From the Department of Ophthalmology, University of
Florida, Gainesville, Florida 55455, the Departments of
¶ Ophthalmology, ** Pharmacology, and
Chemistry, University of Washington, Seattle,
Washington 98195, and the
International Institute of Molecular
and Cell Biology and the Department of Chemistry, University of
Warsaw, PL-02109 Warsaw, Poland
Received for publication, January 6, 2003, and in revised form, January 31, 2003
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ABSTRACT |
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Protein conformational
disorders, which include certain types of retinitis pigmentosa,
are a set of inherited human diseases in which mutant proteins are
misfolded and often aggregated. Many opsin mutants associated with
retinitis pigmentosa, the most common being P23H, are misfolded and
retained within the cell. Here, we describe a pharmacological
chaperone, 11-cis-7-ring retinal, that quantitatively
induces the in vivo folding of P23H-opsin. The rescued
protein forms pigment, acquires mature glycosylation, and is
transported to the cell surface. Additionally, we determined the
temperature stability of the rescued protein as well as the reactivity
of the retinal-opsin Schiff base to hydroxylamine. Our study unveils
novel properties of P23H-opsin and its interaction with the
chromophore. These properties suggest that 11-cis-7-ring retinal may be a useful therapeutic agent for the rescue of P23H-opsin and the prevention of retinal degeneration.
Rhodopsin (Rh)1 is the
visual pigment protein of the rod cell and belongs to a large family of
transmembrane G protein-coupled receptor, which are involved in
numerous physiological functions (1, 2). This superfamily of membrane
glycoprotein receptors is characterized by seven transmembrane
In recent years, more than 100 opsin mutants (apoprotein = opsin,
opsin bound with chromophore = Rh) have been linked to various genetic forms of retinitis pigmentosa, the most common form of hereditary retinal degeneration. Retinitis pigmentosa leads to photoreceptor death and subsequent severe loss of peripheral and night
vision (5, 6). These mutants account for nearly 50% of all the
autosomal dominant retinitis pigmentosa cases with the most frequent
mutation being P23H, which accounts for ~10% of all cases (7).
Although they display distinct biochemical features, the mutant
phenotypes fall mainly into three basic classes (8-10). Class I
mutants are expressed at nearly WT levels and form stable pigment with
11-cis-retinal in the dark. These mutations cluster at the C
terminus of Rh and disrupt vectorial transport to the rod outer
segment. Some mutations also inefficiently activate transducin (5, 11).
Class II mutants do not bind 11-cis-retinal and are retained
in the endoplasmic reticulum. Class III mutants, like P23H, form small
amounts of pigment and mainly remain in the endoplasmic reticulum or
form aggresomes (12, 13). These mutants are targeted for degradation by
the ubiquitin proteasome system (13). The results obtained from the
heterologous expression of the opsin mutants in mammalian cells
correlate well with the findings using transgenic Rh animals (14).
Other phenotypic properties acquired as a result of a mutation may
include the destabilization of the structure formed within the rod
outer segment by mutated Rh molecules (15) or the disruption of other
biological processes unique to the rod outer segment.
Patients with the P23H mutation (Fig.
1A) usually have milder
disease progression than those harboring other Rh mutations. Based on
the crystal structure of Rh, Pro23 is located in the
N-terminal tail within one of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices that are anchored within the lipid bilayer. In contrast to
other G protein-coupled receptors, which respond to diffusible ligands,
Rh (~40,000 daltons) has a covalently bound reverse agonist,
11-cis-retinal, which yields a distinct UV-visible spectrum
with a
max of ~500 nm (3). The crystal structure of Rh
has been previously elucidated (4).
-strands that make up an integral
part of the N-terminal plug (16). The plug keeps the chromophore in its
proper position (3), and mutations within this region result in
improper folding of opsin and poor binding of the chromophore (5).
View larger version (41K):
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Fig. 1.
Models of Rh and structures of
7-locked-retinals. A, two-dimensional model of Rh (4).
White letters on a black background indicate a
mutation associated with retinitis pigmentosa. Pro23 is
shown on a red background. B, chemical structures
of the four 11-cis-7-ring retinal isomers. Carbon atom
numbering is as accepted in retinoid nomenclature. C, WT Rh.
Gln184 binds to Gly182 on the plug between
helices IV and V. Pro23 is located on the first
extracellular loop. D, P23H mutation of Rh.
Gln184 bridges Gly182 and His23 and
stabilizes this portion of the mutant protein. E, molecular
dynamics simulation of Rh and nearby water cap. The same results were
obtained for WT and the P23H mutant. The water cap was put on the
extracellular part of Rh (together with that part buried in membrane in
contact with polar heads of phospholipids). Only water and the
extracellular part of Rh were allowed to move. During 100 ps of
simulations, water found its way toward the cytoplasmic part of Rh
going through the highly hydrophobic (red) surface part of
the membrane of Rh. The hydrophilic surface is colored in
blue, and neutral parts are white. In every
simulation, water took the same route. During the whole course of
simulation, the hydrogen bond involving His23 and
Gln184 was stable. Water is shown as yellow
mesh.
In previous studies, we demonstrated that P23H-opsin (opsin with
proline at position 23 mutated to histidine) forms the pigment poorly,
does not acquire the Golgi-related glycosylation, and is retained
within the cell, collectively providing evidence that it is misfolded
(9). In this paper, we show that P23H-opsin can be induced to properly
fold by providing cells with a seven-membered ring variant of
11-cis-retinal during opsin biosynthesis. Furthermore, the
remarkable affinity and selectivity of mutant opsin for
11-cis-7-ring retinal can be explained based on the crystal
structure of Rh. These findings open the possibility for
pharmacological intervention of this otherwise incurable retinal
degenerative disease.
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EXPERIMENTAL PROCEDURES |
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Synthesis of 11-cis-7-Ring Retinals--
Synthesis of
11-cis-7-ring retinals was performed as described previously
with some modifications (17-19). All of the reactions were performed
in a dried nitrogen atmosphere unless otherwise specified.
2-Cycloheptenone was first converted into allyl acetate by
N-bromosuccinimide bromination in CCl4
followed by treatment with KOAc in hexamethylphosphoramide. Purified
4-acetoxy-2-cycloheptenone (46% from 2-cycloheptenone) was subjected
to a Horner-Emmons reaction with diethyl (2-cyanoethyl)phosphonate,
which gave an isomeric mixture of two trans/cis
(E/Z) cyanoacetates in a 2:1 ratio. The mixture was hydrolyzed
with K2CO3 in MeOH:H2O (5:1), and
then the hydroxy group of the resulting allylic alcohol was protected with tert-butyldimethylsilyl chloride in pyridine (80% from
cycloheptenonyl acetate). The resulting cyano compound was reduced with
diisobutylaluminium hydride in CH2Cl2 to an
aldehyde and purified by flash chromatography on a silica gel (63%).
-Cyclocitral was reduced with NaBH4 to
-cyclogeraniol
and then reacted with triphenylphosphine hydrobromide in MeOH over 3 days to afford
-cyclogeranyltriphenylphosphonium bromide after the
removal of solvent and drying of the residue in vacuum. Wittig reaction
of the silylated aldehyde with an excess of phosphonium salt in the
presence of potassium tert-butoxide and a catalytic amount
of 18-crown-6 in methylene chloride at ambient temperature afforded
protected cyclic alcohol in 75% yield. The
tert-butyldimethylsilyl protecting group was removed by
treatment with tetrabutylammonium fluoride in dry THF, and the
resulting alcohol was oxidized with MnO2 in
CH2Cl2 to a mixture of two (E/Z) cyclic ketones
(2:1 ratio) in 96% yield. This mixture was condensed with triethyl
phosphonoacetate under Horner-Emmons conditions, followed by lithium
aluminum hydride reduction of the resulting isomeric mixture of ethyl
7-ring retinoates and oxidation of retinols with MnO2
(86%) in CH2Cl2.
The following isomers were used: isomer 1 (Fig. 1B), 9,11,13-tri-cis-7-ring retinal: 1.04 (s, 6H, 2xCH3-1), 1.45-1.50 (m, 2H, CH2-2), 1.59-1.67 (m, 2H, CH2-3), 1.74 (s, 3H, CH3-5), 1.90 (m, 2H, J 6.74 Hz, CH2-22), 1.97 (s, 3H, CH3-9), 2.03 (t, 2H, CH2-4), 2.46 (t, 2H, J 7.26 Hz CH2-20), 2.51 (t, 2H, J 6.74 Hz CH2-21), 5.78 (d, 1H, J 7.78 Hz, H-14), 6.20 (d, 1H, J 11.42 Hz, H-7), 6.54 (d, 1H, J 11.42 Hz, H-8), 6.95 (d, 1H, J 16.08 Hz, H-12), 7.08 (d, 1H, J 16.08 Hz, H-11), 10.11 (d, 1H, J 7.78 Hz, H-15); isomer 2 (Fig. 1B), 11,13-di-cis-7-ring retinal: 1.04 (s, 6H, 2xCH3-1), 1.47-1.50 (m, 2H, CH2-2), 1.60-1.67 (m, 2H, CH2-3), 1.73 (s, 3H, CH3-5), 1.86 (m, 2H, J 6.74 Hz, CH2-22), 2.00 (s, 3H, CH3-9), 2.04 (t, 2H, CH2-4), 2.45 (t, 2H, J 7.27 Hz CH2-20), 2.54 (t, 2H, J 6.74 Hz CH2-21), 5.79 (d, 1H, J 7.78 Hz, H-14), 6.32 (d, 1H, J 16.08 Hz, H-7), 6.52 (d, 1H, J 16.08 Hz, H-8), 7.02 (m, 2H, H-11, H-12), 10.12 (d, 1H, J 7.78 Hz, H-15); isomer 3 (Fig. 1B), 11-cis-7-ring retinal: 1.04 (s, 6H, 2xCH3-1), 1.44-1.52 (m, 2H, CH2-2), 1.57-1.67 (m, 2H, CH2-3), 1.74 (s, 3H, CH3-5), 1.86 (m, 2H, J 6.74 Hz, CH2-22), 2.00 (s, 3H, CH3-9), 2.04 (t, 2H, CH2-4), 2.58 (t, 2H, J 6.75 Hz CH2-20), 2.87 (t, 2H, J 6.75 Hz CH2-21), 5.93 (d, 1H, J 8.3 Hz, H-14), 6.22 (d, 1H, J 11.42 Hz, H-12), 6.32 (d, 1H, J 16.08 Hz, H-7), 6.52 (d, 1H, J 16.08 Hz, H-8), 6.91 (s, 1H, J 11.42 Hz, H-11), 10.03 (d, 1H, J 7.79 Hz, H-15); and isomer 4 (Fig. 1B), 9,11-di-cis-7-ring retinal: 1.04 (s, 6H, 2xCH3-1), 1.44-1.52 (m, 2H, CH2-2), 1.57-1.67 (m, 2H, CH2-3), 1.74 (s, 3H, CH3-5), 1.86 (m, 2H, J 6.74 Hz, CH2-22), 2.00 (s, 3H, CH3-9), 2.04 (t, 2H, CH2-4), 2.58 (t, 2H, J 6.75 Hz CH2-20), 2.87 (t, 2H, J 6.75 Hz CH2-21), 5.93 (d, 1H, J 8.3 Hz, H-14), 6.17 (d, 1H, J 11.41 Hz, H-12), 6.21 (d, 1H, J 16.08 Hz, H-7), 6.53 (d, 1H, J 16.08 Hz, H-8), 6.98 (s, 1H, J 11.41 Hz, H-11), 10.03 (d, 1H, J 7.78 Hz, H-15).
Cell Lines and Growth Conditions-- For the construction and selection of tetracycline-inducible opsin cell lines, we used the Invitrogen T-RExTM system. Briefly, wild type opsin and the P23H mutant were excised as EcoRI-NotI fragments from pMT4 and then cloned into the EcoRI-NotI site within the polylinker of pcDNA4. Using opsin-specific forward and reverse primers, the entire gene was sequenced for verification. These plasmids were then separately transfected by calcium-phosphate precipitation into T-RExTM-293 cells that already stably expressed the tetracycline repressor; these cells were routinely grown under blasticidin selection. After transfection, zeocin was added to the culture medium, and surviving colonies of cells were isolated and subsequently expanded into larger 6-well plates. Each of these clones was exposed to tetracycline, and 48 h after induction, the cells were harvested and solubilized with 1% DM. Separately, uninduced cells were also solubilized. The samples were run on SDS-PAGE and immunoblotted with the monoclonal antibody 1D4. Cell lines were chosen on the basis of production of the least amount of opsin (i.e. nondetectable opsin levels on immunoblots) in the absence of tetracycline and moderate amounts of opsin in the presence of the drug. HEK293 cells were grown in Dulbecco's modified Eagle's medium containing high glucose (Invitrogen) at 37 °C in the presence of 5.0% CO2. In all of the experiments, the cells were harvested after 48 h of induction with tetracycline (1 µg/ml).
Cell Culture and Regeneration--
The cells were washed three
times with PBS, harvested, and incubated with different analogs of
11-cis-retinal (50 µM) for 45 min at 4 °C.
The cells were then lysed with 1.0% n-dodecyl--maltoside (DM) (Anatrace) in the presence of protease inhibitors (complete protease inhibitor mixture tablets; Roche Molecular Biochemicals) and
centrifuged at 36,000 rpm in a Beckman ultracentrifuge for 30 min at
4 °C. The supernatant was incubated with 1D4-coupled CNBr-activated
Sepharose 4B (Pharmacia Corp.) beads overnight. The beads were then
washed three times with PBS containing 0.1% DM. Bound pigments were
eluted off the beads using 0.1 mM synthetic peptide (last 9 amino acid residues of the C terminus of Rh) in 0.1% DM. In
experiments when retinoids (50 µM) were added during biosynthesis, a Me2SO solution of retinoids (10 µl of 100 mM) was added directly to the cell culture medium after 2 and 24 h of induction. The cells were harvested at 48 h and
lysed with 1.0% DM. Pigment was purified by immunoaffinity
chromatography. The UV-visible spectra of the eluted pigment samples
were then recorded on a Perkin Elmer Lambda 800 UV-visible
spectrophotometer in the range of 250-650 nm.
SDS Gel Electrophoresis and Immunoblotting-- The samples were loaded on 10% SDS-polyacrylamide gels (20) and transferred to Immobilon-NC membrane (Millipore) according to established protocols. The membranes were blocked with blocking buffer (Licor) for 1 h and incubated at room temperature with 1D4 antibody (1:1000) for 1 h. The membranes were then washed with PBS containing 0.1% Tween 20 and then incubated for 1 h at room temperature with IRDye800TM-conjugated affinity purified goat anti-mouse IgG (Licor), diluted 1:5000. The membranes were then washed with PBS containing Tween 20 and scanned using an Odyssey Infrared imager (Licor).
Glycosylation Status-- Purified 7-Rh and 7-P23H-Rh (Rh and P23H-Rh regenerated with 11-cis-7-ring retinal, respectively) were treated with N-glycanase according to the manufacturer's recommendations (Glyko). The samples were incubated at 37 °C for 16 h and then loaded onto a 10% polyacrylamide gel. The immunoblots were developed as described above.
Photosensitivity Experiments-- Whole cells expressing WT and P23H-opsin treated with 11-cis-7-ring retinals were harvested and exposed for 20 min to light emitted from a Fiber-Lite, MI 150, High Intensity Illuminator (Dolan-Jenner). The cells were then washed and then lysed with 1.0% DM as described above. In a separate experiment, immunoaffinity purified 7-Rh and 7-P23H-Rh samples were exposed to light from Fiber-Lite, and UV-visible spectra were recorded at different times.
Hydroxylamine Sensitivity-- Whole cells expressing WT and P23H-opsin treated with 11-cis-7-ring retinals were harvested and treated with 500 mM neutral solution of hydroxylamine for 45 min. The cells were thoroughly washed with PBS to remove all traces of hydroxylamine. Rh was then purified, and spectra were taken. In a separate experiment, purified 7-Rh and 7-P23H-Rh were treated with 20 mM neutral hydroxylamine, and UV-visible spectra were recorded at different times.
Thermostability of Pigments and Acid Hydrolysis-- Purified 7-Rh and 7-P23H-Rh were incubated at 37 °C, and the spectra were recorded at indicated times. In acid treatment experiments, purified 7-Rh and 7-P23H-Rh were treated with 100 mM sulfuric acid, and spectra were recorded.
HPLC Analysis of Retinoids-- A 450-µl aliquot of either purified 7-Rh, purified 7-P23H-Rh, or cell medium were treated with 50 µl of 10% SDS and 100 µl of 1 M NH2OH (freshly prepared, pH 7.5). The mixture was kept at room temperature for 30 min, and then 400 µl of MeOH and 400 µl of hexane were added. The mixture was shaken on a vortex for 5 min and centrifuged at 14,000 rpm to separate layers. The hexane layer was collected. Another 400 µl of hexane was added to the aqueous layer, and extraction was repeated. Combined hexane layers were dried down and redissolved in 120 µl of hexane, and 100-µl fractions were analyzed by normal phase HPLC as previously described (21).
Immunocytochemistry-- HEK293 cells expressing WT Rh or P23H-opsin under a tetracycline-inducible promoter were cultured in Dulbecco's modified Eagle's medium (Invitrogen). The cells were attached to glass bottom microwell dishes (MatTek Corp.) with Cell-Tak (Becton Dickinson Labware). Expression of opsin or P23H-opsin was induced by the addition of 1 µg/ml tetracycline as recommended by the manufacturer's protocol (Invitrogen). The cells were treated with 50 µM 11-cis-7-ring retinal 2 h after induction. The cells were harvested after 24 h and fixed with 4% paraformaldehyde (Fisher) in PBS (136 mM NaCl, 11.4 mM sodium phosphate, pH 7.4) for 10 min and washed by PBS. To block nonspecific labeling, the cells were incubated in 1.5% normal goat serum (Vector Lab., Inc.) in PBST (136 mM NaCl, 11.4 mM sodium phosphate, 0.1% Triton X-100, pH 7.4) for 15 min at room temperature. The cells were incubated overnight at 4 °C in purified 1D4 antibody diluted with PBST. The sections were rinsed in PBST and incubated with indocarbocyanine (Cy3)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Lab., Inc.) and Hoechst 33342 dye (Molecular Probes). The cells were rinsed in PBST and mounted in 50 µl of 2% 1,4-diazabicyclo-[2.2.2]octane (Sigma) in 90% glycerol to retard photobleaching. For confocal imaging, the cells were analyzed on a Zeiss LSM510 laser scanning microscope (Carl Zeiss, Inc.).
Protein Simulation and Modeling-- Coordinates for Rh were taken from the Protein Data Bank (1HZX) (22). The addition of hydrogen atoms and all of the optimizations were done in Insight II (InsightII release 2000, Accelrys, Inc., San Diego, CA). Crystallographic water was removed, and water molecules were introduced based on the accessible space in the extracellular region. No minimization was performed before water was added.
A water layer (5 Å thick) was used to coat the extracellular part of
Rh as well as residues in contact with polar phospholipids heads. All
of the water molecules were allowed to move freely, as was the
extracellular half of Rh, which contains P23H and retinal. Because no
water cap was put on the cytoplasmic part of Rh, this part of the
molecule was frozen to prevent degradation of the model.
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RESULTS |
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Rationale-- The chromophore of Rh, 11-cis-retinal, plays a central role in the photoactivation process (28) and is also important for the stabilization of the receptor. For example, Rh is stable for months in mild detergents, although opsin precipitates in a few hours (29). The chromophore may also induce a proper folding of Rh mutants (12, 30). The P23H mutation may destabilize the native chromophore-accepting conformation of opsin, leading to aberrant folding and subsequent aggregation (13). However, an analog of 11-cis-retinal may facilitate native-like folding of P23H-opsin and therefore play the role of pharmacological chaperone. Recently, we demonstrated that WT opsin regenerated with 11-cis-7-ring retinal (7-Rh), containing a chromophore with a seven-membered ring to prevent isomerization around the C11=C12 double bond (Fig. 1B), is very stable in vivo and in vitro (31). In contrast, bleaching of the WT opsin regenerated with 11-cis-6-ring retinal (6-Rh), which contains a more rigid chromophore, produces multiple isomers with modest changes in protein conformations (28). These results reveal that 11-cis-7-ring retinals, particularly isomer 3 (Fig. 1B), are easily accepted into the binding site of opsin and provide additional contact sites by virtue of the seven-membered ring and the 9-methyl group with the protein moiety (31). Because of its intrinsic flexibility, this analog could potentially influence/affect protein folding during biosynthesis, which is particularly important for those mutant proteins that fold less efficiently. Clearly, the binding pocket of WT opsin, and possibly that of the P23H mutant as well, adopt specific conformations that allow it to bind preferentially certain retinoids (32). This observation provided the impetus to pursue the experiments described below.
11-cis-7-Ring Retinal Chaperones Folding of
P23H-opsin--
Rh is regenerated with 11-cis-7-ring
retinals in the harvested membranes (Fig.
2B), whereas only a small
amount of pigment was formed when membranes containing P23H-opsin were
treated with 11-cis-retinal (9), 11-cis-6-ring
retinal, or 11-cis-7-ring retinal after the cells
were harvested (Fig. 2B). If the mutant proteins are
structurally unstable, we reasoned that addition of retinal
during the course of protein biosynthesis might induce a
more native-like folding of the protein and its subsequent
stabilization. Our first attempts utilized 11-cis-locked
versions of 11-cis-retinal in stable cell lines expressing
WT and P23H-opsin. The UV-visible spectra of the immunoaffinity
purified samples from 11-cis-6- or 11-cis-7-ring
retinals provided to the cells during biosynthesis showed that both
retinals recombined with WT opsin (Fig. 2C). Supporting our
prediction, P23H-opsin formed significant amounts of pigment when
11-cis-7-ring retinal was added during biosynthesis (Fig.
2D). The max of the pigment was ~490 ± 3 nm, unlike the WT protein (
max = ~500 ± 3 nm) (Fig. 2, C and D). The UV-visible spectra
with a
max of ~440 nm of acid-denatured purified
7-P23H-Rh (Fig. 2D, inset) provided evidence that
the chromophore forms a Schiff base with rescued P23H-opsin.
Importantly, no significant pigment was seen when
11-cis-6-ring retinal, which is photoisomerable along
C9- and C13-C=C double bonds (28), was added to
P23H-opsin-expressing cells (Fig. 2D). Furthermore,
11-cis-9-demethyl-7-ring retinal was also ineffective in
pigment formation with P23H-opsin (data not shown). These experiments
illustrate unique specificity of the binding interaction between
11-cis-7-ring retinal and P23H-opsin.
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Immunoblots showed that the rescued P23H-opsin had a high molecular
weight and a Golgi-associated glycosylation pattern (Fig. 3B, lane 5) similar
to the WT protein (Fig. 3A), suggesting that the protein is
sufficiently folded to proceed along the secretory pathway. This is in
striking contrast to P23H-opsin that was not treated with
11-cis-7-ring retinal during P23H-opsin biosynthesis (Fig.
3B, lanes 1-4). Purified 7-Rh and 7-P23H-Rh were
efficiently deglycosylated by N-glycanase, and the mutant
protein spontaneously aggregated, forming high molecular weight
aggregates (Fig. 3, C and D).
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In control experiments, we tested 9-cis-retinal and
11-cis-retinal for proper folding of P23H-opsin. It should
also be noted that 9-cis-retinal promoted transport of
P23H-opsin to the cell surface as previously shown by another group of
investigators (12). We observed that 11-cis-retinal also
induced the in vivo folding and stabilization of P23H-opsin
forming visual pigments (499 nm and
492 nm, for Rh and P23H-Rh, respectively) (Fig. 2,
C and D). Moreover, these pigments also acquired
a mature state of
glycosylation.2
11-cis-retinoids are unstable in vitro and
in vivo and rapidly undergo "reverse isomerization"
(33). Therefore, locked analogs were used throughout the rest of this study.
Properties and Stability of 7-P23H-Rh--
When the mixture of the
four 7-ring retinal isomers (Fig. 1B) was added to cells
expressing opsin and P23H-opsin during biosynthesis, both WT opsin and
P23H-opsin selectively bound only isomer 3 (Fig. 4, A and D). These
observations further support the extraordinary specificity of WT and
P23H-opsin for binding this 11-cis-retinal isomer (spectrum
in Fig. 4D). Addition of 11-cis-retinal to cell membranes containing either WT or P23H-opsin that were already treated
with the 11-cis-7-ring retinal during their biosynthesis did
not lead to its substitution after photobleaching, as measured spectrophotometrically and by retinoid analysis of the bound
chromophores (data not shown). 7-Rh, purified in the detergent, is
stable to light (Fig. 5A),
whereas purified 7-P23H-Rh undergoes bleaching under the same
conditions (Fig. 5B). In contrast, both Rhs are stable in
the membranes of HEK293 cells (Fig. 5, C and D).
This result suggests that, in detergent, the chromophore in 7-P23H-Rh is more flexible, thus allowing photoisomerization. The mechanism by
which 7-P23H-Rh undergoes bleaching in detergent most likely involves
isomerization of isomer 3 to three other isomers and its subsequent
hydrolysis (Fig. 4F). The membranes apparently stabilized
7-P23H-Rh, thus preventing isomerization of its bound chromophore
(Figs. 4E and 5D). In tissue culture samples,
small amounts of non-Rh-bound retinals eluted between 30 and 40 min and
remained in equilibrium with the vast majority of retinols, governed by
the redox potential of the cells (Fig. 4A,
inset).
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To further characterize the rescued protein, we eluted the affinity bound 7-Rh and 7-P23H-Rh under conditions that selectively released only the folded form of the protein (10 mM phosphate, pH 6.0) (34). The spectra of the eluates (Fig. 2, E and F) and the corresponding immunoblots (Fig. 3, E and F) indicated that ~80% of 7-P23H-Rh folded to form pigment. Additionally, immunoblots of these eluates demonstrated that both contained a mature glycosylated band and unglycosylated bands; however, only the material eluted at pH 6.0 had the Golgi-specific glycosylation pattern.
We also probed the structure of the rescued protein by determining its
sensitivity to neutral hydroxylamine. 7-P23H-Rh is resistant to
hydroxylamine treatment when embedded in the lipid bilayer of HEK293
cells, suggesting that the rescued protein adopts a conformation
that sequesters the Schiff base linkage within the protein and protects
it from chemical modification (Fig.
6B). Unlike the purified WT
protein (Fig. 6A), the mutant chromophore is accessible to
hydroxylamine in detergent (0.1% DM), as evidenced by the formation of
the retinaloximes (max = ~360 nm). These data imply
that the structure of 7-P23H-Rh is less tightly packed than that of the
7-Rh. Finally, Fig. 6 (C and D) shows the
temperature stability of the purified 7-Rh and 7-P23H-Rh. There was no
change in the amount of chromophore after incubation of 7-Rh at either 4 °C (not shown) or 37 °C (Fig. 6C). However, for the
rescued 7-P23H-Rh, there is time-dependent thermal
bleaching that is accelerated by increased temperature, with a
half-life of ~4 min at 37 °C (Fig. 6D) and 10 days at
4 °C in the detergent solution (data not shown).
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Localization of the Rescued
7-Locked-P23H-Rh--
Immunofluorescence microscopy demonstrated that
P23H-opsin is retained intracellularly, predominantly in a perinuclear
distribution with punctuated fluorescence consistent with aggresomes
(Fig. 7C) (9, 12, 13). In
contrast, the rescued 7-P23H-Rh is predominantly found in a diffuse
pattern with significantly greater staining at the cell surface (Fig.
7D) similar to the 7-Rh and WT opsin, which are found
predominantly at the plasma membrane (Fig. 7, A and
B). In summary, this result suggests that the rescued P23H-Rh was not only correctly glycosylated and formed a pigment but
was also correctly routed to the cell membranes.
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DISCUSSION |
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Protein Conformational Disorders--
Dominantly inherited
diseases can result from (a) haploinsufficiency of a gene,
e.g. as for the melanocortin-4 receptor (35, 36);
(b) constitutive activity (gain of function) in some opsin mutants (37) or lack of activity because of somatic mutation (38); and
(c) loss of function because of mutant protein misfolding and aggregation (39). The diseases caused by misfolded proteins are
known as protein conformational disorders and include Alzheimer's disease, Huntington's disease, Parkinson's disease, diabetes
insipidus, cystic fibrosis, prion disease, emphysema, dominant
cataracts, and oculopharyngeal muscular dystrophy (reviewed in Refs. 40 and 41). Gene delivery is one method of correcting haploinsufficiency. Constitutive activity has been successfully blocked by reverse agonist-like compounds (42, 43), and conformation and function can be
rescued by small chemical agents (38). The medically most prevalent
forms of dominant disorders are the protein conformational disorders.
Although they are the most challenging to treat, some successes have
been reported. Examples include the rescue of misfolded cystic
fibrosis transmembrane regulator (CFTR) variant F508 (44) and the
Z-variant of
1-antitrypsin (45). In the instance of CFTR, the osmolytes glycerol and trimethylamino-oxide have been used
(46-48). The protein acquires mature glycosylation and is transported
to the cell surface where it pumps Cl
ions. The chemical
chaperone 4-phenylbutyric acid mediated a marked increase in secretion
of
1-antitrypsin (49). Recent studies, however, suggest
that the rescued protein is less stable than the WT (47). Other
approaches to inducing the folding of proteins have been developed. For
example, mini-chaperones that prevent
-sheet formation prevent toxic
aggregation of amyloid-
found in Alzheimer's disease and the PrP
protein associated with prion disease (50).
P23H Rescue--
We demonstrate that P23H-opsin can be induced to
properly fold and be stabilized with a seven-membered locked ring
version of 11-cis-retinal, the inverse agonist of opsin.
Furthermore, our model system provides a quantitative means to study
the folding of this membrane protein. The rescued 7-P23H-Rh contains a
Schiff base linkage with Lys296, producing pigment with a
max of 490 nm (Fig. 2D) that can be used as a
quantitative measure of the amount of correctly folded protein. Using
chromatographic conditions that selectively elute the folded form of
the protein, 11-cis-7-ring retinal leads to the rescue of
~80% of the P23H-opsin. We have also demonstrated that the rescued
protein is not only folded but also proceeds along the secretory
pathway. Like the WT, the rescued protein acquires the heterogeneous
glycosylation associated with oligosaccharide processing within the
Golgi apparatus. Clearly, the protein is of a sufficiently native-like
conformation and is not sequestered by the cellular quality control
apparatus, at least that which exists at the level of endoplasmic reticulum.
Furthermore, we show that the rescued protein is structurally different from the WT protein. The detergent-purified protein is light-sensitive, displays thermal instability, and is sensitive to neutral hydroxylamine. The bleaching of detergent-solubilized 7-P23H-Rh occurs because of isomerization of double bonds along the polyene chain of the chromophore producing a mixture of isomers. However, in the membranes of the HEK cells, 7-P23H-Rh is stable, and therefore, it is expected this pigment will also be stable in vivo.
Chemical and Structural Considerations of the Rescue-- The mutant P23H protein is less stable because it is misfolded and is thus retained intracellularly. The rescue effect with 11-cis-7-ring retinal leads to increased amounts of the correctly folded mutant and is extraordinarily specific as shown by the lack of activity observed with several structural homologs (11-cis-9-demethyl-ring retinal and 11-cis-6-ring retinal). However, unlike the WT protein, purified 7-P23H-Rh appears to be in a less compact conformation, as evidenced by its sensitivity to neutral hydroxylamine in detergent. We have also observed that the rescued protein spontaneously releases the locked retinoid over time (Fig. 6D). Previous studies have demonstrated that 11-cis-7-ring retinal is inherently more flexible than 11-cis-6-ring retinal (31). Perhaps the rigidity of 11-cis-6-ring retinal does not allow the P23H-opsin binding pocket to accept this chromophore. Alternatively, it is possible that P23H-opsin binds 11-cis-6-ring retinal but that the resulting protein is less stable and maybe more susceptible to water-assisted hydrolysis of the Schiff base linkage, as it is in the case of 9-cis-retinal.
Our molecular modeling studies provide a possible explanation for the
defect caused by P23H mutation. Two alterations appear to be present in
P23H mutant: (a) The mutation disrupts ionic interactions of
Pro23, Gln182, and Glu184 on the
tip of the plug between helices 4 and 5 (Fig. 1, C and D). Mutation of Pro to His leads to formation of conjugated
hydrogen bonding that exposes the chromophore-binding site. This
interaction stiffens the plug and possibly opens the binding site of
opsin, allowing water to hydrolyze the Schiff base between the retinal chromophore and -amino group of Lys296. This prediction
is consistent with a hipsochromic shift in the absorption maximum of
the chromophore and accessibility to hydrolysis in the presence or
absence of hydroxylamine. (b) There is a hydrophilic channel
in the interior of the protein that could accommodate water molecules
(Fig. 1E). In the model, Rh was soaked with water on the
external side, only including the part of the membranes that is charged
and without constraints imposed on water molecules. During simulation,
a route was established where water molecules drifted from one side to
another (cytoplasmic). This route was along helix II and partly along
helices I and III. The key residue facilitating this transport was
Thr93 on helix II (close to Glu113).
Collectively, these two effects may alter the electronic/dipole environment around the retinal, explaining the nearly 10-nm blue shift.
In summary, we have shown using a cell culture model system that the
retinitis pigmentosa mutant P23H-opsin can be induced to properly fold
by providing cells with a seven-membered ring variant of
11-cis-retinal during opsin biosynthesis. Furthermore, we
explain this remarkable affinity and selectivity of mutant opsin for
11-cis-7-ring retinal on the basis of the crystal structure of Rh. These findings open the possibility for pharmacological rescue
of the P23H mutant associated with this incurable retinal degenerative disease.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Robert Molday for providing the 1D4 monoclonal antibody, Dr. M. Gelb for help during retinoid syntheses, Dr. Karen Smith for generating the cell lines, and Yunie Kim for help with the manuscript preparation. Dr. Kaushal especially thanks Sri Sathya Sai Baba for inspiration for this work.
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FOOTNOTES |
---|
* This work was supported by a Career Development Award from the Foundation Fighting Blindness (to S. K.), funds from Research to Prevent Blindness (to S. K.) and the British Retinitis Pigmentosa Society (to S. K.), NEI, National Institutes of Health Grant EY01730 (to S. K. and K. P.), and funds from the Department of Ophthalmology Vision Foundation (to S. K.) and the Institute of Human Genetics (to S. K.). Computational tasks were partly done in the ICM computer center (University of Warsaw, Warsaw, Poland). This work was also supported by National Institutes of Health Grants EY09339 and EY13385 (to K. P.), a grant from Research to Prevent Blindness, Inc. (to the Department of Ophthalmology at the University of Washington), and grants from Foundation Fighting Blindness, Inc. and 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.
§ Both authors contributed equally to this work.
¶¶ Research to Prevent Blindness, Inc. Senior Investigator.
To whom correspondence may be addressed: Dept. of
Ophthalmology, University of Florida, Gainesville, FL 32610. Tel.:
352-846-2124; Fax: 352-392-7839; E-mail: skaushal@eye.ufl.edu.
Published, JBC Papers in Press, February 1, 2003, DOI 10.1074/jbc.M300087200
2 S. M. Noorwez, R. Malhotra, and S. Kaushal, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
Rh, rhodopsin;
DM, n-dodecyl--maltoside;
GPCR, G protein-coupled receptor;
WT, wild type;
PBS, phosphate-buffered saline;
HPLC, high
pressure liquid chromatography;
ICM, Interdisciplinary Center of
Modeling.
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