all-trans-retinyl palmitate were incubated in the absence of RPE65, no 11-cis-retinol was generated (Fig 5E), verifying that nonspecific thermal isomerization did not occur Interestingly, in the absence of liposomes, RPE65 did not generate 11-cis-retinol from nonincorporated all-transretinyl palmitate (Fig 5F) These results indicate that association of RPE65 with liposomes containing the retinyl ester substrate is essential for the efficient isomerohydrolase activity of RPE65 Isomerohydrolase activity of purified RPE65 0.30 0.25 0.20 0.15 0.10 0.05 0 1/S (µM–1) Fig Kinetic analysis of isomerohydrolase activity of purified RPE65 (A) Time course of 11-cis-retinol generation Liposomes containing all-trans-retinyl palmitate (250 lM lipids, 3.3 lM all-transretinyl ester) were incubated with purified RPE65 (25 lg) for the indicated time intervals, and the generated 11-cis-retinol was quantified by HPLC (B) Dependence of isomerohydrolase activity on RPE65 protein concentration Various amounts of purified RPE65, as indicated, were incubated with liposomes (250 lM lipids, 3.3 lM all-trans-retinyl palmitate) for h The 11-cis-retinol generated from the reaction was calculated from the area of the 11-cis-retinol peak (mean ± standard deviation, n = 3) (C) Lineweaver–Burk plot of 11-cis-retinol generation by RPE65 Liposomes with increasing concentrations (S) of all-trans-retinyl palmitate were incubated with equal amounts of purified RPE65 (25 lg) Initial rates (V) of 11-cis-retinol generation were calculated according to 11-cis-retinol production recorded by HPLC kinetic parameters kcat and Km for this reaction: the Michaelis constant (Km) was 3.7 lm and the turnover number (kcat) was 1.45 · 10)4 s)1 for purified RPE65 (Fig 6C) FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS 3025 Isomerohydrolase activity of purified RPE65 O Nikolaeva et al Discussion A key step of the retinoid visual cycle is the conversion of all-trans-retinyl ester to 11-cis-retinol, which is catalyzed by isomerohydrolase Although the isomerohydrolase activity was first reported over 20 years ago [25], the enzyme has eluded definite identification until now Recently, we and others [16–18] have shown that cell lysates coexpressing RPE65 and LRAT can generate 11-cis-retinol from all-trans-retinol, assuming that the long-sought isomerohydrolase in the visual cycle is RPE65 As the isomerohydrolase activity has never been demonstrated in purified RPE65, some researchers in this field are still not convinced that RPE65 is the isomerohydrolase [20] The present study established a novel in vitro isomerohydrolase assay that utilizes all-trans-retinyl ester incorporated into liposomes as substrate for the isomerohydrolase Using this assay, we demonstrated that purified RPE65, when reassociated with lipid membranes, directly converts all-trans-retinyl ester to 11-cis-retinol, leading to the conclusion that RPE65 is the isomerohydrolase Furthermore, this enzymatic activity assay allowed us to measure the kinetic parameters of purified RPE65 A major reason why previous studies failed to demonstrate isomerohydrolase activity using purified RPE65 is that the isomerohydrolase activity of the protein is highly sensitive to all detergents that were previously used for solubilization of RPE65 [26] In addition, early attempts to detect the isomerohydrolase activity of RPE65 were complicated because both all-trans-retinol and retinyl ester were proposed as possible substrates for the isomerase [8,27] Although we later established that all-trans-retinyl ester is the substrate for isomerohydrolase [24], its insolubility in hydrophilic milieu limits its application in isomerohydrolase assays Consequently, experiments employing ectopic coexpression of LRAT and RPE65 in mammalian cells previously provided only indirect evidence of the isomerohydrolase activity of RPE65 [16–18] We have also shown that colocalization of LRAT and RPE65 in the same membrane is essential for isomerohydrolase activity [17] This represents another challenge to reconstituting the isomerohydrolase activity of RPE65 in vitro, as LRAT has not been purified as a full-length protein To overcome this difficulty, the present study established a novel assay with which to characterize the enzymatic activity of purified RPE65 by embedding the highly hydrophobic substrate – all-trans-retinyl palmitate – into liposomes that serve as a carrier of the substrate to the enzyme Our results showed that utilization of liposomes dramatically enhanced the magnitude of RPE65 isomerohydrolase activity 3026 Solubilization of membrane-associated proteins is the critical first step in their purification Although the amounts of solubilized RPE65 increase with increasing concentrations of Chaps, higher concentrations of Chaps also abolished the enzymatic activity of RPE65 By careful titration, we found that Chaps at a concentration of 0.1% was optimal for solubilizing RPE65 while preserving its catalytic activity Interestingly, several previous studies reported that RPE65 efficiently binds retinyl ester substrate even at 1% Chaps [20,21] It is likely that high concentrations of Chaps (e.g 0.5%) may partially disturb the RPE65 conformation, abolishing its catalytic activity, while leaving its substrate-binding ability intact In this case, the all-transretinyl ester is probably bound nonproductively and cannot be converted to 11-cis-retinol Nonproductive binding has been previously reported in other enzymes [28] Retinyl ester is a hydrophobic substance and does not freely exchange between membranes [29] In RPE cells, retinyl esters are confined either to microsomal membranes or lipid droplets [30] It is unlikely that the hydrophobic substrate diffuses from the membrane to the aqueous phase to interact with the protein Therefore, it is necessary for RPE65 to interact with the lipid membrane to extract the hydrophobic substrate Indeed, it has been previously reported that RPE65 demonstrates high affinity for phospholipid vesicles [31] RPE65 may bind to phospholipids through an attached palmitoyl group [32] or through a hydrophobic patch on the protein surface [33] In the current work, we confirmed, using the liposome flotation assay, that RPE65 efficiently binds to liposomes containing retinyl ester It is possible that RPE65 regains its catalytically active conformation upon binding to the liposomes Other examples of this phenomenon exist, such as the recent finding that binding to lipid membranes induces a conformational change in Bax protein [23] This could explain why RPE65 cannot catalyze the conversion of all-trans-retinyl ester substrate alone but displays robust activity when it is incorporated into liposomes The current study shows that no 11-cis-retinol was generated by purified RPE65 when N,N-dimethylformamide-solubilized all-trans-retinyl palmitate was added in the absence of liposomes This may seem to contradict previous results published by us and others, showing that a small amount of 11-cis-retinol was generated when N,N-dimethylformamide-solubilized all-trans-retinyl palmitate was added to an isomerohydrolase assay system using RPE65 in bovine [24] or mouse [34] RPE microsomes However, the low level of isomerohydrolase activity observed in those assays could be explained by the presence of lipid-containing microsomes, which not only served to contain RPE65 FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS O Nikolaeva et al in its membrane-bound, active conformation, but could also allow a small proportion of retinyl ester to be incorporated into the lipids of the microsomal membrane to serve as a substrate for RPE65 In contrast, the present study was performed using purified RPE65 in a membrane-free environment Therefore, this disparity can be ascribed to the lack of microsomal membranes in the present study Interestingly, previous attempts to reconstitute RPE65 in proteoliposomes have been unsuccessful; that is, isomerohydrolase activity has not been restored [20] It is possible that retinyl palmitate incorporated into liposomes can promote the formation of the catalytically active conformation of RPE65 upon its reassociation with liposomes The data presented in this article suggest that interaction of RPE65 with lipid membrane is essential for its isomerohydrolase activity Previously, it has been proposed that light can regulate RPE65 function, switching it between inactive soluble and active membrane-associated forms using a palmitoylation mechanism [32] At that time, the authors interpreted RPE65 as a retinyl ester-binding protein that presents its substrate to an unknown isomerohydrolase [32] We assume that the membrane association is probably essential for extracting highly hydrophobic retinyl ester substrate from the membrane Although a crystal structure of RPE65 is not yet available, a computer model using a carotenoid oxygenase as a template suggests that retinyl ester is bound inside a hydrophobic tunnel [35] It is likely that RPE65 binds to the retinyl ester-containing membrane in such a manner that the entrance of the tunnel would be located close to the membrane surface Such an interaction would allow for substrate to transfer from the hydrophobic milieu of the membrane to the hydrophobic tunnel of the RPE65 active site This transfer would be energetically favorable, as it would allow the hydrophobic substrate to avoid unfavorable interactions with water The exact mechanism for the interaction of RPE65 with the membrane is currently unknown It has been suggested that palmitoylation of the three Cys residues may be responsible for the membrane association [32] However, it was later shown that these Cys residues are not palmitoylated [34] Recently, a new palmitoylation site (Cys112) was found to be essential for membrane association of RPE65 [12] It has also been shown that a fragment of RPE65 containing residues 126–250 interacts with the lipid monolayer substantially more strongly than other fragments [33], suggesting that the sequence of RPE65 located between residues 126 and 250 residues might be very important for binding to the membrane Isomerohydrolase activity of purified RPE65 The isomerohydrolase activity of purified RPE65 obeyed classic Michaelis–Menten kinetics for a singlesubstrate enzyme-catalyzed reaction Thus, kcat and Km values for the purified RPE65 were determined and compared with those of the other enzymes that process retinoids and carotenoids enzymes The kcat value was calculated to be 1.45 · 10–4 s)1 Although this value seems low, it is still higher than the kcat for the purified truncated form of LRAT (4.8 · 10–5 s)1) [36] The kcat for full-length LRAT has not been determined, as it has never been purified The kcat for human b-carotene oxygenase was reported to be 0.011 s)1, which is 75-fold higher than that of RPE65 [37] However, it should be taken into account that the purified RPE65 in the assay was not completely bound to liposomes and, furthermore, liposome-bound RPE65 may be incompletely refolded into its active conformation The Km value for purified recombinant chicken RPE65 was approximately 10-fold higher than that for LRAT [36] and two-fold lower than the Km measured for unpurified human RPE65 [16] Previously, it has been estimated that RPE65 has a specific activity at least 25 000-fold lower than that of LRAT [16], suggesting that the high abundance of RPE65 in the RPE may be necessary to compensate for its low catalytic capacity It is likely that the kcat value for RPE65 measured in this work is a lower estimate of RPE65 isomerohydrolase activity in the RPE, which can be higher for several reasons First, a change in conformation of purified RPE65 upon reassociation with liposomes may limit the reaction rate Second, retinyl ester might adopt various physicochemical forms in the complex mixtures in the RPE (i.e emulsions, membrane vesicles, mixed micelles) This may also affect the enzymatic activity of RPE65 in RPE cells In summary, the present study demonstrates that purified RPE65 possesses intrinsic isomerohydrolase activity, and provides conclusive biochemical evidence that RPE65 is the isomerohydrolase of the visual cycle It also reveals that retinyl ester must be incorporated into the phospholipid membrane to serve as a substrate for RPE65 isomerohydrolase This finding opens new opportunities to study the specificity of RPE65 for modified retinyl esters and to elucidate the chemical mechanism of the isomerohydrolase reaction Experimental procedures Construction of Ad-RPE65 with a His-tag The chicken RPE65 cDNA was cloned as described previously [22] A DNA sequence encoding a histidine-hexamer FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS 3027 Isomerohydrolase activity of purified RPE65 O Nikolaeva et al (6 · His) was inserted at the N-terminus of the chicken RPE65 cDNA by PCR, using the following primers: forward primer, 5¢-GCGGCCGCCACCATGCATCATCACCA TCACCATTACAGCCAGGTGGAGC-3¢ containing a NotI site (underlined) and the Kozak sequence (bold); and reverse primer, 5¢-AAGCTTCATGCTCTTTTGAAGAGTC CATGG-3¢, containing a HindIII site (underlined) Preparation, amplification and titration of the recombinant adenovirus (Ad-RPE65) were performed as described previously [17] Evaluation of the effect of Chaps concentration on the efficiency of RPE65 solubilization Recombinant RPE65 was expressed as described previously [22] The 293A-LRAT cells [35] expressing RPE65 were harvested, resuspended in Buffer R (10 mm BTP, pH 8.0, 100 mm NaCl), homogenized by sonication, and aliquoted Each portion was supplemented with various Chaps concentrations (0%, 0.001%, 0.01%, 0.1%, 0.3%, and 0.5%) After incubation for h, each homogenate was centrifuged at 200 000 g for 30 at °C to obtain the solubilized fractions Two micrograms of protein from each fraction was used for western blot analysis with the antibody against RPE65 [11] to quantify RPE65 Purification of recombinant RPE65 The cells expressing chicken RPE65 were resuspended in Buffer A (50 mm sodium phosphate, pH 8.0), lysed by three freeze–thaw cycles, and centrifuged at 100 000 g for 30 at °C The pellet was resuspended in 35 mL of Buffer B (50 mm sodium phosphate, pH 8.0, 150 mm NaCl, 10% glycerol, 0.1% Chaps), sonicated, incubated for h at °C, and centrifuged at 125 000 g for h at °C The supernatant was loaded onto an Ni2+-nitrilotriacetic acid agarose (Qiagen Inc., Valencia, CA, USA) column The column was washed with Buffer C (50 mm sodium phosphate, pH 8.0, 300 mm NaCl, 10% glycerol, 0.1% Chaps) containing 10 mm imidazole Protein was eluted with Buffer C containing 250 mm imidazole The RPE65 elution pattern and the purity of RPE65 were examined by Coomassie Brilliant Blue staining and western blot analysis The RPE65 protein-enriched fractions were pooled, concentrated, and dialyzed at °C against Buffer D (50 mm sodium phosphate, pH 8.0, 100 mm NaCl, 10% glycerol, 0.1% Chaps) The concentration of the purified RPE65 was determined by Bradford assay [38] Western blot analysis The same amount of total protein (20 lg) was blotted with antibody against RPE65 (1 : 1000 dilution) or antibody against His-tag (Sigma-Aldrich, St Louis, MO, USA) as 3028 previously described [24] The membrane was briefly washed with the stripping buffer (Pierce, Rockford, IL, USA) and reblotted with a monoclonal antibody for b-actin (Abcam, Cambridge, MA, USA) where it was specified (1 : 2500 dilution) Western blot images were captured with the imager Chemi-Genius2 (Syngene, Frederick, MD, USA) Liposome preparation All phospholipids used in this study were purchased from Avanti Polar Lipids (Alabaster, AL, USA) Chloroform stocks of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dilauroyl-sn-glycero-3-phosphocholine were mixed at 85 : 15 (mol ⁄ mol) and supplemented with all-trans-retinyl palmitate to produce a 75 : lipid ⁄ retinyl ester ratio l-a-1Palmitoyl-2-arachidonyl-phophatidylcholine ([14C]PC) was used to label liposomes in proportions of 0.1 lCiỈmL)1 The organic solvent was removed by argon flow under dim red light, and the dried lipids ⁄ all-trans-retinyl palmitate film was dispersed in Buffer R by vortexing This mixture was exposed to five freeze–thaw cycles and passed through a polycarbonate membrane (0.1 lm) with a Mini-Extruder (Avanti Polar Lipids) The total lipid concentration of the resulting liposome suspension was mm Liposome flotation assay to detect membrane binding of purified RPE65 The purified recombinant RPE65 (25 lg) was incubated with 20 lL of liposomes (100 lm lipid, 1.3 lm all-trans-retinyl palmitate) in Buffer R for h at 37 °C in the dark The mixture (50 lL) was adjusted to a final sucrose concentration of 1.8 m (final volume 450 lL), placed at the bottom of a 3.5 mL ultracentrifuge tube, and overlaid consecutively with 850 lL portions of 1.35, 0.8 and 0.25 m sucrose in the same buffer The gradient was centrifuged at 250 000 g for h at 10 °C, and 500 lL fractions were then drawn from the top The pellets were resuspended in 100 lL of Laemmli sample buffer to detect aggregated and sedimented protein Aliquots of each fraction (30 lL) and pellets (6 lL) were analyzed by immunoblotting with the antibody against RPE65 The RPE65 content in each fraction was analyzed by densitometry The lipid distribution was determined by liquid scintillation counting of [14C]PC radioactivity In vitro isomerohydrolase activity assay The 293A-LRAT cells expressing RPE65 were lysed in Buffer R For each reaction, the liposomes (250 lm lipids, 3.3 lm all-trans-retinyl palmitate) and either 500 lg of total proteins of cell lysates, 250 lg of Chaps-solubilized supernatant proteins or 25 lg of the purified RPE65 was added to 200 lL of Buffer R containing 0.5% BSA and 25 lm FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS O Nikolaeva et al cellular retinaldehyde-binding protein After h of incubation at 37 °C in the dark, the generated retinoids were extracted with 300 lL of methanol and 300 lL of hexane and 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acyltransferase Biochemistry 42, 6090–6098 Lindqvist A & Andersson S (2002) Biochemical properties of purified recombinant human beta-carotene 15,15¢-monooxygenase J Biol Chem 277, 23942–23948 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 FEBS Journal 276 (2009) 3020–3030 ª 2009 The Authors Journal compilation ª 2009 FEBS ... Journal compilation ª 2009 FEBS O Nikolaeva et al for the following RPE65 purification and enzymatic assays Isomerohydrolase activity of purified RPE65 A kDa Purification of recombinant RPE65 To facilitate... of the isomerohydrolase activity on Chaps concentration was measured for total cell lysates (4) and Chaps-soluble fractions ( ) and plotted The activity was calculated from the peak areas of the... has historically hindered its use as a substrate for assays of isomerohydrolase activity In this study, a novel isomerohydrolase activity assay was developed in which alltrans-retinyl ester was