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J. Biol. Chem., Vol. 276, Issue 51, 48483-48493, December 21, 2001
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From the Departments of
Received for publication, June 22, 2001, and in revised form, October 1, 2001
The regeneration of 11-cis-retinal,
the universal chromophore of the vertebrate retina, is a complex
process involving photoreceptors and adjacent retinal pigment
epithelial cells (RPE). 11-cis-Retinal is coupled to opsins
in both rod and cone photoreceptor cells and is photoisomerized to
all-trans-retinal by light. Here, we show that RPE
microsomes can catalyze the reverse isomerization of
11-cis-retinol to all-trans-retinol (and
13-cis-retinol), and membrane exposure to UV light further
enhances the rate of this reaction. This conversion is inhibited when
11-cis-retinol is in a complex with cellular
retinaldehyde-binding protein (CRALBP), providing a clear demonstration
of the protective effect of retinoid-binding proteins in retinoid
processes in the eye, a function that has been long suspected but never
proven. The reverse isomerization is nonenzymatic and specific to
alcohol forms of retinoids, and it displays stereospecific preference
for 11-cis-retinol and 13-cis-retinol but is
much less efficient for 9-cis-retinol. The mechanism of reverse isomerization was investigated using stable isotope-labeled retinoids and radioactive tracers to show that this reaction occurs with the retention of configuration of the C-15 carbon of
retinol through a mechanism that does not eliminate the hydroxyl group, in contrast to the enzymatic all-trans-retinol to
11-cis-retinol reaction. The activation energy for the
conversion of 11-cis-retinol to
all-trans-retinol is 19.5 kcal/mol, and 20.1 kcal/mol for isomerization of 13-cis-retinol to
all-trans-retinol. We also demonstrate that the reverse
isomerization occurs in vivo using exogenous
11-cis-retinol injected into the intravitreal space of wild
type and Rpe65 The regeneration of 11-cis-retinal is critical for
sustaining vision in vertebrates (for a review, see Ref. 1). The visual pigments of rod and cone photoreceptors require
11-cis-retinal, as their chromophores complex via
Schiff base to opsin molecules. Upon absorption of light,
11-cis-retinal photoisomerizes to
all-trans-retinal, triggering the phototransduction process
through a G-protein cascade, ultimately leading to neuronal signaling
(2-4). Eventually, all-trans-retinal is released by opsin
and converted back to 11-cis-retinal through a series of
enzymatic steps, termed the retinoid cycle, occurring in both
photoreceptor cells and adjacent
RPE1 (1).
In the currently described model, the chromophore undergoes several
chemical transformations. First, all-trans-retinal is released from the opsins by hydrolysis of the protonated Schiff base.
It is then transported out of the rod outer segment (ROS) disks by
either an ATP-binding cassette transporter (5, 6) or by simple
diffusion and finally is reduced by all-trans-retinol dehydrogenase (7-10) in the reaction that appears to be the
rate-limiting step in pigment regeneration (11).
All-trans-retinol then diffuses into the RPE, where it is
esterified to insoluble all-trans-retinyl esters by
lecithin-retinol acyl transferase (LRAT) (12, 13). Either retinyl
esters, retinol, or another intermediate is then employed as substrate
for an isomerization reaction that converts the
all-trans-retinoid to 11-cis-retinol (for a
review, see Ref. 1) (Fig. 1), with the highly expressed RPE-specific
protein RPE65 currently believed to play a critical role in this
process (14-16). After isomerization to 11-cis-retinol (for
details, see below), the retinol is oxidized to
11-cis-retinal by an NAD-specific 11-cis-retinol
dehydrogenase (11-cis-RDH) and other, yet unidentified NADP-specific enzymes (17-22) (Fig. 1). Finally,
11-cis-retinal diffuses back into the ROS and cone outer
segments, where it is reincorporated into opsin molecules to regenerate
rhodopsin and cone pigments. 11-cis-Retinal could
alternatively be produced by direct photoisomerization of
all-trans-retinal with the help of retinal G-protein-coupled
receptor protein in RPE and Müller cells (23, 24). The overall
production of 11-cis-retinal by photoisomerization could be
sufficient to drive cone pigment regeneration but insufficient to fully
regenerate rod rhodopsin. Furthermore, this process would be absent in
the dark-phase recovery of light-sensitive visual pigments.
As described above, a crucial reaction of the retinoid cycle is the
enzymatic isomerization to 11-cis-retinol. It has been proposed that the energy required for the formation of
11-cis-retinol ( The production of 13-cis-retinol by alternative retinoid
binding proteins, demonstrates the need for stringent control of retinoids, particularly the 11-cis-retinoids that are
sensitive to heat and light because of their difference in free energy
from that of all-trans- and 13-cis-retinoids
( Other threats to the control of retinoid flow include environmental
factors, particularly damage to photoreceptors and RPE from
light-induced action. Studies have shown that intense and long duration
exposure to excessive light can induce damage to both the retina and
RPE (43-47), with the greatest susceptibility to injury arising from
wavelengths of light near the maximum absorption region of rhodopsin
(~500 nm) and in the blue/UV-A region (320-450 nm) (45, 46).
Evidence suggests that green light exposure is most responsible for
damage incurred through chronic low level exposure, while blue/UV-A
light-induced damage is incurred through short intense exposures
(i.e. sun gazing, arc welding), which are able to penetrate
the UV light-absorbing ability of the lens (<400 nm) and produce
damage to the retina and RPE (45, 46, 48, 49). Furthermore, it is
believed that the damage is largely because of oxidation within the
photoreceptor and RPE. Crockett and Lawwill showed that damage to
cultured RPE from blue light (435 nm) is directly proportional to the
oxygen concentration within the culture medium (44). Pautler
et al. (47) demonstrated that blue light exposure to
isolated RPE affects the molecular transport capabilities of the RPE
and lowers their capacity for oxygen uptake. The use of antioxidants,
including dimethylthiourea (50), the spin-trapping reagent
phenyl-N-tert-butylnitrone (51), and, more
importantly, ascorbic acid, which is naturally found in abundance in
the retina but decreases during intense light exposure (52), appears to
help alleviate light damage within the retina and RPE (53-56).
Recently, Sparrow and Cai (57) showed that illumination of RPE with
blue light triggers apoptotic processes. These cells contain the
fluorophore pyridinium bisretinoid (A2E), a condensation
product of all-trans-retinal and phosphoethanolamine that
absorbs blue light.
Traditionally, the measure of retinal damage was performed by measuring
rhodopsin or DNA levels (43). However, these measurements are typically
not performed until ~2 weeks after the initial visual insult. In
recent years, it has been possible to characterize the effects of
light-induced retina and RPE damage at the molecular level. For
example, Sun and Nathans (58) demonstrated that in the presence of
all-trans-retinal, exposure of the ATP-binding cassette
transporter to UV light induces its aggregation and loses activity.
In this study, we show that RPE microsomes catalyze the isomerization
of 11-cis-retinol to all-trans-retinol (and
13-cis-retinol) and that the rate of isomerization is
significantly augmented by exposure of RPE microsomes to UV light.
However, if 11-cis-retinol is allowed to bind to CRALBP in
the presence of RPE microsomes, the reaction is substantially impeded.
Therefore, our results demonstrate a protective effect of
retinoid-binding proteins in this process. Moreover, we demonstrated
that when 11-cis-retinol is injected into the vitreal space,
reverse isomerization occurs in vivo in wild type mice.
Similarly, mice with defective forward isomerization (disrupted
RPE65 gene (14)) also display reverse isomerization. These
results suggest that, similar to in vitro conditions, the
reaction is nonenzymatic in vivo as well. This study
provides in depth analysis in vitro and in vivo
of the novel reverse isomerization activity of cis-retinol
in RPE microsomes and in the eye.
Materials
Bovine eyes were obtained from a local slaughterhouse (Schenk
Packing Co., Inc., Stanwood, WA), and RPE microsomes were prepared as
described previously (28). Additionally, mouse liver, kidney, and brain
microsomes were prepared using similar methods. Microsomes were
suspended in 10 mM MOPS, pH 7.0, containing 1 µM leupeptin and 1 mM dithiothreitol at a
protein concentration of ~5 mg/ml as determined by the Bradford
method (59). Aliquots were stored at Rpe65 Mice
All experiments involving animals were approved by the
University of Washington Animal Care Committee and conformed with the recommendations of the American Veterinary Medical Association Panel on
Euthanasia. All animals were maintained in complete darkness, and all
manipulations were done under dim red light (Eastman Kodak Co. number 1 Safelight filter (transmittance >560 nm)). Typically, 2-3-month-old mice were used in experiments. RPE65-deficient mice were
derived from a 129/SV R1 embryonic stem cell line generated by
homologous recombination with the targeting vector pPNT-RPE65 (14).
This resulted in the replacement, in the targeted allele, of 1 kb of
5'-flanking region; exons 1, 2, and 3; and intervening introns of the
RPE65 gene by the neo gene. The original
chimeric founder was crossed to C57Bl/6 mice, and the line was
maintained as a recombinant inbred line. Progeny of matings were
genotyped by RPE65-specific three-primer PCR (60).
Retinoid Synthesis
[15-2H,18O]13-cis-retinol
and [15-2H,18O]11-cis-retinol were
synthesized based on previously published methods (31). Briefly, either
11-cis-retinal or 13-cis-retinal (1 µmol) was
dissolved in 300 µl of
CH3CN/H218O at a ratio of 3:1
(v/v). Next, 2 mg of p-toluenesulfonic acid or 2 mg of MOPS
acid were added for 13-cis-retinol and
11-cis-retinol synthesis, respectively, and the mixture was
incubated overnight at room temperature. An excess of NaBD4
was added (~5 mg), the reaction mixture was incubated on ice for 20 min and diluted with 500 µl of H2O. The retinoids were
extracted with hexane (1 ml) and then purified by HPLC (HP1100; Beckman
Ultrasphere-Si, 5µ; 4.6 × 250 mm; 10% ethyl acetate, 90%
hexane; flow rate 1.4 ml/min). Concentrations were determined
spectrophotometrically, and isotopic exchange was determined by MS
(Kratos profile HV-3 direct probe).
Synthesis of
Pro-R,S-[15-3H]11-cis-Retinol
To a vial containing 100 µl of 16 mM
11-cis-retinal in DMF, 500 µl of 9.6 mM
[3H]NaBH4 (520 mCi/mmol; PerkinElmer Life
Sciences) in DMF was added. The vial was flushed with argon, capped,
and left on ice for 40 min with occasional vortexing. The reaction was
stopped by adding 750 µl of H2O, and retinoids were
extracted with hexane and purified by HPLC (see above).
Synthesis of [15-3H]11-cis-Retinal
To a 1.5-ml polypropylene tube containing 5 mg of
MnO2 in 100 µl of DMF, 253 µl of 3.33 mM
pro-R,S-[15-3H]11-cis-retinol
was added. The reaction mixture was flushed with argon, capped, and
placed on a mixer at room temperature for 30 min. The reaction mixture
was then chilled on ice and mixed with 300 µl of ice-cold
H2O. Retinoids were extracted with hexane and purified by
HPLC as above.
Synthesis of Stereospecific Pro-R- or
Pro-S-[15-3H]11-cis-retinol
Synthesis of pro-R- or
pro-S-[15-3H]11-cis-retinol was
achieved by utilizing the pro-R-specific enzyme, horse liver
alcohol dehydrogenase (HLADH; Sigma) and the pro-S-specific
enzyme, the purified recombinant 11-cis-RDH, as described
previously (10). HLADH was first purified on a Mono Q column
equilibrated with 10 mM BTP, pH 7.4, using a linear
gradient from 0 to 500 mM NaCl over 60 min at a flow rate
of 0.7 ml/min. The HLADH fraction (eluted at 1-3 min; 0.6 mg/ml) was
dialyzed against 10 mM BTP, pH 7.4, and then used for the study.
Reverse Isomerization Assay
The reverse isomerization assay was performed using 10 µl of
microsomes (RPE, liver, kidney, or brain; ~50 µg of protein) and 90 µl of 100 mM MES, pH 5.5. The reaction was initiated by the addition of 0.5 µl of 11-cis-retinol in DMF (4 mM), was incubated for 20 min at 37 °C, and was quenched
by the addition of 300 µl MeOH, and the retinoids were extracted with
300 µl of hexane. The samples were vortexed for 2 min followed by
centrifugation at 14,000 RPM on a microcentrifuge for phase separation.
A 100-µl aliquot of the hexane extract was analyzed by HPLC (Beckman
Ultrasphere-Si, 5µ, 2.0 × 250 mm, or Alltech solvent miser
5µ, 2.1 × 250 mm; 10% ethyl acetate in hexane; flow rate 0.5 ml/min), and concentrations were determined spectrophotometrically at
325 nm. The above assay conditions were used for all experiments unless
mentioned otherwise. Boiled controls were obtained by boiling
microsomes for at least 8 min.
Protection of 11-cis-Retinol by CRALBP from Isomerization
CRALBP (125 µg) or boiled CRALBP (~28 µM), in
90 µl of 100 mM MES, pH 6.0, was added to 10 µl of RPE
microsomes treated with or without 5-min UV light.
11-cis-Retinol (0.5 µl, 4 mM in DMF) was added
to a total volume of 125 µl. After incubation, the retinoids were
extracted as before, or the microsomes were pelleted at 150,000 × g for 1 h at 4 °C. The supernatant and pellet were extracted as
described previously.
UV and Boiling Analysis of RPE Microsomes
For the UV treatment time course, 200 µl of RPE microsomes
were UV-treated. At 5, 10, 20, and 45 min of UV exposure, an aliquot of
20 µl was drawn from the cuvette and tested for reverse isomerization activity. For temperature inactivation, 200 µl of RPE microsomes were
first UV-treated for 5 min, and a 20-µl aliquot was removed for
testing. The remaining microsomes were then boiled for at least 8 min,
and another 20-µl aliquot was removed. The cycle of UV treatment and
boiling was repeated once more, followed by one more UV treatment,
where each time 20 µl was removed for testing. The data are presented
with S.D.
Substrate Specificity
Substrate specificity was obtained by performing time courses
from 0 to 20 min using 9-cis-retinol (4 mM in
DMF), 13-cis-retinol (4 mM in DMF),
11-cis-retinol (4 mM in DMF), or
all-trans-retinol (4 mM in DMF). Retinols and
retinals were analyzed by HPLC as described previously.
pH Profile of Forward Versus Reverse Isomerization
pH Profiles were performed using UV-treated RPE microsomes for
both the reverse isomerization assay and the forward isomerization assay at pH ranges of 5.5-9.5 for the former and 6.5-9.5 for the latter, both in 0.5 pH increments. The reverse isomerization assay was
performed as before, except with a 90 mM concentration of the appropriate buffer (MES for pH 5.5-6.5, BTP for pH
7.0-9.5). The forward isomerization assay used 1% bovine serum
albumin, 1 mM ATP, 25 µM rCRALBP, 60 mM buffer of appropriate pH (MES for pH 6.5 and BTP for pH
7.0-9.5), 100 µg of total protein from RPE microsomes (20 µl), and
0.5 µl of all-trans-retinol (4 mM in DMF). The
forward isomerization assay was incubated for 2 h at 37 °C, and
retinoids were extracted and analyzed by the same method as in the
reverse isomerization assay.
Determination of Activation Energies for Isomerization of
11-cis-Retinol and 13-cis-Retinol to All-trans-retinol
The reverse isomerization assay was performed at temperatures
ranging from 6 to 53 °C in 6 °C increments. Time courses were performed at each temperature ranging from a maximum of 0-30 min in
5-min increments for lowest temperatures to 0-12 min for highest temperatures in 3-min increments. Each measurement was performed in
duplicate, and the experiment was repeated for both
11-cis-retinol and 13-cis-retinol. Rates were
determined from the amounts of all-trans-retinol produced,
and initial rates were calculated by linear regression across all
points measured for the time course. Arrhenius plots for
11-cis- and 13-cis-retinol were produced.
MS Analysis of Retinoids
The reverse isomerization assay was performed using either
[15-2H,18O]11-cis-retinol or
[15-2H,18O]13-cis-retinol (both 4 mM in DMF) in ~30 vials simultaneously. The hexane
extract was pooled, dried down under argon, reconstituted in 200 µl
of hexane, and separated on HPLC. The all-trans-retinol, 11-cis-retinol, and 13-cis-retinol fractions were
collected, dried down under argon, reconstituted in 10 µl of hexane,
and analyzed by MS.
Inhibition of Forward and Reverse Isomerization by Alcohols
Comparisons of inhibition between the forward isomerization
assay and reverse isomerization assay were tested in a variety of
conditions. To the reverse isomerization assay and forward isomerization assay, MeOH, EtOH, isopropyl alcohol, or isobutyl alcohol
(2% (v/v) final concentration) was added. Retinoid analysis was done
as described previously.
Inhibition of Forward and Reverse Isomerization by
Protein-modifying Agents and Proteases
RPE microsomes were modified with the following reagents: acetic
anhydride, N-ethylmaleimide (NEM; Sigma), phospholipase
A2 (Sigma), and Pronase (Sigma).
Acetic Anhydride--
RPE microsomes (50 µl, ~250 µg) were
added to 40 µl of 100 mM sodium borate, pH 9.0, and 10 µl of 1 M acetic anhydride in CH3CN. The
mixture was allowed to incubate on ice for 10 min.
NEM--
10 µl of 1 M NEM solution in 100 mM BTP, pH 7.5, was added to 90 µl of RPE microsomes
(~450 µg) and incubated for 1 h at 37 °C while shaking.
Phospholipase A2--
To 50 µl of RPE microsomes,
~1 µg of PLA2 was added (150 µl of total volume in 50 mM BTP, pH 7.5) and incubated for 1 h at 37 °C.
Pronase--
To 50 µl of RPE microsomes, 1 µg of Pronase was
added (150-µl total volume in 50 mM BTP, pH 7.5) and
incubated for 1 h at 37 °C.
Following each modification, the microsomes were centrifuged at
150,000 × g for 1 h at 4 °C. The microsomes
were washed with 3 × 200 µl of either 100 mM MES,
pH 5.5, for reverse isomerization assay or 100 mM BTP, pH
7.5, for forward isomerization and then resuspended in 50 µl of the
appropriate buffer, and 10 µl was used for reverse isomerization
assay, and 20 µl was used for forward isomerization assays.
Determination of the Stereoconfiguration of
[15-3H]All-trans-retinol Generated by Reverse
Isomerization Assay
The reverse isomerization assay was performed using either
pro-R-[15-3H]11-cis-retinol (2.74 mM in DMF) or
pro-S-[15-3H]11-cis-retinol (2.98 mM in DMF). The reaction was initiated by the addition of
1.0 µl of [15-3H]11-cis-retinol to the
reverse isomerization assay. Retinoids were purified by HPLC, and the
all-trans-retinol fractions were collected, dried down under
a stream of argon, and reconstituted in DMF. The stereoconfiguration
was examined with HLADH and prRDH in ROS, which are
pro-R-specific enzymes with respect to the specificity of
all-trans-retinol at the prochiral methylene hydroxyl group. The assay was carried out as follows (8, 10). The reaction mixture (100 µl) for the ROS assay included 83 mM sodium phosphate, pH
7.5, ROS (16 µg of rhodopsin), 1 mM dithiothreitol, 30 µM bovine serum albumin, and 600 µM NADP.
The HLADH assay included BTP (62 mM with 0.05% Tween 80, pH 8.6), 21 µg of HLADH, 1 mM dithiothreitol, and 600 µM NAD. The
[15-3H]all-trans-retinol (15-30
µM final concentration in the assay) generated from the
reverse isomerization assay was added last to initiate the reaction.
The reaction was incubated at 33 °C for 45 min (60 min for HLADH)
and stopped with 400 µl of MeOH, 150 µl of 1 M NaCl,
and 50 µl of 0.1 M NH2OH. After 6 min at room temperature on a mixer, 400 µl of CH2Cl2 was
added. After mixing and centrifuging to separate the phases, the lower
phase was removed, and the upper phase was extracted three more times
with 400 µl of CH2Cl2. Radioactivity was
measured in 450 µl of the upper phase by scintillation counting.
Retinoid Extraction from Mouse Eye and Analysis
All procedures were performed under dim red light as described
previously (22). Retinoid analysis was performed using Beckman Ultrasphere Si 5µ (2.0 × 250 mm) and an isocratic solvent
system of 0.5% ethyl acetate in hexane (v/v) for 0-10 min followed by 10% ethyl acetate in hexane from 10 to 25 min at a flow rate of 0.5 ml/min (total of 25 min). This allowed the separation of
11-cis-, 13-cis-, and
all-trans-retinyl esters. Typically, two mouse eyes were
used per assay and repeated two or three times. All experimental procedures related to the analyses of dissected mouse eyes and derivitization have been described in detail previously (16, 61).
Intravitreal Administration of 11-cis-Retinol
25 µg of 11-cis-retinol in 1 µl of
Me2SO (87.4 mM) was administered into the
vitreal space employing a 10-µl syringe (model 701RN, Hamilton) with
a 32-gauge × 9.37-mm needle, point style 2 (Hamilton). Mice
were kept in the dark, and analysis was performed 2 weeks
postintravitreal administration.
Protection of 11-cis-Retinol by CRALBP from Isomerization to
All-trans-retinol--
11-cis-Retinol was added to
untreated RPE microsomes, and the reverse isomerization to
all-trans-retinol was observed (Fig. 2A, trace
a). The assay was performed at low pH to inhibit activity of
LRAT, which shows maximal activity at high pH. The high level of
all-trans-retinyl esters is a result of endogenous
accumulation and partial esterification of exogenous retinol. To
eliminate interference from endogenous retinoids in typical in
vitro assays, they are destroyed by UV treatment (25). When RPE
microsomes were treated with UV light for 5 min,
all-trans-retinol was formed in more significant amounts
(Fig. 2A, trace b), including
formation of 9-cis-retinol (indicated by an
asterisk). Finally, when 11-cis-retinol was added
to UV-untreated (data not shown) and UV-treated microsomes in the
presence of 28 µM CRALBP, isomerization to
all-trans-retinol decreased dramatically (Fig.
2A, trace c, and Fig. 2B,
bars a and b), suggesting that CRALBP
protects 11-cis-retinol from reverse isomerization catalyzed
by RPE microsomes exposure to UV light. The protective mechanisms most
likely involve the binding of 11-cis-retinol into a
hydrophobic pocket of CRALBP. To test this possibility, the reaction
mixture was fractionated into soluble and insoluble fractions and
individually analyzed. 11-cis-Retinol was present in the
CRALBP-containing supernatant (Fig. 2B, bar
c), and only trace amounts were found in the pellet (Fig.
2B, bar d). In contrast, when soluble
and insoluble fractions were separated and analyzed in the absence of
CRALBP, only trace amounts of retinoids were present in the supernatant
fraction, and the pellet displayed significant formation of
all-trans-retinol (data not shown). These results suggest
that CRALBP plays a protective role for 11-cis-retinol, preventing its isomerization back to all-trans-retinol, as
required by the flow of retinoids in physiological conditions (reviewed in Ref. 1).
Evidence of Light-induced Damage to RPE Microsomes Caused by UV
Light Exposure--
Studies reveal that photo-oxidative damage to
biological systems is dependent on the wavelength of light and
intensity and the duration of light exposure (43). Therefore, to
demonstrate UV damage to RPE, a time course of UV treatment was
performed on RPE microsomes. After exposure of 5, 10, 20, or 45 min, an aliquot of RPE microsomes was taken and tested. Fig.
3A shows the HPLC analysis at
5, 10, 20, and 45 min (traces a, b,
c, and d, respectively), demonstrating that
increasing duration of UV light exposure to RPE microsomes increases
the rate of isomerization of 11-cis-retinol to other
isomers. Note that the formation of 13-cis-retinol (marked
by an asterisk in trace d) was
apparent as retinol isomers began to reach equilibrium.
Next, to determine whether the highly reactive species created by
light-induced damage could be disabled by heat, RPE microsomes were
alternatively UV-treated and boiled for 8 min, this process was
repeated once more, and then microsomes were subjected to a final 5-min
UV treatment. After each treatment, an aliquot was removed and tested
(Fig. 3B). Traces a, c, and
e in Fig. 3B show the effects of UV treatment,
and traces b and d show the effects of
boiling for 8 min. In traces b and d,
the amount of all-trans-retinol formed was greatly reduced,
indicating that boiling greatly reduced isomerization catalyzed by the
light-induced damage to RPE microsomes. The gradual increase in
formation of all-trans-retinol and other isomers including
formation of 13-cis-retinol (asterisk), following UV-treatment (traces a, c, and
e), indicated that boiling for 8 min did not completely
destroy all effects of light-induced damage; thus, the additional UV
exposure was accumulative. These results suggest that light is
responsible for generation of active species that augment the reverse isomerization.
Retinoid Substrate Specificity of UV-induced Isomerization--
To
test the isomeric specificity, time courses were performed to evaluate
formation of all-trans-retinol from
11-cis-retinol, 13-cis-retinol, and
9-cis-retinol and the formation of
13-cis-retinol from all-trans-retinol in the
presence of UV-treated RPE microsomes and boiled UV-treated RPE
microsomes. The results of isomerization for retinols are shown in Fig.
4, and they indicate that the rate of
all-trans-retinol formation from 11-cis-retinol
and 13-cis-retinol was virtually identical, while the
formation of all-trans-retinol from 9-cis-retinol
was only slightly higher than the result obtained from boiled
UV-treated RPE microsomes. Additionally, all-trans-retinol showed some conversion to 13-cis-retinol (Fig. 4).
Isomerization of 11-cis-retinal was measured to determine
whether isomerization also occurs on the aldehyde level. The amounts of
all-trans-retinal were measured by the amount of
syn- and anti-oximes produced after treatment
with NH2OH. The results showed that isomerization to
all-trans-retinal was more than 5 times slower than that at the alcohol level. Additionally, no UV-dependent
enhancement was observed for the isomerization of
11-cis-retinal to all-trans-retinal. Furthermore,
11-cis-retinyl palmitate was resistant to isomerization to
all-trans-retinyl esters (data not shown).
Comparison of Forward Enzymatic Isomerization of All-trans-retinol
with 11-cis-Retinol to Reverse Isomerization: pH Profile of
Isomerization of 11-cis-Retinol--
To determine whether reverse
isomerization of 11-cis-retinol operated at a preferential
pH, UV-treated microsomes were incubated with 11-cis-retinol
at various pH values ranging from 5.5 to 9.5 in steps of 0.5 pH units
(Fig. 5A). The greatest amount
of all-trans-retinol formed at pH ranges of 5.5-6.0. At
higher pH, the amount of all-trans-retinol formed declined
sharply. Additionally, at higher pH, formation of retinyl esters
increased steadily (Fig. 5B, inset). These
results suggest that the low activity of the reverse isomerization at high pH could not be entirely a consequence of retinol esterification in conditions that promote higher LRAT activity. When the RPE microsomes were partially inactivated by heat treatment at 100 °C
for 10 min to denature LRAT and the reverse isomerization activity was
reactivated by UV light, a similar pH profile was observed (data not
shown), demonstrating that the contribution of LRAT activity to affect
this pH profile was minimal. For comparison, a pH profile was performed
on the enzymatic isomerization of all-trans-retinol to
11-cis-retinol at a pH range of 6.5-9.5 in steps of 0.5. As expected, the peak formation of 11-cis-retinol occurs at pH
7.5, which is very close to physiological pH (Fig. 5B).
Again, with higher pH, there was a corresponding increase in the
formation of retinyl esters (inset). These results suggest
that the two isomerization reactions may operate by different
mechanisms.
Determination of the Activation Energy of the Isomerization of
11-cis-Retinol and 13-cis-Retinol to All-trans-retinol in UV-treated
RPE Microsomes--
To understand the energetics of the isomerization
to all-trans-retinol, we measured the rate of isomerization
of 11-cis-retinol and 13-cis-retinol to
all-trans-retinol at temperatures ranging from 5 to 53 °C
in 6 °C increments. At each temperature, a time course was performed
to determine the rate of isomerization. Arrhenius plots were generated
by graphing the logarithm of initial rates of reaction
versus 1/T (Fig. 6,
A and B). The activation energy was determined by
a linear least-squares fitting of the data points, with the slope used
in Equation 1,
Isomerization of cis-Retinols to All-trans-retinol in UV-treated
Microsomes of Other Tissues--
To test if isomerization could be
observed in tissues other than those from bovine RPE, microsomes were
produced from mouse liver, kidney, and brain, subjected to 5-min UV
treatment, and measured for their ability to isomerize
11-cis-retinol, 13-cis-retinol, or
9-cis-retinol (Fig.
7A). Microsomes from these
other tissues showed similar isomeric specificity as that of RPE
microsomes with 9-cis-retinol, again showing little
activity. Additionally, mouse liver, kidney, and brain microsomes were
compared with bovine RPE microsomes for their ability to isomerize
11-cis-retinol with no UV treatment, UV treatment for 5 min,
or 8-min boiling of UV-treated microsomes. Although liver, kidney, and
brain microsomes showed slightly higher abilities to isomerize
11-cis-retinol to all-trans-retinol in
untreated microsomes, upon UV treatment, the amount of
all-trans-retinol in all microsomes increased dramatically
(Fig. 7B). Again, boiling of UV-treated microsomes reduced
the formation of all-trans-retinol. This provided further
evidence that the reverse isomerization is nonenzymatic and
significantly augmented by light treatment.
Comparison of Enzymatic Versus Nonenzymatic Isomerization by
Inactivation Methods Using Alcohols, Protein-modifying Agents, and
Ascorbic Acid--
To demonstrate that isomerization of
11-cis-retinol to all-trans-retinol is a
nonenzymatic reaction and different from the forward
all-trans-retinol to 11-cis-retinol enzymatic
isomerization, UV-treated RPE microsomes were treated with alcohols,
protein-modifying agents, and vitamin C and measured for both
isomerization reactions. Treatments were conducted with 2% methanol,
2% ethanol, 2% isopropyl alcohol, 2% isobutyl alcohol (v/v), 100 mM NEM, 100 mM acetic anhydride, 1 mg/ml
phospholipase A2, and 1 mg/ml Pronase (Fig. 8A). The results show that
isomerization to all-trans-retinol (gray
bars) is not particularly sensitive to any of the
modifications. In contrast, enzymatic isomerization to
11-cis-retinol (black bars) is
significantly affected by all modifications. Furthermore, an addition
of 10 mM ascorbic acid to UV-treated RPE and liver microsomes greatly reduced the amount of all-trans-retinol
formed, indicating the antioxidant activity of vitamin C (Fig.
8B). The reverse isomerization was 50% inactivated by
incubation of the RPE microsomes at 50 °C for 30 min, while the
forward reaction was 100% inhibited by similar treatment for 5 min
(data not shown). Together, these data suggest that the forward
reaction is catalytic, while the reverse reaction does not involve an
enzyme.
MS Analysis of the Reverse Isomerization in RPE
Microsomes Using [15-2H,18O]11-cis-Retinol
and [15-2H,18O]13-cis-Retinol and
Stereospecificity of Isomerization Using
Pro-R-[15-3H]11-cis-retinol and
Pro-S-[15-3H]11-cis-retinol--
Reverse isomerization
can proceed via retention, inversion, or mixed configuration with
respect to the C-15 methylenehydroxyl group. To probe the
mechanism by which isomerization of cis-retinols to
all-trans-retinol occurs, retinols labeled with stable
isotopes were prepared. All-trans-retinol was collected by
HPLC after the addition of
[15-2H,18O]11-cis-retinol or
[15-2H,18O]13-cis-retinol to
UV-treated RPE microsomes and analyzed by MS. Fig.
9, A and C, show MS
fragmentation patterns of
[15-2H,18O]13-cis-retinol and
[15-2H,18O]11-cis-retinol,
respectively. Note the shift to 289 (M+) because of
incorporation of stable isotopes. Fig. 9, B and
D, show MS fragmentation patterns of
all-trans-retinol collected after isomerization of the
substrates shown in Fig. 9, A and C, respectively. There was no difference in the fragmentation patterns. These results indicate that no loss of 18O occurred,
suggesting that isomerization occurs because of catalysis at a site
other than the alcohol functional group. Next,
11-cis-11-fluororetinol was employed as substrate (data not
shown). No isomerization was observed, the same result as the forward
reaction (31), suggesting that electron-withdrawing groups prevent
isomerization in either direction.
To further determine that the alcohol functional group was not the site
of reaction, the stereospecificity of isomerization was evaluated using
pro-R-[15-3H]11-cis-retinol (Fig.
9E, bars 1 and 2 and
bars 5 and 6, respectively) or
pro-S-[15-3H]11-cis-retinol, both
prepared using either HLADH or 11-cis-RDH (bars
3 and 4 and bars 7 and
8, respectively) (see "Experimental Procedures"). The
all-trans-retinol produced after the addition of the above
substrates to UV-treated RPE microsomes was collected and oxidized
using ROS/NADP (bars 1, 3,
5, and 7) or with HLADH/NAD (bars
2, 4, 6, and 8), to
stereospecifically eliminate a hydrogen atom from the C-15
position. The activity was measured by scintillation counting of the
aqueous layer containing the cofactor. The results show that
pro-R-[15-3H]11-cis-retinol
converts to all-trans-retinol with retention of
configuration by the UV-treated RPE microsomes. This was demonstrated when the isomerization product, all-trans-retinol, was
oxidized by either ROS or HLADH, which have both been shown to be
pro-R-specific with respect to the retinoid (22). The
radioactive label was transferred to the NADP or NAD cofactor (Fig.
9E, bars 1 and 2 and
bars 5 and 6). This indicates that
pro-R-[15-3H]11-cis-retinol
isomerizes to
pro-R-[15-3H]all-trans-retinol,
demonstrating that the configuration is retained. In contrast, when
pro-S-[15-3H]11-cis-retinol was
isomerized to all-trans-retinol and oxidized, no radiolabel
was transferred to the cofactor, indicating that the
pro-S-[15-3H]all-trans-retinol is
formed during isomerization.
Isomerization of 11-cis-Retinol to All-trans-retinol in Vivo--
To explore if reverse isomerization occurs in vivo,
11-cis-retinol was injected intravitreally into wild type
mice (Rpe65+/+). In the dark, it was anticipated that
11-cis-retinol would be converted to
11-cis-retinyl esters if reverse isomerization did not occur in vivo. Compared with the control eyes, the treated eyes,
which were never exposed to light, contained significantly elevated levels of all-trans-retinyl esters (Fig.
10, peak 3) and
13-cis-retinyl esters (peak 1) at the
expense of 11-cis-retinol (peak
6) or 11-cis-retinyl esters (peak
2). 11-cis-Retinal bound to opsin was identified as syn- and anti-11-cis-retinal oximes
(peaks 4 and 4') and was not
significantly altered in treated and untreated Rpe65+/+
mice. Very similar results were obtained in four independent
experiments. These results suggest that the reverse isomerization
occurs in vivo.
Two possible mechanisms of reverse isomerization in
vivo would involve the membrane-catalyzed isomerization observed
in vitro or could isomerize through enzymatic catalysis of
all-trans-retinol to 11-cis-retinol. To test
between these two possibilities, 11-cis-retinol was injected
intravitreally into Rpe65-/- mice, whose forward isomerization is absent (14, 16). Compared with the control eyes,
significant increases in the level of all-trans-retinyl esters (Fig. 10, lower panel) and
13-cis-retinyl esters (peak 1) were
observed at the expense of 11-cis-retinol, while
11-cis-retinyl esters were below detectable levels.
11-cis-Retinal bound to opsin and identified as
syn- and anti-11-cis-retinal oximes
(peaks 4 and 4') was only observed in
mice treated with 11-cis-retinol, suggesting that once the
binding pool of 11-cis-retinoids, composed of opsin, CRALBP,
and interphotoreceptor retinoid binding protein (IRBP), is saturated,
the remaining amounts are converted to all-trans-retinol and
esterified. Comparable results were obtained from four independent experiments. Together, these results demonstrate that reverse isomerization occurs in vivo most likely through the
mechanisms extensively characterized in vitro and described above.
Protective Effect of CRALBP on the Reverse
Isomerization--
The importance of maintaining the integrity of
11-cis-retinal production in RPE is critical for the proper
functioning of vision. Disruption of the retinoid metabolism either by
genetic or environmental factors often leads to severe visual disorders (19, 21, 35-42). The roles of retinoid-binding proteins in this
process, although not fully understood, are believed to be important
for proper transport and protection of the retinoid moiety. Some
retinoid-binding proteins are better defined than others. For example,
RBP is involved in retinol transport in the blood stream (62); CRBP I
functions intracellularly to partition vitamin A into a soluble form or
as insoluble retinyl esters (62); and CRALBP has been initially
proposed to have dual modifying effects on LRAT and
11-cis-RDH activities (63), although the interpretation of
these results should be considered with caution, since these effects
could be an artifact of lowering the apparent substrate concentration
and the higher affinity of CRALBP for the aldehyde than alcohol of
11-cis-isomer, respectively. The most profound stimulatory
effect of CRALBP is on the rate of isomerization reaction through mass
action (31). In this study, we investigated the effects of RPE
microsomes on the isomerization of 11-cis-retinol and
13-cis-retinol to all-trans-retinol. These
reactions were further augmented by UV treatment of RPE membranes
in vitro. When CRALBP is added to UV-treated microsomes,
11-cis-retinol is protected from isomerization by the
resulting UV-induced products, probably shielded within a hydrophobic
pocket in the core of the CRALBP protein (Fig. 2). The hypothesis of a
hydrophobic pocket to protect retinoids is based on analogy to the
crystal structures of CRBP and RBP (reviewed in Ref. 62), since the
high resolution structure of CRALBP is not yet known.
Only a limited number of studies have shown direct protective roles for
retinoid-binding proteins. One investigation of UV light exposure on
hairless mice found that it greatly reduced the concentrations of
retinyl esters, although all-trans-retinol was largely
unaffected. This suggested that retinoid-binding proteins such as CRBP
I protect all-trans-retinol from the damaging effects of UV
light (64, 65). Although more recent work has suggested that CRBP's
primary role in UV-mediated damage is more related to retinoid
mobilization in response to the light-induced injury (66), other
studies of UV exposure to human skin and to Gecko lens
further support the protective role of CREB I (64, 67). In the case of
IRBP, Crouch et al. (68) showed that
11-cis-retinol could be protected from both oxidation and
isomerization if bound to IRBP. Our studies add to these observations,
showing the important protective role of CRALBP for proper flow of
retinoids in RPE microsomes.
Chemistry of the Reverse Isomerization--
We performed several
experiments to demonstrate that the isomerization of
11-cis-retinol in RPE microsomes is not an enzymatic process
that is further augmented by a by-product of UV treatment. The extended
duration of UV treatment accelerates the formation of
all-trans-retinol and 13-cis-retinol (Fig.
3A), and by alternating UV exposure and boiling, reverse
isomerization activity is gained and diminished, although not
completely in the boiling stage, allowing cumulative damage during UV
treatment. This is documented by the formation of
13-cis-retinol in the third cycle (Fig. 3B). Similar activity is found when microsomes were produced from the liver,
kidney, and brain of mouse (Fig. 7). Chemical modifications that either
reduced or eliminated the enzymatic isomerization of
all-trans-retinol to 11-cis-retinol had little
effect on the nonenzymatic reverse isomerization, and the addition of
10 mM ascorbic acid to UV-treated RPE or liver microsomes
quenched isomerization (Fig. 8B). This supports the previous
studies on the protective effects of ascorbic acid from light-induced
damage in vivo (69).
Substrate specificity experiments showed that while
11-cis-retinol and 13-cis-retinol isomerized to
all-trans-retinol at similar rates, 9-cis-retinol
was less affected, and all-trans-retinol isomerized to
13-cis-retinol at a slower rate than expected. Experimental calculation of the activation energy of isomerization of
11-cis-retinol and 13-cis-retinol to
all-trans-retinol confirmed their similarity with 19.5 kcal/mol and 20.1 kcal/mol, respectively. These values were also in
good agreement with the 17-25 kcal/mol calculated for the enzymatic
isomerization to 11-cis-retinol (31). Additionally, MS
analysis and stereospecificity experiments indicate that the hydroxyl
group is not disturbed during reverse isomerization (Fig. 9). These
results eliminate a number of possible mechanisms by which
isomerization could occur, most notably dehydration and formation of
retinyl carbocation.
The formation of a carbocation by the addition of an electrophile
(proton) to the polyene chain might be a more likely mechanism of
isomerization that would preserve the OH group. This mechanism is
favorable at low pH, as we observed experimentally.
11-cis-Retinol and 13-cis-retinol are preferable
possibly because of nonlinear electron distribution along the polyene
chain. These reactions described here are different from earlier
studies of the much slower cis-trans isomerization of
retinals in the presence of phosphatidylethanolamine (70), crude tissue
extracts (71), lipid dispersion (72), or ROS (73).
Alternatively, the mechanism may involve thio radicals, as demonstrated
for cis-trans isomerization of polyunsaturated fatty acid
residues in phospholipids (74). Although we observed only a ~50%
reduction in reverse isomerization when membranes were NEM-treated, the
UV-treated RPE membranes were active even after a few days of storage
at 4 °C (data not shown). These findings could be explained by a
stable intermediate deep in the membranes that is not accessible for
NEM modification. All together, we favor this mechanism less than
electrophilic addition to the polyene chain, because thio radicals are
predicted to be very unstable.
Implication of Reverse Isomerization to Biochemical Studies of
Forward Isomerization--
Our studies also provide new insights into
isomerization in vitro for the forward reaction. Over the
years, the isomerization of all-trans-retinol to
11-cis-retinol in vitro was hampered by exceedingly low production of 11-cis-retinol (25, 75, 76). A
major enhancement of isomerization was observed in just the last few
years when the retinoid-binding proteins CRALBP and/or CRBP were
included in the assays (1, 27-29, 31). The explanation for low
activity in the earlier studies could be accounted for by two
processes: 1) UV-treated membranes were used that promoted reverse
isomerization, and 2) the lack of retinoid-binding proteins pooled
thermodynamically unfavorable reaction products (28, 29, 31).
Implication of Reverse Isomerization in Vivo--
In wild type
mice, reverse isomerization also occurs in vivo. When
11-cis-retinol was injected into the eye of dark-adapted mice, more than 95% was converted to the all-trans-isomer.
Due to very high activity of LRAT in the RPE, retinols are stored in
the form of fatty acid esters (reviewed in Ref. 1). This observation
explains why mice have a very low pool of 11-cis-retinyl esters and lends additional support to the idea that the different pools of retinoid in the eye are in chemical and thermodynamic equilibria rather than driven by energetically driving coupled reactions. From several potential mechanisms of reverse isomerization, two appear to have the highest probability. One involves reverse isomerization through the enzymatic steps present in the RPE that normally produce 11-cis-retinol from
all-trans-retinol but run in reverse. The second is that the
11-cis-retinol conversion to all-trans-retinol is
a result of the reverse isomerization similar to that characterized in
the study using in vitro assays. The strong support of a
nonenzymatic reaction comes from the analysis of Rpe65
Although a mechanistic correlation between the reverse isomerization
in vivo and in vitro cannot be confirmed, a
number of facts suggest that they are similar. First, both reactions
produce all-trans, and subsequently 13-cis
isomers2 from
11-cis-retinol. Second, both reactions appear to proceed independently of the enzymatic steps for all-trans-retinol
to 11-cis-retinol isomerization, because the in
vitro reaction is nonenzymatic, and the in vivo
reaction does not require RPE65. Third, previous studies have shown
that activation of the only functional group on
11-cis-retinol (and all-trans-retinol) often causes rearrangements resulting in the formation of anhydroretinol or
retroretinols (33). The carbocation mechanism proposed for the
enzymatic forward isomerization is plausible because the transition state can be stabilized in the active site of the enzyme, preventing anhydroretinol formation. However, in our in vivo retinoid
analysis of wild type and RPE65
In summary, we show that exposure of RPE microsomes to UV light
generates products that catalyze the isomerization of
11-cis-retinol to all-trans-retinol when
11-cis-retinol is unprotected. This reaction is offset
physiologically by the requirement to convert all-trans-retinol to 11-cis-retinol. This reverse
isomerization is inhibited when 11-cis-retinol is protected
in a complex with CRALBP. The reverse isomerization is nonenzymatic and
specific to alcohol forms of retinoids and displays a stereospecific
preference. It occurs with the retention of configuration at the
methylenehydroxyl C-15 carbon of retinol through a mechanism
that does not eliminate the hydroxyl group; in contrast, the forward
reaction undergoes a mechanism that eliminates the hydroxyl group. The
Arrhenius plots show that the activation energy was found to be 19.5 kcal/mol for 11-cis-retinol to all-trans-retinol
and 20.1 kcal/mol for the 13-cis-retinol to
all-trans-retinol isomerization reaction, suggesting that an
energetically similar intermediate is formed as the proposed
carbocation for the forward reaction. Finally, the reverse
isomerization occurs in vivo, with and without functional enzymes of forward isomerization.
We thank Drs. Rosalie K. Crouch, David
S. Papermaster, and Vladimir Kuksa for comments on the manuscript;
Dr. Michael Redmond for Rpe65 mice; Dr. Angel Rodriguez de
Lera for 11-cis-11-fluororetinal; and Dr. Martin Sadilek for
assistance with MS analysis.
*
This work was supported by National Institutes of Health
Grants EY07031 (vision training grant; to J. K. M.), EY09339 and EY66-3988 (Research to Prevent Blindness, Inc. (RPB) to the
Department of Ophthalmology at the University of Washington), by the
Ruth and Milton Steinbach Fund, by the Alcon Research Institute, and by
the E. K. Bishop Foundation.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, October 16, 2001, DOI 10.1074/jbc.M105840200
2
It is worth speculating that formation of
13-cis-retinoids in RDH5 knockout mice (23) could be
a result of the lower affinity of CRALBP toward
11-cis-retinol as compared with 11-cis-retinal. Thus, CRALBP protects against reverse isomerization in native conditions, but this process is modified in conditions of lower oxidation power in RDH5 knockout mice, leading to production of 13-cis-retinoids (Fig. 1).
The abbreviations used are:
RPE, retinal pigment
epithelial cell(s);
CRALBP, cellular retinaldehyde-binding protein;
CRBP, cellular retinol-binding protein;
LRAT, lecithin-retinol
acyltransferase;
MES, 4-morpholineethanesulfonic acid;
MOPS, 3-[N-morpholino]propanesulfonic acid;
MS, mass
spectrometry;
NEM, N-ethylmaleimide;
ROS, rod outer
segments;
RDH, retinol dehydrogenase;
HPLC, high pressure liquid
chromatography;
BTP, 1,3-bis[tris(hydroxymethyl)-methyl-amino]propane);
DMF, N,N-dimethylformamide.
Isomerization of 11-cis-Retinoids to
All-trans-retinoids in Vitro and in
Vivo*
§,,, and§¶
Ophthalmology,
¶ Pharmacology, and § Chemistry, University of
Washington, Seattle, Washington 98195
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ mice, which have defective forward
isomerization. This study demonstrates an uncharacterized activity of
RPE microsomes that could be important in the normal flow of retinoids
in the eye in vivo during dark adaptation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G = ~4 kcal/mol)
is provided by hydrolysis of the fatty acid chain of
all-trans-retinyl esters in a reaction that involves an
enzymatic nucleophilic attack at the C-11 position and alkyl cleavage of the ester, allowing free rotation around the
C-11-C-12 bond and removal of the nucleophile with concomitant
hydration to form 11-cis-retinol (25, 26). More recently, it
has been found that apo-CRALBP greatly enhances the formation of
11-cis-retinol in vitro upon addition of
all-trans-retinol to RPE microsomes (27-30). Additionally,
by substituting CRBP for CRALBP, not only are
all-trans-retinol and 11-cis-retinol produced,
but also 13-cis-retinol in amounts that cannot be explained
through spontaneous thermal isomerization (31). CRBP preferentially
binds all-trans-retinol but will bind other isomers with
lower affinity. These observations and others have led to the proposal
that isomerization to 11-cis-retinol progresses through mass
action, with binding of 11-cis-retinol to CRALBP
(G > 8 kcal/mol) driving the reaction, and
that the transition state exists as a carbocation where electron
delocalization across the polyene chain reduces the bond order,
allowing isomerization to occur with the isomeric specificity of
the binding protein (1, 31). Thus, this hypothetical reaction is, in
part, similar to the mechanism proposed for the much more extensively
studied retinol dehydratase (32).
G = ~3-4 kcal/mol). Biochemical defects, often
caused by mutations in enzymes and retinoid-binding proteins involved
in the retinoid cycle, can break down the carefully protected retinoid
metabolic processes, causing a number of eye diseases. Such mutations
include defects in the ATP-binding cassette transporter (Stargardt
disease, recessive retinitis pigmentosa, and age-related macular
degeneration) (33, 34), RPE65 (Leber congenital amaurosis) (35-37),
CRALBP (autosomal recessive retinitis pigmentosa and retinitis punctata
albescens) (38, 39), LRAT (early onset severe retinal dystrophy) (40), and 11-cis-RDH (fundus albipunctatus) (19, 21, 41, 42). Studies of 11-cis-RDH knockout mice (disruption of the
RDH5 gene) have shown that loss of the
11-cis-retinol-specific dehydrogenase can be compensated for
by other dehydrogenases within the RPE (yet to be identified),
causing only minor phenotypes; however, on the biochemical level, the
loss of retinoid protection and control leads to the accumulation of
11-cis-retinol/13-cis-retinol and
11-cis-retinyl esters/13-cis-retinyl esters (Fig.
1). This suggests that
11-cis-RDH is necessary to prevent perturbation of retinoid
flow, and its loss results in the production of
13-cis-retinoids for which there is no known biological
function (20).
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Fig. 1.
Conversion of
all-trans-retinol in RPE.
All-trans-retinol diffuses to the RPE from ROS, and it is
isomerized to 11-cis-retinol (forward reaction) and then
oxidized to 11-cis-retinal by 11-cis- retinol
dehydrogenases (11-cis-RDHs). Retinoids are protected by
binding proteins (CRALBP and CRBP) to facilitate transport of
hydrophobic substrates and also to prevent inadvertent isomerization to
nonfunctional isomers such as 13-cis-retinol or unproductive
conversion to all-trans-retinol (reverse isomerization).
Lack of the RDH5 gene product leads to accumulation of
13-cis-retinol (22).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C and were typically used
within 1 month of preparation. UV treatment of microsomes was performed
in 200-µl aliquots in a quartz cuvette on a ChromatoUVE
transilluminator (model TM-15 from UVP Inc.) at 0 °C for 5 min
unless mentioned otherwise. All experiments were carried out under dim
red light.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Protection of 11-cis-retinol
by CRALBP. A, HPLC analysis of retinoids 20 min after
the addition of 0.5 µl of 11-cis-retinol (4 mM
in DMF) to untreated RPE (a), RPE microsomes treated for 5 min with UV light (b), and UV-treated microsomes with the
addition of 28 µM CRALBP (c). Retinoids were
extracted with hexane as described under "Experimental Procedures."
In addition to all-trans-retinol and
11-cis-retinol, a minor production of
9-cis-retinol (denoted by asterisk) after UV
treatment was also observed in the absence of CRALBP, suggesting a lack
of isomeric specificity for this reaction. B, the
bars indicate the relative amount of
all-trans-retinol and 11-cis-retinol after
incubation of 11-cis-retinol in UV-treated RPE microsomes
with no CRALBP (a) or the addition of ~28 µM
CRALBP (b). Additional samples containing CRALBP were
centrifuged to pellet membrane fractions, and the supernatant
(c) and pellet (d) were analyzed for retinol
content.
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Fig. 3.
UV-treatment time course and temperature
inactivation. A, HPLC traces a-d
show the effect of increasing UV exposure to RPE microsomes. Added
11-cis-retinol (peak 2) isomerizes to
all-trans-retinol (peak 3) and then to
9-cis-retinol and 13-cis-retinol (denoted by an
asterisk) as the retinoids reach equilibrium. B,
temperature inactivation of UV damage to RPE microsomes.
Traces a-e show the effects of alternating
exposure of RPE microsomes to UV exposure (5 min) (traces
a, c, and e) and boiling (8 min)(traces b and d).
13-cis-Retinol (asterisk) was also formed upon
final exposure to UV treatment, suggesting accumulation of
light-induced damage.
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Fig. 4.
Substrate specificity of reverse
isomerization. Reverse isomerization was conducted in the presence
of 11-cis-retinol, 13-cis-retinol,
9-cis-retinol, or all-trans-retinol for a 0-20
min time course. The top panels show
chromatograms for boiled UV-treated RPE microsomes after 20 min of
incubation (bottom trace) and UV-treated RPE
microsomes incubated for 20 min (top trace). The
peaks are identified as follows: 9-cis-retinol
(peak 1), all-trans-retinol
(peak 2), 11-cis-retinol
(peak 3), and 13-cis-retinol
(peak 4). The bottom panels compare formation of
all-trans-retinol (or 13-cis-retinol in the case
of the all-trans-retinol panel) in UV-treated RPE microsomes
(circles) versus boiled UV-treated RPE microsomes
(squares). Insets show the UV-visible spectra of
selected retinoids.
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Fig. 5.
pH profile of reverse and forward
isomerization. A, pH profile of reverse isomerization
to all-trans-retinol. This reaction optimizes at pH 5.5-6.0
and then dramatically drops off at higher pH partially because of the
increasing rate of retinyl ester formation at higher pH
(inset), and the preference for isomerization to
all-trans-retinol at low pH (see "Results").
B, pH profile of isomerization of
all-trans-retinol to 11-cis-retinol. This
enzymatic reaction optimizes at pH 7.5. The inset shows the
increase in formation of retinyl esters with increasing pH.
View larger version (15K):
[in a new window]
Fig. 6.
Arrhenius plots of isomerization
of 11-cis-retinol and 13-cis-retinol
to all-trans-retinol. The rate of formation of
all-trans-retinol was measured in duplicate at temperatures
ranging from 5 to 53 °C in 6 °C increments. The log of the rate
was plotted against 1/T * 1000. Linear least-squares
measurement was performed, and the activation energies were calculated
from the slopes and were found to be 19.5 kcal/mol for
11-cis-retinol (A) and 20.1 kcal/mol for
13-cis-retinol (B).
where R is the gas constant (1.98 kcal/K·mol),
and Ea is the activation energy. The calculated
activation energies were found to be 19.5 kcal/mol for
11-cis-retinol (Fig. 6A) and 20.1 kcal/mol for
13-cis-retinol (Fig. 6B). These values are
comparable with Ea = ~17-25 kcal/mol for
all-trans-retinol to 11-cis-retinol isomerization
and are in general agreement, indicating that although both the forward
and reverse isomerization process proceed through different mechanisms,
the intermediates could be comparable in terms of their energetics
because both have the ability to lower the isomerization barrier from
~45 kcal/mol to about half (31).
(Eq. 1)
View larger version (47K):
[in a new window]
Fig. 7.
Isomerization of
cis-retinols to all-trans-retinol in
UV-treated RPE microsomes. A, relative amounts of
all-trans-retinol formed from cis-retinols in the
presence of bovine RPE, mouse liver, kidney, and brain microsomes.
Black, 11-cis-retinol; gray,
13-cis-retinol; white, 9-cis-retinol.
B, relative amounts of all-trans-retinol
formed from 11-cis-retinol in bovine RPE, mouse liver,
kidney, and brain microsomes with no UV treatment (white), 5 min of UV treatment (gray), or 8 min of boiling following UV
treatment (black). Amounts are normalized to total retinoid
content measured by HPLC and spectrophotometry.
View larger version (19K):
[in a new window]
Fig. 8.
Inactivation of forward and reverse
isomerization by alcohols, protein-modifying reagents, and ascorbic
acid. A, the effects of modifying reagents on reverse
isomerization (gray) and forward isomerization
(black). a, control; b, 2% MeOH;
c, 2% EtOH; d, 2% isopropyl alcohol;
e, 2% isobutyl alcohol; f, 100 mM
NEM; g, 100 mM acetic anhydride; h, 1 mg/ml phospholipase A2; i, 1 mg/ml Pronase.
UV-treated RPE microsomes were exposed to these reagents and then
tested for activity. B, loss of reverse isomerization
activity in the presence of 10 mM ascorbic acid in bovine
RPE and liver microsomes that were UV-treated for 5 min.
View larger version (22K):
[in a new window]
Fig. 9.
MS analysis and stereospecificity of
isomerization of
[15-2H,18O]11-cis-retinol
and
[15-2H,18O]13-cis-retinol to
all-trans-retinol. A and C,
the fragmentation patterns of
[15-2H,18O]13-cis-retinol and
[15-2H,18O]11-cis-retinol,
respectively, indicating the shift to 289 resulting from the presence
of 2H and 18O labels. B and
D, the MS fragmentation pattern of
all-trans-retinol collected from reverse isomerization,
indicating that there is no loss of 18O. E,
retention of configuration during reverse isomerization.
Pro-R-[15-3H]11-cis-retinol was
used as substrate in bars 1 and 2 and
bars 5 and 6, respectively.
Pro-S-[15-3H]11-cis-retinol
prepared using HLADH or 11-cis-RDH was used as substrate in
bars 3 and 4 and bars
7 and 8, respectively.
All-trans-retinol generated from reverse isomerization was
collected and oxidized using ROS/NADP for bars 1,
3, 5, and 7, and with HLADH/NAD for
bars 2, 4, 6, and
8.
View larger version (26K):
[in a new window]
Fig. 10.
Reverse isomerization of
11-cis-retinol to all-trans-retinol
in vivo. Upper panel, HPLC
separation of retinoids from Rpe65+/+ and
Rpe65+/+ mice administered 11-cis-retinol into
the intravitreal space. Peak 1,
13-cis-retinyl esters; peak 2,
11-cis-retinyl esters; peak 3,
all-trans-retinyl esters; peaks 4 and
4', syn- and
anti-11-cis-retinal oxime; peaks
5 and 5', syn- and
anti-all-trans-retinal oxime; peak 6,
11-cis-retinol; peak 7,
all-trans-retinol. Lower panel, HPLC
analysis of retinoids for Rpe65 / and
Rpe65/ mice administered 11-cis-retinol into
the intravitreal space.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice, which have a defective forward isomerization reaction (14, 16).
Almost complete isomerization of 11-cis-retinol injected
into the eye in the dark was observed, suggesting that the forward
reaction is not essential for the reverse isomerization. Importantly,
higher levels of 13-cis-retinyl esters were observed in
treated Rpe65+/+ and Rpe65/ mice, showing resemblance to the in vitro reverse isomerization.
/ mice, no anhydroretinol
or retroretinol was observed. This suggested that in vivo
the hydroxyl group of 11-cis-retinol was not involved in the
reverse isomerization process.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Ophthalmology, University of Washington, Box 356485, Seattle, WA
98195-6485. Tel.: 206-543-9074; Fax: 206-221-6784; E-mail:
palczews@u.washington.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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M. L. Batten, Y. Imanishi, T. Maeda, D. C. Tu, A. R. Moise, D. Bronson, D. Possin, R. N. Van Gelder, W. Baehr, and K. Palczewski Lecithin-retinol Acyltransferase Is Essential for Accumulation of All-trans-Retinyl Esters in the Eye and in the Liver J. Biol. Chem., March 12, 2004; 279(11): 10422 - 10432. [Abstract] [Full Text] [PDF]
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S. M. Noorwez, V. Kuksa, Y. Imanishi, L. Zhu, S. Filipek, K. Palczewski, and S. Kaushal Pharmacological Chaperone-mediated in Vivo Folding and Stabilization of the P23H-opsin Mutant Associated with Autosomal Dominant Retinitis Pigmentosa J. Biol. Chem., April 11, 2003; 278(16): 14442 - 14450. [Abstract] [Full Text] [PDF]
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F. Haeseleer, G.-F. Jang, Y. Imanishi, C. A. G. G. Driessen, M. Matsumura, P. S. Nelson, and K. Palczewski Dual-substrate Specificity Short Chain Retinol Dehydrogenases from the Vertebrate Retina J. Biol. Chem., November 15, 2002; 277(47): 45537 - 45546. [Abstract] [Full Text] [PDF]
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J. P. Van Hooser, Y. Liang, T. Maeda, V. Kuksa, G.-F. Jang, Y.-G. He, F. Rieke, H. K. W. Fong, P. B. Detwiler, and K. Palczewski Recovery of Visual Functions in a Mouse Model of Leber Congenital Amaurosis J. Biol. Chem., May 17, 2002; 277(21): 19173 - 19182. [Abstract] [Full Text] [PDF]
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