The results showed that purified recombinant chicken RPE65 had a high affinity for all-trans-retinyl palmitate-containing lipo-somes and demonstrated a robust isomerohydrolase activity.. I
Trang 1reassociation 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
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-pro-tein 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
photo-activation 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-trans-retinol is esterified by lecithin:all-trans-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 isomeri-zation 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 anal-ysis 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
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 3 February 2009, revised 23
March 2009, accepted 24 March 2009)
doi:10.1111/j.1742-4658.2009.07021.x
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 contro-versial Here, we developed a novel liposome-based assay for isomerohy-drolase activity The results showed that purified recombinant chicken RPE65 had a high affinity for all-trans-retinyl palmitate-containing lipo-somes and demonstrated a robust isomerohydrolase activity Furthermore,
we found that all-trans-retinyl ester must be incorporated into the phos-pholipid 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
Abbreviations
Ad-RPE65, adenovirus expressing RPE65; LRAT, lecithin:retinol acyltransferase; MOI, multiplicity of infection; PC, phosphatidylcholine; RPE, retinal pigment epithelium.
Trang 2visual 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
isom-erohydrolase [16–18] However, as isomisom-erohydrolase
activity has never been shown using purified RPE65,
there is skepticism about whether RPE65 is indeed the
isomerohydrolase [19] Two groups have reported
inde-pendently 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, 293A-LRAT cells were infected with different titers of adeno-virus expressing RPE65 (Ad-RPE65) [multiplicity of infection (MOI) 5–500] and harvested 24 h after infec-tion 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 concentra-tions of 0.1–0.5% solubilized significant amounts of recombinant RPE65 in the cells, whereas lower concen-trations 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 Fig 1 Scheme of retinoid visual cycle.
Trang 3assay with liposomes containing all-trans-retinyl ester
was developed (see Experimental procedures) In the
absence of Chaps, incubation of the total cell
homoge-nate 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
activ-ity, we measured the dependence of isomerohydrolase
activity on Chaps concentration, both for total
293A-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 isomero-hydrolase assay, an initial plateau of enzymatic activity was observed up to 0.1% of Chaps In line with previ-ous 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
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Fig 2 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%)-sol-ubilized 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 1 h at 4 C, and then centrifuged at 200 000 g for 30 min 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 l M lipids, 0.66 l M 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 2 h The generated retinoids were analyzed by HPLC, and peaks were identified as follows: 1, ret-inyl 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).
Trang 4for the following RPE65 purification and enzymatic
assays
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
antibod-ies against RPE65 (Fig 3B) and the His-tag (Fig 3C)
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
centri-fugal force causes liposomes to float to the top of the
sucrose gradient, owing to inherent buoyancy,
separat-ing liposomes from unbound protein, which remains in
the bottom fractions [23] All-trans-retinyl palmitate
was incorporated into
1,2-dioleoyl-sn-glycero-3-phos-phocholine⁄ 1,2-dilauroyl-sn-glycero-3-phosphocholine
liposomes at a lipid⁄ retinyl palmitate ratio of 75 : 1
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
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
his-torically hindered its use as a substrate for assays of
isomerohydrolase activity In this study, a novel
isom-erohydrolase activity assay was developed in which
all-trans-retinyl ester was incorporated into liposomes,
and all-trans-retinyl ester-containing liposomes were
then used as the substrate for measuring the
isomero-hydrolase activity of purified RPE65 As shown in Fig 5A, incubation of purified RPE65 with the lipo-somes containing all-trans-retinyl palmitate generated a
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Fig 3 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
Ni 2+ –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
1 h Lane 3: flow-through fraction not bound to the Ni 2+ –nitrilotri-acetic 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 5 lg for lane 4 For western blot analysis, the amount of protein was
500 ng for each lane.
Trang 5A
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Fig 4 Interaction of purified RPE65 with liposomes Purified RPE65 protein (25 lg) was incubated with the 14 C-labeled lipo-somes (100 l M lipids, 1.3 l M all-trans-retinyl palmitate) for 2 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 [ 14 C]PC and expressed as a percentage of the total amount of [ 14 C]PC in the gradient (means ± standard deviation, n = 3) (B–E) Purified RPE65 was incubated with (B, C) and without (D, E) 14 C-labeled liposomes, and centrifuged in the gradient as described above The same volumes from each frac-tion (30 lL) and pellets (6 lL) were exam-ined 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 inde-pendent experiments (mean ± standard deviation, n = 3).
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Fig 5 Isomerohydrolase activity of purified RPE65 reconstituted in liposomes Purified RPE65 (25 lg) was incubated with the following substrates in the presence of
25 l M cellular retinaldehyde-binding protein and 0.5% BSA for 2 h at 37 C The gener-ated retinoids were analyzed by HPLC (A) Ten microliters of liposomes (250 l M lipids, 3.3 l M 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 l M all-trans-retinol; (E) 3.3 l M all-trans-retinyl palmitate added in 2 lL of N,N-dimethylformamide without liposomes; (F) 10 lL of liposomes (250 l M lipids, 3.3 l M all-trans-retinyl palmi-tate) 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.
Trang 6significant 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
coe-lution 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
pos-sibility 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-reti-nol 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
Inter-estingly, in the absence of liposomes, RPE65 did not
generate 11-cis-retinol from nonincorporated
all-trans-retinyl 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
Kinetics of the isomerohydrolase activity
of purified RPE65
To determine the steady-state kinetics of RPE65
activ-ity, 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-trans-retinyl 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
there-fore conducted within this range Second, to establish
the dependence of 11-cis-retinol production on the
con-centration of purified RPE65, the liposomes containing
all-trans-retinyl palmitate were incubated with
increas-ing 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 lgÆmL)1
RPE65 (Fig 6B) Finally, to analyze the substrate
dependence of the RPE65 isomerohydrolase, we
mea-sured the initial reaction velocity using different
concen-trations of retinyl ester incorporated into the liposomes
Lineweaver–Burk analysis of these data yielded the
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)4s)1 for purified RPE65 (Fig 6C)
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Fig 6 Kinetic analysis of isomerohydrolase activity of purified RPE65 (A) Time course of 11-cis-retinol generation Liposomes containing all-trans-retinyl palmitate (250 l M lipids, 3.3 l M all-trans-retinyl ester) were incubated with purified RPE65 (25 lg) for the indicated time intervals, and the generated 11-cis-retinol was quan-tified by HPLC (B) Dependence of isomerohydrolase activity on RPE65 protein concentration Various amounts of purified RPE65,
as indicated, were incubated with liposomes (250 l M lipids, 3.3 l M all-trans-retinyl palmitate) for 2 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.
Trang 7A key step of the retinoid visual cycle is the conversion
of all-trans-retinyl ester to 11-cis-retinol, which is
cata-lyzed by isomerohydrolase Although the
isomerohy-drolase 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
gener-ate 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
research-ers in this field are still not convinced that RPE65 is
the isomerohydrolase [20] The present study
estab-lished a novel in vitro isomerohydrolase assay that
uti-lizes 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
demon-strate 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
isomero-hydrolase [24], its insolubility in hydrophilic milieu
limits its application in isomerohydrolase assays
Conse-quently, experiments employing ectopic coexpression of
LRAT and RPE65 in mammalian cells previously
pro-vided only indirect evidence of the isomerohydrolase
activity of RPE65 [16–18] We have also shown that
colocalization of LRAT and RPE65 in the same
mem-brane is essential for isomerohydrolase activity [17] This
represents another challenge to reconstituting the
iso-merohydrolase 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
hydropho-bic substrate – all-trans-retinyl palmitate – into
lipo-somes that serve as a carrier of the substrate to the
enzyme Our results showed that utilization of liposomes
dramatically enhanced the magnitude of RPE65
iso-merohydrolase activity
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 concen-tration 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 sub-strate-binding ability intact In this case, the all-trans-retinyl 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 There-fore, 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 con-firmed, 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 con-formational 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-dimethyl-formamide-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 isomero-hydrolase 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
Trang 8in 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
con-trast, the present study was performed using purified
RPE65 in a membrane-free environment Therefore,
this disparity can be ascribed to the lack of
micro-somal 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
interac-tion of RPE65 with lipid membrane is essential for its
isomerohydrolase activity Previously, it has been
pro-posed that light can regulate RPE65 function, switching
it between inactive soluble and active
membrane-associ-ated 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
extract-ing 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
hydro-phobic tunnel of the RPE65 active site This transfer
would be energetically favorable, as it would allow the
hydrophobic substrate to avoid unfavorable
interac-tions 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
palmitoyla-tion site (Cys112) was found to be essential for
mem-brane association of RPE65 [12] It has also been
shown that a fragment of RPE65 containing residues
126–250 interacts with the lipid monolayer
substan-tially more strongly than other fragments [33],
suggest-ing that the sequence of RPE65 located between
residues 126 and 250 residues might be very important
for binding to the membrane
The isomerohydrolase activity of purified RPE65 obeyed classic Michaelis–Menten kinetics for a single-substrate enzyme-catalyzed reaction Thus, kcatand Km values for the purified RPE65 were determined and compared with those of the other enzymes that process retinoids and carotenoids enzymes The kcatvalue was calculated to be 1.45· 10–4s)1 Although this value seems low, it is still higher than the kcatfor the purified truncated form of LRAT (4.8· 10–5s)1) [36] The kcat for full-length LRAT has not been determined, as it has never been purified The kcatfor 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 Kmmeasured 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], sug-gesting 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 isomero-hydrolase 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 sub-strate for RPE65 isomerohydrolase This finding opens new opportunities to study the specificity of RPE65 for modified retinyl esters and to elucidate the chemi-cal mechanism of the isomerohydrolase reaction
Experimental procedures Construction of Ad-RPE65 with a His-tag The chicken RPE65 cDNA was cloned as described previ-ously [22] A DNA sequence encoding a histidine-hexamer
Trang 9(6· His) was inserted at the N-terminus of the chicken
RPE65 cDNA by PCR, using the following primers:
for-ward 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)
Prepara-tion, amplification and titration of the recombinant
adeno-virus (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
con-centrations (0%, 0.001%, 0.01%, 0.1%, 0.3%, and 0.5%)
After incubation for 1 h, each homogenate was centrifuged
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 min
(50 mm sodium phosphate, pH 8.0, 150 mm NaCl, 10%
glycerol, 0.1% Chaps), sonicated, incubated for 1 h at
agarose (Qiagen Inc., Valencia, CA, USA) column The
col-umn was washed with Buffer C (50 mm sodium phosphate,
pH 8.0, 300 mm NaCl, 10% glycerol, 0.1% Chaps)
contain-ing 10 mm imidazole Protein was eluted with Buffer C
con-taining 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
phos-phate, 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
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
1,2-dilauroyl-sn-glycero-3-phosphocholine were mixed at
The organic solvent was removed by argon flow under dim
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 5 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
mixture (50 lL) was adjusted to a final sucrose concentra-tion 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
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
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 super-natant proteins or 25 lg of the purified RPE65 was added
to 200 lL of Buffer R containing 0.5% BSA and 25 lm
Trang 10cellular retinaldehyde-binding protein After 2 h of
extracted with 300 lL of methanol and 300 lL of hexane
and analyzed by normal-phase HPLC as described
previ-ously [24]
Acknowledgements
This study was supported by NIH grants EY012231
and ET015650, grant P20RR024215 from the National
Center for Research Resources, a research award from
JDRF, a grant from ADA, and a research grant from
OCAST HR07-067
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