From the Departments of a Medical Biosciences and g Ophthalmology, Umeå University, S-901 85 Umeå, Sweden, the c Cole Eye Institute, and f Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the e Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, and the d Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115
Received for publication, July 19, 2002, and in revised form, January 16, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutations in the human cellular
retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To
understand the molecular basis of impaired CRALBP function, we have
characterized human recombinant CRALBP containing the disease causing
mutations R233W or M225K. Protein structures were verified by amino
acid analysis and mass spectrometry, retinoid binding properties were
evaluated by UV-visible and fluorescence spectroscopy and substrate
carrier functions were assayed for recombinant
11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was
less soluble than the R233W mutant and lacked retinoid binding
capability and substrate carrier function. In contrast, the R233W
mutant exhibited solubility comparable to wild type rCRALBP and bound
stoichiometric amounts of 11-cis- and
9-cis-retinal with at least 2-fold higher affinity than
wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type
rCRALBP. Analyses by heteronuclear single quantum correlation NMR
demonstrated that the R233W protein exhibits a different conformation
than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more
constrained structure than that of the wild type protein. Overall, the
results show that the M225K mutation abolishes and the R233W mutation
tightens retinoid binding and both impair CRALBP function in the visual
cycle as an 11-cis-retinol acceptor and as a substrate carrier.
Mutations in the human gene RLBP1 encoding the cellular
retinaldehyde-binding protein
(CRALBP)1 cause retinal
pathology and have been associated with autosomal recessive retinitis
pigmentosa (1), Bothnia dystrophy (2, 3), retinitis punctata albescens
(4), fundus albipunctatus (5), and Newfoundland rod-cone dystrophy (6).
These diseased phenotypes are all characterized by photoreceptor
degeneration and night blindness (delayed dark adaptation) but differ
in age of onset, rate of progression, and severity. The molecular basis for the clinical differences in these related retinal dystrophies is
not well understood and no effective therapies exist for the pathology
resulting from impaired CRALBP function.
CRALBP is an abundant, 36-kDa protein in the cytosol of the retinal
pigment epithelium (RPE) and Müller cells of the retina where it
carries endogenous 11-cis-retinol and
11-cis-retinal (7). The CRALBP ligand binding cavity is
mapped in the accompanying report (8). In vivo studies (9)
show that CRALBP serves as a major 11-cis-retinol acceptor
in the isomerization step of the visual cycle (7, 10, 11), stimulating
the enzymatic isomerization of all-trans- to
11-cis-retinol in the rod visual cycle. However, CRALBP
appears to function within an RPE protein complex (12) and to serve
multiple functions. In vitro, CRALBP facilitates the
oxidation of 11-cis-retinol to 11-cis-retinal by
11-cis-retinol dehydrogenase (12, 13), retards
11-cis-retinol esterification in the RPE by lecithin:retinol
acyltransferase (13), and is required for hydrolysis of endogenous RPE
11-cis-retinyl ester (14).
Six CRALBP mutations have been linked with retinal
pathology, including three missense mutations (R150Q, M225K,
and R233W), a frameshift mutation and two predicted splice junction
alterations (1, 2, 4, 6). Recombinant CRALBP (rCRALBP) containing the
R150Q mutation lacks the ability to bind 11-cis-retinal and exhibits low solubility (1). Toward a better understanding of the
molecular basis of retinal pathology associated with RLBP1 gene defects, we report here characterization of rCRALBP containing the
M225K or R233W disease-causing mutations (Fig.
1).
Materials--
11-cis-Retinal was obtained from the
NEI, National Institutes of Health, and 9-cis-retinal was
purchased from Sigma. Tritiated 11-cis-retinol was produced
by reduction of 11-cis-retinal with NaB[3H]4 (15).
Mutagenesis and Production of rCRALBP Mutants M225K and
R233W--
Mutant rCRALBP cDNA carrying either the R233W or M225K
substitutions were created using The QuikChange site-directed
mutagenesis method (Stratagene). Briefly, WT human CRALBP cDNA in
the pET19b vector (16) was cleaved with XbaI and
HindIII and the coding region subcloned into pBlueSK.
The following complimentary oligonucleotides were used to substitute a
Lys for a Met at residue 225 (underlined) in mutant M225K: sense,
5'-GAAGATGGTGGACAAGCTCCAGGATTCCTT-3'; antisense,
5'-AAGGAATCCTGGAGCTTGTCCACCATCTTC-3'. To substitute a Trp
for an Arg at residue 233 (underlined) in the R233W mutant, complimentary oligonucleotides were also used: sense,
5'-ATTCCTTCCCAGCCTGGTTCAAAGCCATCC-3'; antisense,
5'-GGATGGCTTTGAACCAGGCTGGGAAGGAAT-3'. Each
mutagenesis mix was transformed into Escherichia coli strain
XL1-Blue (Stratagene), mutant clones identified by restriction mapping
with NspI for M225K and MspI for R233W, were
amplified, cleaved with XbaI and HindIII, and
ligated back into expression vector pET19b (Novagen). Each insert was
sequenced in both directions using the ABI PRISM Dye Terminator Cycle
Sequencing kit and the model 377 DNA sequencer (PerkinElmer Life
Sciences, Applied Biosystems). WT and rCRALBP mutants M225K and R233W
were expressed in E. coli strain BL21(DE3)LysS with a
N-terminal His tag and purified using nickel-nitrilotriacetic acid-agarose columns (Qiagen) (16). Recombinant protein was quantified
according to Bradford (17) using WT rCRALBP previously quantified by
amino acid analysis for the standard reference protein.
Mass Spectrometry, Amino Acid Analysis, and
Electrophoresis--
The masses of the intact mutant proteins were
determined by LC ESMS using a PerkinElmer Life Sciences Sciex API 3000 triple quadrupole electrospray mass spectrometer, a Vydac C4 column
(1.0 × 150 mm), an Applied Biosystems model 140D high-performance
liquid chromatography system and aqueous acetonitrile/trifluoroacetic acid solvents at a flow rate of 50 µl/min (18, 19).
Phenylthiocarbamyl amino acid analysis was performed with an Applied
Biosystems model 420H/130/920 automated system and vapor phase HCl
hydrolysis (20). Purified rCRALBP mutants M225K and R233W (~100 pmol
each) were digested overnight with trypsin, and the peptide digests
were analyzed with a PE Biosystems Voyager DE Pro MALDI-TOF mass
spectrometer using Analysis of Retinoid Binding Function--
Retinoid labeling of
purified apo-rCRALBP with 11-cis-retinal or
9-cis-retinal, removal of excess retinoid, bleaching, and analysis by UV visible spectroscopy and fluorescence spectroscopy were
performed in the dark, under dim red illumination as previously described (8, 19).
Analysis of 11-cis-Retinol Dehydrogenase Activity--
Human
recombinant 11-cis-retinol dehydrogenase (rRDH5) was
expressed in Hi-5 insect cells using a baculovirus vector kindly provided by Dr. K. Palczewski (12, 23) and purified to apparent homogeneity by nickel affinity chromatography. rRDH5 oxidation activity
was measured at pH 7.5 (8, 24) and reduction activity was measured at
pH 5.5 (25) using purified mutant or WT rCRALBP or equimolar amounts of
free 11-cis-retinol or 11-cis-retinal as
substrate (8). Control assays with free retinoid as substrate were
done in the absence of any carrier protein.
Solution State Heteronuclear Single Quantum Correlation
NMR--
15N uniformly labeled WT and mutant R233W rCRALBP
were prepared by biosynthetic incorporation in E. coli
strain BL21(DE3)LysS grown in defined minimal media (8, 26). Purified
mutant and WT rCRALBP with bound 11-cis-retinal (~0.3
mM) were adjusted to 8% D2O (v/v) and
transferred to 250 µl of microcell NMR tubes (Shigemi Inc., Allison
Park, PA) (8). All NMR experiments were performed at 25 °C with a
Varian INOVA 500-MHz spectrometer equipped with a triple resonance
probe. Sensitivity enhanced two-dimensional 1H-15N heteronuclear single quantum correlation
experiments were recorded using water-flip-back for water suppression.
Data was processed on a Sun UltraSPARC workstation using NMRPipe and
Pipp software (8, 27, 28). Holo-protein preparations were maintained in
the dark or under dim red illumination to prevent retinoid isomerization.
Expression and Structural Integrity of rCRALBP Mutants M225K and
R233W--
WT and mutant rCRALBPs were produced in bacteria and
SDS-PAGE of the crude soluble bacterial lysates and re-suspended pellet fractions showed that the M225K mutant was less soluble than the R233W
rCRALBP mutant (Fig. 2). The R233W mutant
was present in the soluble lysate fraction in amounts comparable to
that of the WT protein (Fig. 2). The purified
mutant proteins were characterized by
amino acid analysis (Table I) and by LC ESMS and the determined compositions and intact masses found to be in excellent agreement with
the sequence calculated values (M225K, Mobs = 39,110 ± 3, Mcalc = 39,107; R233W,
Mobs = 39,145 ± 4, Mcalc = 39,140). About 73% of each mutant
protein sequence was confirmed by MALDI-TOF MS peptide mass mapping,
including the peptides containing the M225K and R233W substitutions
(Supplemental Fig. S1).
Retinoid Binding Properties of Mutants M225K and
R233W--
UV-visible spectral analysis of purified mutant R233W and
WT rCRALBP with bound 11-cis-retinal were essentially
identical, however, with bound 9-cis-retinal the ligand
absorbance is slightly red-shifted for the R233W mutant (Fig.
3). Upon exposure to bleaching illumination, the chromophore absorbance for both holo-proteins shifts
to ~380 nm due to the formation of unbound
all-trans-retinal. In contrast, UV-visible spectra of
purified mutant M225K exhibit no evidence for binding of either
11-cis or 9-cis-retinal (Fig. 3). Retinoid
labeling performed in bacterial lysates prior to protein purification
yielded the same UV-visible spectral results (data not shown).
Apparent equilibrium dissociation constants (Kd) for
mutant R233W rCRALBP complexed with 11-cis- or
9-cis-retinal were determined by fluorescence titration of
the apo-protein, monitoring the decrease in the
intrinsic fluorescence of the protein upon ligand binding (Table II). The determined
Kd values reveal significantly greater affinity of
the R233W mutant for both 11-cis-retinal
(Kd ~ 10 nM) and
9-cis-retinal (Kd ~ 24 nM)
relative to WT rCRALBP (Kd ~ 21 nM for
11-cis-retinal and ~53 nM for
9-cis-retinal). The average number of binding sites extracted from the mutant R233W titration data was about 0.6 for either
retinoid.
Mutant Substrate Carrier Function for 11-cis-Retinol
Dehydrogenase--
When rRDH5 was assayed in the presence of the
M225K rCRALBP mutant plus free retinoid, about 3-fold greater
Km values were obtained relative to WT rCRALBP for
both oxidation and reduction (Table III).
When rRDH5 was assayed using R233W-bound retinoid as substrate,
determined Km values were 4- to 7-fold higher than
WT rCRALBP (Table III). Relative to the free retinoid controls, neither
mutation appeared to significantly effect Vmax (Table III). In vivo CRALBP is thought to facilitate the
RDH5-catalyzed oxidation of 11-cis-retinol to
11-cis-retinal (13) and in vitro the
Vmax for WT rCRALBP was ~20% greater in the
oxidation reaction relative to free 11-cis-retinol and
~10-16% greater than the mutants. No significant difference in
Vmax was observed using WT rCRALBP or free
11-cis-retinal in the reduction reaction. The results support impaired substrate carrier function in the RDH5 oxidation reaction for the M225K and R233W rCRALBP mutants.
Conformational Differences between WT rCRALBP and Mutant
R233W--
The 1H-15N heteronuclear single
quantum correlation (HSQC) NMR spectra for 15N uniformly
labeled holo-R233W rCRALBP and WT holo-rCRALBP were recorded in the
dark and overlaid for comparison (Fig.
4). Resonances for Trp-165,
Trp-244, Met-208, Met-222, and Met-225 in HSQC NMR spectra of WT
rCRALBP were assigned in the accompanying report (8). Tentative
assignments have also been made for the other four Met in WT rCRALBP
(8) and randomly designated Ma, Mb, Mc, and Md (Fig. 4). Mutant R233W has a total
of three Trp, and Trp-233 was assigned in Fig. 4 based on the one
additional resonance in the downfield chemical shift region
characteristic of Trp side-chain NH. Conformational differences in
holo-protein structures between WT rCRALBP and mutant R233W are
demonstrated (Fig. 4) by the WT (blue) and R233W
(red) resonances that do not superimpose, such as
Md. Structural differences within the retinoid binding
cavity are exemplified by Met-222, an apparent retinoid binding pocket component (8), which clearly does not align with any other signal in
the R233W HSQC spectrum. In separate experiments, HSQC NMR spectra were
recorded before and after exposure of 15N uniformly labeled
holo-R233W rCRALBP to bleaching illumination. The results (Fig.
5A) show that the majority of
resonances remain unaffected by ligand isomerization. A few chemical
shift changes are apparent in the R233W HSQC spectra upon light-induced
retinoid isomerization, but the changes are much less extensive than
those observed upon bleaching the WT protein (Fig. 5B).
Functionally impaired CRALBP was first associated with autosomal
recessive retinitis pigmentosa (arRP) in 1997 when the missense mutation R150Q was found in the RLBP1 gene from a family in India (1).
Retinitis pigmentosa is a family of inherited diseases with many forms
and causative genes and classification of the disease types continue to
evolve (29). Since 1997, five other recessive defects in the
RLBP1 gene have been found to cause retinal dystrophies,
including the two missense mutations M225K and R233W associated with
retinitis punctata albescens and Bothnia dystrophy (2, 5). Pathological
mutations in the CRALBP gene have now been associated with other
phenotypes and detected in pedigrees from Europe, the Middle East,
Newfoundland, and India (1-6). RLBP1 gene defects are
thought to be a rare cause of retinal disease (4); however, in northern
Sweden the high prevalence of Bothnia dystrophy caused by the R233W
mutation constitutes a significant medical problem for which therapies
are sought (2, 3). To better understand the molecular basis of retinal
pathology associated with impaired CRALBP and possibilities for
therapeutic intervention, we have pursued structure-function studies of
the mutant rCRALBPs containing the disease causing substitutions M225K
and R233W.
The primary structural integrity of the purified M225K and R233W mutant
recombinant proteins was confirmed by amino acid analysis and mass
spectrometry. In contrast to the largely insoluble R150Q rCRALBP
associated with arRP (1), the R233W mutant exhibits solubility
comparable to that of WT rCRALBP, whereas the M225K mutant is less
soluble than WT rCRALBP but significantly more soluble than the R150Q
mutant. With regard to retinoid binding properties, UV-visible spectral
analysis revealed that the M225K mutant resembled the R150Q mutant and
completely lacked the ability to bind cis-retinoids (1). In
contrast, mutant R233W bound stoichiometric amounts of
11-cis- or 9-cis-retinal based on absorbance spectral ratios (19).
Fluorescence titrations yielded apparent equilibrium dissociation
constants for the R233W mutant that demonstrated nanomolar affinities
for 9-cis- and 11-cis-retinal that were about
2-fold tighter than determined for WT rCRALBP. The variability of the retinoid affinity data (relative standard error of the mean was ~15-23%) was within the limits of experimental error of the
titration methodology and due, in part, to the low aqueous solubility
of retinoids and variable apo-protein stability. Furthermore, the protein concentration (0.5 µM) used in the fluorescence
titrations was significantly higher than the apparent
Kd values, and, under these conditions,
Kd values should be considered to be upper limits
for the actual values (30). The difference between the binding
affinities of WT rCRALBP and its R233W mutant may therefore be larger
than observed here.
Studies with crude extracts from bovine RPE microsomes (13), and
purified proteins (12) strongly support a substrate carrier interaction
between CRALBP and RDH5. The kinetic analyses performed here with
purified recombinant proteins demonstrate that rCRALBP harboring either
the M225K or the R233W mutations decrease the apparent affinity of
rRDH5 for 11-cis-retinol and 11-cis-retinal relative to the WT protein. The Km of rRDH5 for
11-cis-retinoid in the presence of the M225K mutant
approximated that for the enzyme reaction with free retinoid (12). An
even higher Km was obtained for rRDH5 using the
holo-R233W mutant as substrate, reflecting lower affinity between
enzyme and retinoid due to the tighter binding of retinoid by R233W.
The R233W mutant likely hinders movement of the hydrophobic substrate
to the active site of enzyme. These data are consistent with the notion
that CRALBP affects the activity of RDH5 by "channeling" of
retinoids to the enzyme, much like cellular retinoic acid binding
protein II facilitates delivery of retinoic acid to the retinoic acid
receptor (31, 32).
Clues to the structural basis for the tighter retinoid binding
properties of the R233W mutant were obtained by two-dimensional NMR.
HSQC NMR spectra showed that the three-dimensional structure of the
R233W mutant with bound ligand was significantly different than that of
WT holo-rCRALBP, including different retinoid-binding cavity
conformations. Furthermore, conformational changes observed by NMR
after photoisomerization of 11-cis-retinal in the ligand binding pocket were much more pronounced for the WT protein (8) than
for the R233W mutant (Fig. 5). Residues Arg-233 and Met-225 are
conserved within human, bovine, and mouse CRALBP (Fig. 1), and the
present results are consistent with ligand interactions with both
residues. Replacing positively charged Arg-233 with apolar tryptophan
likely strengthens nonionic interactions within the hydrophobic ligand
binding pocket, resulting in a more constrained, less flexible R233W
protein structure. Likewise, inserting charged lysine in place of
apolar Met-225 appears to disrupt critical interactions within the
retinoid binding cavity, perhaps by opening the hydrophobic region to
greater solvent accessibility, which in turn precludes specific ligand
interaction and lowers M225K protein solubility.
The function of CRALBP in the visual cycle depends upon the rapid
association and dissociation of retinoid from the ligand binding
pocket. The results of this study implicate impairment of both retinoid
binding and release as causes of the night blindness and retinal
pathology reported for human patients with CRALBP mutations. CRALBP
serves as the major 11-cis-retinol acceptor in the
isomerization reaction of the rod visual cycle, therefore the lack of
retinoid binding by M225K rCRALBP significantly slows the enzymatic
conversion of all-trans to 11-cis-retinol, as
observed for the CRALBP knockout mouse (9). Binding of
11-cis-retinol by apoCRALBP, coupled with its oxidation to
11-cis-retinal by RDH5, provides a strong driving force for
isomerization (13, 33). Interactions with other proteins likely
facilitate the release of ligand from the CRALBP binding pocket,
promoting catalytic rather than stoichiometric retinoid binding (9,
12). The R233W mutation results in tighter rCRALBP retinoid binding and lower rRDH5 affinity for rCRALBP bound retinoid. The overly tight retinoid binding caused by the R233W mutation appears to hinder the
rapid release of ligand, resulting in a "full house" effect that
impairs 11-cis-retinol acceptor function and slows the
isomerization of all-trans to 11-cis-retinol.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
[in a new window]
Fig. 1.
Location of the M225K and R233W mutations in
the CRALBP retinoid binding pocket. Amino acid sequence
surrounding residues Met-225 and Arg-233 in the CRALBP retinoid binding
cavity are shown for human (h), bovine (b), and
mouse (m) CRALBP (8, 19, 34). Species differences are
shaded, and residues influencing CRALBP retinoid binding are
shown in boldface.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamic acid as matrix (21, 22).
SDS-PAGE was performed on 10% or 12% acrylamide gels using the
Bio-Rad Mini-Protein II system (8).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (98K):
[in a new window]
Fig. 2.
SDS-PAGE analysis of rCRALBP mutants
M225K and R233W. Crude bacterial lysates (~15 µg of total
protein) and purified recombinant proteins (1-2 µg of each) from
cells expressing wild type rCRALBP, mutant M225K, or mutant R233W were
analyzed by SDS-PAGE on a 12% gel and stained with Coomassie Blue.
S, crude soluble lysate; RP, re-suspended pellet
(inclusion body) fraction; P, purified protein; and
MW, molecular weight markers.
Amino acid compositions of human rCRALBP mutants
View larger version (25K):
[in a new window]
Fig. 3.
Retinoid binding analysis of CRALBP mutants
M225K and R233W. UV-visible absorption spectra are shown before
and after exposure to bleaching illumination following retinoid
labeling with either 11-cis- or 9-cis-retinal and
removal of excess retinoid. With bound 11-cis-retinal, the
ligand absorbance maxima for mutant R233W ( max = 425.3 ± 0.6 nm, n = 3) and the WT protein are
indistinguishable. With bound 9-cis-retinal, the ligand
absorbance maxima for R233W (
max = 408.3 ± 1.2 nm,
n = 3) is slightly red-shifted compared with the human
WT protein (
max = 400 nm) (15). The absorption spectra
from mutant M225K shows no chromophore absorbance near 425 or 400 nm
indicating no bound retinoid; the ~380-nm absorbance is from free
retinoid and/or nonspecific interaction with retinoid.
Equilibrium dissociation constants of rCRALBP with retinoids
Kinetic parameters of mutant CRALBP substrate carrier function
View larger version (32K):
[in a new window]
Fig. 4.
Heteronuclear single quantum correlation NMR
spectra of uniform 15N-labeled WT and mutant R233W
rCRALBP. HSQC NMR spectra for WT holo-rCRALBP
(blue) and holo-R233W rCRALBP (red) were recorded
separately in the dark then overlaid. These experiments correlate
directly bonded 1H-15N pairs within the protein
structures and show that substantial conformational differences exist
between the WT and mutant R233W holo-proteins. Met-222 in the WT
spectra does not overlay with any resonance in the R233W spectra,
demonstrating that the proteins differ in retinoid binding pocket
conformation. Trp-233 in the R233W spectra was assigned by an
additional characteristic Trp resonance in the downfield chemical shift
region relative to WT rCRALBP. Other residue assignments were
determined elsewhere (8).
View larger version (19K):
[in a new window]
Fig. 5.
Heteronuclear single quantum correlation NMR
spectra of uniform 15N-labeled mutant R233W and WT rCRALBP
before and after bleaching. The 1H-15N
correlation spectrum for the proteins with bound
11-cis-retinal was recorded in the dark (red),
and the sample was then exposed to bleaching illumination and
re-analyzed (blue) by the same
1H-15N correlation experiment. A,
mutant R233W HSQC spectra. The vast majority of the resonances overlay
in both experiments, indicating very little conformational change
occurs upon ligand isomerization in the R233W ligand binding pocket.
B, WT rCRALBP HSQC spectra. Although most residues remain
unchanged upon bleaching (8), more chemical shift changes are apparent
than in the R233W spectra.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. John C. Saari for useful discussions and for reviewing the manuscript prior to publication.
![]() |
FOOTNOTES |
---|
* This study was supported in part by National Institutes of Health Grants EY6603, EY14239, HL58758, and CA68150, by a Research Center grant from The Foundation Fighting Blindness, by funds from the Cleveland Clinic Foundation, and by grants from the Swedish Medical Research Council (Projects 10866 and 9745). A preliminary report of this work was presented at The Annual Meeting of the Association for Research in Vision and Ophthalmology, April 29 through May 4, 2001, Ft. Lauderdale, FL (35).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.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Fig. S1.
b To whom correspondence may be addressed: Clinical Genetics, Norrlands University Hospital, Umeå University, S 901 85 Umeå, Sweden. Tel.: 46-90-785-1781; Fax: 46-90-128-163; E-mail: irina.golovleva.us@vll.se.
h Present address: Dept. of Molecular Biology, Astra Zeneca, S-431 83 Mölndahl, Sweden.
i To whom correspondence may be addressed: Cole Eye Institute (i31), Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-0425; Fax: 216-445-3670; E-mail: crabbj@ccf.org.
Published, JBC Papers in Press, January 20, 2003, DOI 10.1074/jbc.M207300200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CRALBP, cellular retinaldehyde-binding protein; arRP, autosomal recessive retinitis pigmentosa; HSQC, heteronuclear single quantum correlation; LC ESMS, liquid chromatography electrospray mass spectrometry; RPE, retinal pigment epithelium; WT, wild type; MALDI-TOF, matrix-assisted laser desorption time of flight.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Maw, M. A., Kennedy, B., Knight, A., Bridges, R., Roth, K. E., Mani, E. J., Mukkadan, J. K., Nancarrow, D., Crabb, J. W., and Denton, M. J. (1997) Nat. Genet. 17, 198-200[Medline] [Order article via Infotrieve] |
2. | Burstedt, M. S. I., Sandreg, O., Holmgren, G., and Forsman-Semb, K. (1999) Invest. Ophthalmol. Vis. Sci. 40, 995-1000[Abstract] |
3. |
Burstedt, M. S. I.,
Sandreg, O.,
Forsman-Semb, K.,
Golovleva, I.,
Janunger, T.,
Wachtmeister, L.,
and Sandgren, O.
(2001)
Arch. Ophthalmol.
119,
260-267 |
4. | Morimura, H., Berson, E. L., and Dryja, T. P. (1999) Invest. Ophthalmol. Vis. Sci. 40, 1000-1004[Abstract] |
5. | Katsanis, N., Shroyer, N. F., Lewis, R. A., Cavender, J. C., Al-Rajhi, A. A., Jabak, M., and Lupski, J. R. (2001) Clin. Genet. 59, 424-429[CrossRef][Medline] [Order article via Infotrieve] |
6. | Eichers, E. R., Green, J. S., Stockton, D. W., Jackman, C., Whelan, J., McNamara, J. A., Johnson, G. J., Lupski, J. R., and Katsanis, N. (2002) Am. J. Human Genet. 70, 955-964[CrossRef][Medline] [Order article via Infotrieve] |
7. | Saari, J. C. (1994) in The Retinoids (Sporn, M. A. , Roberts, A. B. , and Goodman, D. S., eds) , pp. 351-385, Raven Press, Ltd., New York |
8. |
Wu, Z.,
Yang, Y.,
Shaw, N.,
Bhattacharya, S.,
Yan, L.,
West, K.,
Roth, K.,
Noy, N.,
Qin, J.,
and Crabb, J. W.
(2003)
J. Biol. Chem.
278,
12390-12396 |
9. | Saari, J. C., Nawrot, M., Kennedy, B. N., Hurley, J. B., Garwin, G. G., Huang, J., and Crabb, J. W. (2001) Neuron 29, 739-748[Medline] [Order article via Infotrieve] |
10. | Crouch, R. K., Chader, G. J., Wiggert, B., and Pepperberg, D. R. (1996) Photobiology 64, 613-621 |
11. | Rando, R. R. (2001) Chem. Rev. 101, 1881-1896[CrossRef][Medline] [Order article via Infotrieve] |
12. | Bhattacharya, S. K., Wu, Z., Jin, Z., Yan, L., Miyagi, M., West, K., Nawrot, M., Saari, J. C., and Crabb, J. W. (2002) FASEB J. 16, A14 |
13. | Saari, J. C., Bredberg, D. L., and Noy, N. (1994) Biochemistry 33, 3106-3112[Medline] [Order article via Infotrieve] |
14. |
Stecher, H.,
Gelb, M. H.,
Saari, J. C.,
and Palczewski, K.
(1999)
J. Biol. Chem.
274,
8577-8585 |
15. | Garwin, G. G., and Saari, J. C. (2000) Methods Enzymol. 316, 313-324[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Crabb, J. W.,
Carlson, A.,
Chen, Y.,
Goldflam, S.,
Intres, R.,
West, K. A.,
Hulmes, J. D.,
Kapron, J. T.,
Luck, L. A.,
Horwitz, J.,
and Bok, D.
(1998)
Protein Sci.
7,
746-757 |
17. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
18. | Crabb, J. W., Chen, Y., Goldflam, S., West, K., and Kapron, J. (1998) Methods Mole. Biol 89, 91-104 |
19. |
Crabb, J. W.,
Nie, Z.,
Chen, Y.,
Hulmes, J. D.,
West, K. A.,
Kapron, J. T.,
Ruuska, S. E.,
Noy, N.,
and Saari, J. C.
(1998)
J. Biol. Chem.
273,
20712-20720 |
20. | Crabb, J. W., West, K. A., Dodson, W. S., and Hulmes, J. D. (1997) in Current Protocols in Protein Science, Unit 11.9, Supplement 7 (Coligan, J. E. , Ploegh, H. L. , Smith, J. A. , and Speicher, D. W., eds) , pp. 11.9.1-11.9.42, John Wiley & Sons, Inc., New York |
21. | West, K. A., Yan, L., Miyagi, M., Crabb, J. S., Marmorstein, A. D., Marmorstein, L., and Crabb, J. W. (2001) Exp. Eye Res. 73, 479-491[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Miyagi, M.,
Sakaguchi, H.,
Darrow, R. M.,
Yan, L.,
West, K. A.,
Aulak, K. S.,
Stuehr, D. J.,
Hollyfield, J. G.,
Organisciak, D. T.,
and Crabb, J. W.
(2002)
Mol. Cell. Proteomics
1,
293-303 |
23. |
Jang, G. F.,
McBee, J. K.,
Alekseev, A. M.,
Haeseleer, F.,
and Palczewski, K.
(2000)
J. Biol. Chem.
275,
28128-28138 |
24. | Saari, J. C., Bredberg, D. L., Garwin, G. G., Buzzylko, J., Wheeler, T., and Palczewski, K. (1993) Anal. Biochem. 213, 128-132[CrossRef][Medline] [Order article via Infotrieve] |
25. | Futterman, S., and Saslaw, L. D. (1961) J. Biol. Chem. 236, 1652-1657[Medline] [Order article via Infotrieve] |
26. | Luck, L. A., Barrows, S. A., Venters, R. A., Kapron, J., Roth, K. A., Paradis, S. A., and Crabb, J. W. (1997) in Techniques in Protein Chemistry VIII (Marshak, D, ed) , pp. 439-448, Academic Press, San Diego, CA |
27. | Delaglio, F., Gresiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277-293[Medline] [Order article via Infotrieve] |
28. | Garrett, D. S., Powers, R., Gronenborn, A. M., and Clore, G. M. (1991) J. Magn. Reson. 95, 214-220 |
29. | Phelan, J. K., and Bok, D. (2000) Mol. Vis. 6, 116-124[Medline] [Order article via Infotrieve], http://www.molvis.org/molvis/v6/a16 |
30. | Noy, N. (1999) in Handbook of Experimental Pharmacology (Nau, H. , and Blaner, W. S., eds), Vol. 139 , pp. 3-29, Springer-Verlag, Heidelberg |
31. |
Dong, D.,
Ruuska, S.,
Levinthal, D. J.,
and Noy, N.
(1999)
J. Biol. Chem.
274,
23695-23698 |
32. | Budhu, A. S., Gillilan, R., and Noy, N. (2001) J. Mol. Biol. 305, 939-949[CrossRef][Medline] [Order article via Infotrieve] |
33. | McBee, J. K., Kuksa, V., Alvarez, R., de Lera, A. R., Prezhdo, O., Haeseleer, F., Sokal, I., and Palczewski, K. (2000) Biochemistry 39, 11370-11380[CrossRef][Medline] [Order article via Infotrieve] |
34. | Kennedy, B. N., Huang, J., Saari, J. C., and Crabb, J. W. (1998) Mol. Vis. 4, 14[Medline] [Order article via Infotrieve], http://www.molvis.org/molvis/v4/p14 |
35. | Crabb, J. W., Andrabi, K., Shaw, N., Wu, Z., Bhattacharya, S., West, K., Burstedt, M., Sandgren, O., Noy, N., and Golovleva, I. (2001) Invest. Ophthalmol. Vis. Sci. 42, 3524 (Abstr. S655) |