From the NEI and the §§ NIDDK,
National Institutes of Health, Bethesda, Maryland 20892-2740, the
Department of Crystallography, Birkbeck College, Malet Street,
London, the ** Medical Research Council, Harwell, Didcot OX11 0RD, and
the
Medical Research Council Human Genetics
Unit, Edinburgh EH4 2XU, United Kingdom, and the
¶¶ Institute of Mammalian Genetics, GSF-National Research
Center for Environment and Health, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany
Received for publication, November 22, 2000, and in revised form, December 18, 2000
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ABSTRACT |
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In Opj, an inherited cataract
in mice, opacity is associated with a mutation in Crygs,
the gene for Cataract is an almost obligatory consequence of aging in humans
and, despite the availability of surgical intervention, is a major
cause of blindness worldwide (1). In addition to "normal" age-related opacification of the lens, there are several inherited genetic cataracts in both humans and animal models (2). Many of these
involve the crystallins, the bulk structural components of the lens
cell cytoplasm (2-13). Crystallins, through their abundance, play a
major role in defining the optical properties of the lens. It has also
become clear that they are not simple "filler," but proteins with
specific functional roles in the cell. Some major crystallins in
non-human species are enzymes, whereas the ubiquitous The transparency of the lens depends upon order, at both the molecular
and cellular levels. Aggregates of structurally perturbed proteins or
localized phase separations within the fiber cells can form
light-scattering centers capable of randomly dispersing incident light,
fogging the lens (19-22). Light scattering may also result from
disruption of the well ordered array of the fiber cell layers and the
junctions between them (23-29). The most interesting genetic cataracts
are those that shed some light both on the processes of opacification
in normal aging and on the functions of crystallins and other lens components.
The Opj (opacity due to poor secondary fiber junctions)
mutant was found among the offspring of a male mouse treated with ethylnitrosourea in a mutagenesis experiment (30, 31). The Opj locus was mapped to mouse chromosome 16, 9.5 ± 1.5cM distal to md (mahoganoid) (32, 33). Here we describe the identification of a coding sequence mutation in
Crygs which is associated with opacity in Opj and
show that the mutation destabilizes the mutant protein in a
temperature-sensitive manner and leads to severe disruption of cortical
fiber cell morphology and organization.
Breeding of Opj Mutant--
The original mutant was found at the
Institute for Mammalian Genetics, Neuherberg, among the offspring in a
mutagenesis experiment with ethylnitrosourea (30). After transfer to
the MRC Mammalian Genetics Unit, Harwell, further breeding was carried
out following Medical Research Council guidelines and under Home Office
project licenses 30/00875 and 30/1517.
The mutant was at first maintained by crosses of Opj/+
heterozygotes to the inbred strain C3H/HeH. It was preserved on this background as frozen embryos. Later live mice were recovered, and
breeding was then continued by crosses of heterozygotes to strain
102/ElH and by intercrosses of Opj/+ × Opj/+ to
obtain homozygotes.
Ophthalmological Examination--
The eye phenotype in live mice
was examined at 5-6 weeks of age or later. The pupils were dilated
with a drop of 1% atropine sulfate applied at least 10 min before
examination. The eyes were then examined by slit-lamp microscopy using
a Zeiss 30SL/M at × 20 magnification or a Nikon FS-3V slit-lamp
microscope (Young Optical, London). Photographs were taken with an
attached Kodak DCS420 still digital camera (Young Optical). Diffuse
illumination was used from the microscope, and the flash setting was
adjusted for each mouse to optimize the exposure. For isolated lenses, microscopy was carried out using a Leica MZ APO, × 20 magnification with dark-field illumination. Photographs were taken using a Leica WILD
MPS48 camera and Kodak 64T film. In both cases images were imported
into Adobe Photoshop.
DNA and Sequence Analysis--
Samples for DNA studies were
obtained from spleens snap-frozen in liquid nitrogen or from tail
snips. DNA was extracted by standard methods (37). PCR primers were
designed from the Crygs gene sequence (35) and were used to
amplify exon sequences from the genomic DNA samples. Sequences are
available on request. PCR products were sequenced directly, both by
"manual" methods, using 33P labeling (Amersham
Pharmacia Biotech) and on an ABI 370 automated sequencer using
the PRISM dye terminator cycle sequencing kit and AmpliTaq polymerase
FS (Applied Biosystems, Foster City, CA), following manufacturer's protocols.
Molecular Modeling--
Mouse Recombinant Proteins--
Clones for the complete coding
sequence of wild type (wt) and Opj mutant
Wild type and Opj mutant clones were restricted with
NdeI and HindIII, subcloned into pET-17b
(Novagen, Madison WI), and transformed into Escherichia coli
strain BL21(DE3)pLysS. Colonies were grown in LB broth with
carbenicillin (50 µg/ml) and chloramphenicol (34 µg/ml in ethanol)
(Sigma). A starter culture was used to inoculate 1 liter of LB broth
with the same concentrations of antibiotics. This was incubated at
37 °C in a rotating shaker to an A600
of 0.6. The culture was cooled to room temperature and induced with isopropylthio- Protein Purification--
Approximately 250 ml of whole cell
lysate was divided into 50-ml batches and dialyzed for 24 h in 4 liters of 50 mM Tris, pH 8.5, 1 mM
dithiothreitol, and 1 mM EDTA at 4 °C, with at least four changes of buffer, using Snake Skin Dialysis tubing, MWCO 10K
(Pierce Chemical Co.). Dialyzed protein was loaded on a Hi Load 26/10
Q-Sepharose FF column (Amersham Pharmacia Biotech) and washed with 1 column volume (~50 ml) of 50 mM Tris, pH 8.5, 1 mM dithiothreitol, and 1 mM
EDTA using the AKTA Explorer 100 (Amersham Pharmacia Biotech)
purification system. Wild type and Opj Mass Spectroscopy--
Samples were dissolved in 0.1%
trifluoroacetic acid to a concentration of about 50-100
µM, and then 50-100 pmol was injected into a Zorbax
CB300 2.1-mm x 15-cm C3 column in an initial solvent of 5%
acetonitrile and 95% of 5% acetic acid. The column was maintained with this solvent for 25 min while the salts and buffers were washed
off and then ramped to 100% acetonitrile over a 20-min time period.
The HP1100 liquid chromatographic mass spectrometer was scanned over a
600-1,700 m/z window every 4 s. The
molecular weight of the protein was deduced from the
m/z data by deconvolution.
Spectrophotometry--
Light scattering experiments were
performed using the PerkinElmer Life Sciences Lambda 2 UV/visible
spectrophotometer and data recorded using the PECSS software
(PerkinElmer Life Sciences Computerized Spectroscopy Software, version
4.2). The absorbance of protein samples at 600 nm was measured at
different temperatures by placing the cuvette into a water-jacketed
cell holder. The required temperatures were maintained using a Haake D1
circulating water bath. The actual sample temperature inside the
cuvette was determined by using a calibration curve generated by
directly measuring the temperature of an equal volume of water (600 µl) over a range of set temperatures. Temperatures were measured
using a Kiethley 871 digital thermometer fitted with a type k
thermocouple. Protein samples at 0.1 and 1 mg/ml were heated using
increments of 4-5 °C with an equilibration time of 15 min.
Circular Dichroism Spectroscopy--
Circular dichroism spectra
were recorded on an Aviv 62DS spectropolarimeter controlled by Aviv
60DS version 4.1i software. The data were processed using the SUPER3
software package (38). Spectra were normalized to the mean residue
ellipticity by applying the scale factor
All far UV CD experiments were performed using a 1-mm path length
cuvette (Hellma, type 100-QS) and protein concentrations of 0.1 mg/ml.
Data were collected over the wavelength range 300-190 nm at 0.2-nm
intervals. All spectra shown were derived from three repeat scans of 20 min so that the time interval between different temperature
measurements was 1 h. Scans were averaged, smoothed, and base line
subtracted (buffer correction).
Near UV CD spectra were recorded over the range 320-250 nm at 0.2-nm
intervals, using a 2-mm path length cuvette (Hellma, type 100-QX) and
protein concentrations of 2.5 and 2.8 mg/ml for the wt and mutant
proteins, respectively. 5 repeat scans were averaged, smoothed, and
base line corrected.
Histology and Electron Microscopy--
For histology, whole eyes
of animals of various ages were fixed in 10% neutral formalin. Both
paraffin and glycol methacrylate sections were taken and visualized
with standard hematoxylin and eosin stain. For electron microscopy,
eyes were fixed in 2.5% glutaraldehyde and 6% sucrose buffered at pH
7.2 with 50 mM sodium cacodylate for 2 h at room
temperature and processed for ultrastructural studies using standard
procedures (39).
Derivation of Opj--
The original mutant, a female expressing
secondary lens fiber discontinuity (designation ENU-410), was recovered
in the offspring of a male mouse exposed to 250 mg of
ethylnitrosourea/kg of body weight (30). An outcross of the presumed
mutation to a homozygous wt yielded 20 offspring expressing the deviant
phenotype and 21 wt offspring. Male to male transmission of the
mutation was observed in outcrosses of male carriers to wt partners.
Intercrosses of heterozygotes yielded 14 wt offspring, 21 offspring
expressing secondary lens fiber discontinuity, and 15 offspring
expressing a more severe lens opacity. The presumed homozygous mutants
with a more severe phenotype were genetically confirmed and indicated that homozygous mutants were viable and fertile. Together, the results
indicated a semidominant autosomal mutation.
Appearance and Progression of Opj Cataract--
Both
Opj/+ and Opj/Opj mice have lens opacities that
increase in severity with age (Fig. 1).
In Opj/+ the cataract was very mild and appeared as fine
opaque striations in parallel with the secondary fibers of the lens.
Crosses of Opj/+ × Opj/+ were made to obtain
homozygotes. For the first few weeks after birth, little difference
could be detected among affected offspring. However, by DNA typing some
homozygotes were found, and at later ages the cataracts in homozygotes
became more severe (Fig. 1).
By 2.5 months one homozygote showed total opacity of the lens, and two
others had milky opacity. By 3.5 months all three had total opacity in
both eyes. By contrast, relatively little progression of the phenotype
was seen in heterozygotes. In heterozygotes aged 7-8 months the
cataracts as seen in the slit-lamp were no more severe than in younger
mice. In three heterozygotes aged 12-15 months the cataracts had
become somewhat more severe. In two the lenses had become milky with
small dense nuclear opacities. In the third, the striations seen in
younger animals had become somewhat more marked.
A Mutation in Crygs in Opj Mice--
Both Opj and
Crygs, the gene encoding Molecular Modeling--
Mouse Recombinant Proteins--
To test the consequences of the
Opj Phe
Both forms of protein were isolated by fast protein liquid
chromatography and were assayed by SDS-polyacrylamide gel
electrophoresis and Western blotting. Mass spectroscopy was used to
confirm their identity, and major species corresponding to the expected
size (20,718 kDa for wt and 20, 658 kDa for Opj) were
obtained, along with minor fractions corresponding to proteins
retaining the initiator methionine.
Heat Stability--
Solutions of wt and Opj Circular Dichroism--
At room temperature, both wt and mutant
proteins at a concentration of 0.1 mg/ml exhibited far UV CD spectra
typical of the Histology of Cataract--
Although both Opj/Opj and
Opj/+ lenses exhibit opacity (Fig. 1), their histological
appearance is quite different (Fig. 7). Up to 9 months in age, Opj/+ lenses are generally similar to
those of +/+ in terms of cellular organization, although they show
signs of unusual protein staining inclusion bodies, presumably protein aggregates, within cells. Similar bodies are also apparent in Opj/Opj lenses but absent from +/+ lenses at any age. In
addition to these potential light-scattering centers,
Opj/Opj lenses, from early stages, show dramatic swelling
and disorganization in the cortical fibers. The smooth gradient in
staining between the outer cortical fiber layer and the inner fibers
seen in +/+ and Opj/+ lenses is extremely uneven in
appearance and shows as a sharp, distorted boundary while cortical
fibers are misshapen and vacuolated. In addition, nuclear staining
suggests that the orderly loss of cell nuclei in maturing fiber cells
is also disrupted, with nuclei-like particles scattered within the
inner layers of fiber cells. Some vacuoles are also apparent in
epithelial cells in the homozygotes.
Electron microscopy of Opj/Opj lenses also showed unusual
features absent from Opj/+ and +/+ at the same ages. Most
notably, this included unusual tangled, fibrous masses, which were
apparent only in Opj/Opj mice even at 23 days of age (Fig.
8). These peculiar features were observed
in multiple lenses of different ages and under different conditions of
fixation and sectioning. They do not appear to be artifacts, although
their nature is not yet known. They were observed at all ages in
Opj/Opj. Only by 16 months in age did one Opj/+
begin to exhibit some of the same features, including the appearance of
vacuoles in epithelial cells and some of the tangled masses seen by
electron microscopy. Even so, it was still much less affected than
Opj/Opj at any age.
Opacity and lens malformation in the inherited murine cataract
Opj are associated with a single base mutation in the coding sequence of the Crygs gene, which encodes the major lens
structural protein Even though the mutant is capable of folding correctly, it is markedly
less stable than the wt, showing a sharply increased tendency to
precipitate with increasing temperature. This occurs in a
concentration-dependent manner, suggesting that
structurally perturbed proteins are aggregating. At a concentration of
1 mg/ml, the mutant protein precipitates out of solution at about
47 °C. In the lens, where the overall protein concentration may be
much higher (17), this is likely to occur at even lower, closer to physiological, temperatures. This temperature-dependent loss of solubility is also reflected in the CD spectra, which show a decrease in the signal at 218 nm, corresponding to organized One of the favored models for senile cataract is the age-related
accumulation of damaged crystallins (17, 22) and the consequent gradual
increase of light-scattering aggregates (41). In recent years attention
has focused on the possibility that transitory structured intermediates
with exposed surface hydrophobic residues play an important role in
protein-folding diseases (42) and that in the eye lens destabilized
proteins are captured and solubilized by the small heat-shock protein
However, although both Opj/Opj and Opj/+ lenses
share the problem of protein aggregate formation, the homozygotes have
much more severe defects that may provide some insights into another question, the function of the So, although unfolded protein and inclusion bodies are common to both
Opj/+ and Opj/Opj, cell structure in the
heterozygote is essentially normal, whereas that of the homozygote is
severely perturbed. This suggests that the disruption of cell
organization and other effects seen in Opj/Opj may be caused
through loss of function of It seems that all crystallins arose by gene recruitment of preexisting
proteins with specific functions to additional structural roles in the
lens (14, 15). In terms of sequence and structure, The severity of the Opj/Opj phenotype may thus reflect a
role for Because new cells expressing S-crystallin, the first mutation to be associated with
this gene. A single base change causes replacement of Phe-9, a key
hydrophobic residue in the core of the N-terminal domain, by serine.
Despite this highly non-conservative change, mutant protein folds
normally at low temperature. However, it exhibits a marked,
concentration-dependent decrease in solubility, associated
with loss of secondary structure, at close to physiological
temperatures. This is reminiscent of processes thought to occur in
human senile cataracts in which normal proteins become altered and
aggregate. The Opj cataract is progressive and more severe
in Opj/Opj than in Opj/+. Lens histology shows
that whereas fiber cell morphology in Opj/+
mice is essentially normal, in Opj/Opj, cortical fiber cell
morphology and the loss of maturing fiber cell nuclei are both severely
disrupted from early stages. This may indicate a loss of function of
S-crystallin which would be consistent with ideas that members of
the
-crystallin superfamily may have roles associated with
maintenance of cytoarchitecture.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-crystallins
belong to the small heat-shock protein superfamily and can act as
"junior chaperones" (14-17). The important
- and
-crystallin
families are evolutionarily and structurally related to each other and
belong to a diverse superfamily of proteins in prokaryotes and
eukaryotes which may be stress-inducible and may play roles related to
maintenance of cell morphology (14, 18).
S-crystallin
(
S)1 is a major component
of the adult mammalian lens, essentially replacing the expression of
other
-crystallins (
A-F), which are expressed from early
embryonic development and are major components of the primary lens
fibers and the earliest secondary fibers (17, 34-36).
S expression
increases after birth and continues as the lens grows throughout life.
Thus high levels of
S mark the cortical fibers of the mature lens,
the same cells affected by the Opj cataract.
Crygs, the gene for
S, was mapped to mouse chromosome 16, close to the locus of Opj (35).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S was modeled using the program
Quanta (Molecular Systems, Waltham MA, courtesy of the NIH Molecular
Modeling Center), running on a Silicon Graphics O2 work
station, and was based on the experimentally derived coordinates of
bovine
B-crystallin (PDB: 4GCR), essentially as described previously
(3).
S were obtained
by PCR, using the cloned cDNA (35) as template. Primers were
designed, incorporating an NdeI site at the 5'-end of the
5'-primer and an HindIII site at the 5'-end of the reverse
complement 3'-primer, to facilitate subsequent cloning (sequences
available on request). Two versions of the 5'-primer were synthesized,
one corresponding to the wt sequence and the other with a single base
change corresponding to the Opj mutant sequence. PCR was
performed using GeneAmp and Taq polymerase (Applied
Biosystems) according to manufacturer's protocols, using an annealing
temperature of 60 °C. Products of the expected size (546 base pairs)
were subcloned into the TOPO PCR II vector (Invitrogen, Carlsbad, CA)
and checked by restriction digest and DNA sequencing.
-D-galactoside (Life Technologies, Inc.)
at a final concentration of 1 mM. Following induction, the
culture was incubated on a rotating shaker, 150 rpm, for 3 h at
room temperature. Bacteria were pelleted by centrifuging at 4 °C for
10 min and stored at
80 °C prior to analysis. Pellets were lysed
by freeze/thawing on ice and resuspended in 0.25 volume of ice-cold 50 mM Tris, pH 8.0 (Digene, Beltsville, MD), 1 mM
EDTA (Research Genetics, Huntsville, AL), and 1 mM
dithiothreitol (Sigma). Complete lysis was achieved by repeatedly
sonicating each suspended pellet with five 20-s bursts. Cellular debris
was removed by centrifugation. The soluble and insoluble fractions of
the whole cell lysate were analyzed for protein expression using 14%
Tris-glycine gels (Novex, San Diego).
S were eluted
using the column gradient profile shown in Table
I,. Fractions were monitored by UV
absorbance and collected in a range of 20-30% NaCl. Combined
fractions were dialyzed against 2 liters of 50 mM MES
hemisodium salt (Sigma) at 4 °C with at least two changes over
24 h. Dialyzed protein was loaded onto a Mono S HR 5/5 cationic
exchange column (Amersham Pharmacia Biotech). Wild type and
Opj
S were eluted using the same gradient profile, and
fractions were again collected in a range of 20-30% NaCl. The
fractions were analyzed using gel electrophoresis, and the presence of
S was confirmed by Western blotting using antiserum GSp1 (35) and
reagents from Vector Laboratories (Burlingame, CA).
Gradient profile in Wt and Opj s elution column
0.1 × MRW/([mg/ml] × pl), where pl is the cuvette path length in centimeters, and MRW is
the mean residue weight (molecular weight divided by number of
residues). For wt and mutant proteins mean residue weight values of
116.4 and 116.1, respectively, were used. Temperature calibration was
performed as above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Lens opacity associated with the
Opj mutation. Top panel shows eyes of
live mice. For the first 2-3 months of life there is little difference
in appearance between Opj/+ and Opj/Opj, but in
the homozygote, opacity increases in severity with age. The lower
panel shows isolated lenses for +/+, Opj/+, and
Opj/Opj at 5weeks and Opj/Opj at 8 months. At
early stages the mutation causes secondary fiber discontinuity, most
notably in Opj/Opj.
S, were mapped to the same
region of mouse chromosome 16 (32, 33, 35). Specific PCR primers were
designed from the sequence of Crygs and used to amplify the
three exons of the gene from genomic DNA prepared from +/+, Opj/+ and Opj/Opj mice. PCR products were
sequenced directly, in both directions, using an automated sequencer. A
potential mutation was identified in exon 2. This was confirmed by
"manual" sequencing using 33P labeling and
autoradiography. As shown in Fig. 2, the
results clearly showed a single base difference between +/+ and
Opj/Opj DNA, whereas Opj/+ showed an intermediate
pattern, with both variants present at ~50% intensity. The
Opj-associated sequence variant is in coding sequence,
changing the codon TTC, corresponding to Phe-9 to TCC, encoding
serine.
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Fig. 2.
A mutation in Crygs
associated with opacity. Top, autoradiographs of
directly sequenced PCR fragments amplified from coding sequence of exon
2. Wild type and Opj/Opj samples have a single base
difference, whereas the Opj/+ sample shows equal intensity
of both bands at about half the intensity of those surrounding. This
figure shows sequence of the reverse strand. Bottom,
position of the sequence change and its consequences. The wt and mutant
codons are shown above the protein sequence. Secondary structure
elements are indicated. turn
indicates a region of folded hairpin turn characteristic of members of
the
-crystallin superfamily.
S was modeled using the
experimentally derived coordinates of bovine
B-crystallin (PDB:
4GCR). The sequences and structures of
-crystallins are well
conserved, allowing simple modeling based on residue replacement (3,
14, 21). Many residues, particularly core hydrophobics, were identical
between the two sequences and were left unchanged. Phe-9 is one such
core hydrophobic residue, occupying a key position in the core of the N-terminal domain (Fig. 3). As shown,
substitution of serine leaves unfilled space and buries a hydrophilic
group in the core.
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Fig. 3.
Illustration of the consequences of the
mutation on side chain packing in the core of the N-terminal domain
of S. The left panel shows a
slice through the model for wt mouse
S, based on experimental
coordinates for bovine
B (PDB: 4GCR). Dotted surfaces
represent Van der Waals radii as an indication of space filling. In the
right panel, serine is simply substituted for Phe-9,
illustrating the difference in size and showing that a hydrophilic side
chain is buried in a hydrophobic core. Models were built using Quanta
(Molecular Systems).
Ser sequence change, wt and Opj
proteins were synthesized using the pET17b expression system. High
levels of protein expression were obtained for both forms (Fig.
4). This included substantial expression
in the soluble fraction, suggesting that both wt and mutant proteins
were able to fold correctly. During expression and isolation of the
proteins, temperatures never exceeded room temperature.
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Fig. 4.
Expression of recombinant wt and
Opj mutant S.
SDS-polyacrylamide gel electrophoresis of soluble protein extracts from
bacteria bearing the empty expression vector (pET) and the
expression vector containing the coding sequence for
S
(pET
S) after induction by
isopropylthio-
-D-galactoside is shown. Lanes
S and Opj show the purified soluble
proteins for both wt and mutant.
S
(0.1-1 mg/ml) were examined by light scattering in heated cuvettes
(Fig. 5). At 0.1 mg/ml, wt protein showed
no significant tendency to precipitate below 59 °C. Increasing the
concentration to 1 mg/ml decreased the precipitation temperature to
55 °C. For the mutant protein at 0.1 mg/ml, precipitation began at
50 °C, whereas at 1 mg/ml the precipitation temperature was reduced
to 46 °C.
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Fig. 5.
Mutant S aggregates
at a lower temperature than wt. Protein samples were incubated in
a heated cuvette at increasing temperature. The absorbance was
measured at 600 nm and was normalized by dividing by the sample
absorbance at 280 nm. Increases in absorbance reflect aggregation of
denatured proteins. For both wt (triangles) and
Opj mutant (squares) proteins two concentrations
of 0.1 and 1 mg/ml were used. Open symbols are for the 0.1 mg/ml concentration, and filled symbols are for 1 mg/ml.
-crystallin family, corresponding to a predominantly
-sheet secondary structure (Fig.
6a). Near UV spectra were also
similar but showed some differences in tryptophan environment,
consistent with a change in core structure in the mutant (Fig.
6b). With increasing temperature, far UV CD signal decreased
in both wt and mutant in a time-dependent manner, although
the effect was more marked in the mutant (Fig. 6c). There
was no indication of a transition to a random coil signal in either
protein, rather the secondary structure signal diminished. This was
made most apparent by following the loss of signal at 218 nm with
increasing temperature over 25 min-intervals (Fig. 6d). Both
proteins showed a similar decrease in signal from 25 to 47 °C
followed by a marked drop in signal for the mutant between 47 and
55 °C associated with a loss of solubility. In the case of the
native protein, the abrupt loss of signal was delayed until around
60 °C.
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Fig. 6.
CD spectroscopy of wt and Opj
mutant S. In traces
a, b, and d the wt is shown in
red, and the mutant in blue. Panel a, far UV
spectra for both proteins at 24 °C. Panel b, near UV
spectra for both proteins at 24 °C. Panel c, far UV CD at
higher temperatures, thick lines, wt; thin lines,
Opj. Blue, 43 °C; green, 51 °C;
black, 55 °C; and red, 59 °C. Panel d, loss of ellipticity at 218 nm with
increasing temperature for 0.1-mg/ml protein solutions. An
equilibration time of 25 min was used between each measurement. Mutant
protein loses secondary structure more rapidly than wt.
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Fig. 7.
Histology of +/+, Opj/+, and
Opj/Opj lenses. Micrographs of transected lenses
from a 6-month-old +/+ mouse (panel A), a 5-month-old
Opj/Opj mouse (panel B), and a 5-month-old
Opj/+ mouse (panel C) showing epithelium
(e), lens fibers (f), and nuclei (n)
near the lens equator. Note in the homozygote, the gross
disorganization of nuclei, swollen and vacuolated lens fibers
(asterisks), irregularly shaped inclusion bodies with
protein-like staining properties (arrows), and those with
nucleic acid-like staining properties (arrowheads).
Magnification in panels A, B, and C
is × 206; calibration bar, 50 µm.
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Fig. 8.
Electron microscopy. Ultrastructure of a
transected lens from a 23-day-old Opj/Opj mouse, showing
swollen and disarrayed lens fibers (f) in both the outer
cortex (OC) of low electron opacity and the inner cortex
(IC) of higher electron opacity. Note in both layers of the
cortex, the irregularly shaped inclusion bodies composed of fiber-like
material of either thin (t) or coarse (c) nature.
The heterozygote shows no such alterations at this age. Magnification, × 15,000; calibration bar, 1.0 µm.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S. This results in a non-conservative sequence
change of Phe
Ser at residue 9 in the protein. Phe-9, and residues at equivalent positions in all four repeated structural motifs in all
members of the
-crystallin superfamily, is an important part of
the closely packed, hydrophobic core of one domain, in this case the
N-terminal domain (21, 40). However, this change does not prevent
correct folding of the protein; evidently, the evolutionarily conserved
tertiary fold pathway is able to overcome the defect in core packing so
that both mutant and wt recombinant
S proteins express well in
bacterial hosts and remain soluble at room temperature. Furthermore,
both of them exhibit essentially similar CD spectra, consistent with
the familiar, predominantly
-sheet structure of the
-crystallins.
Near UV spectra are also similar, although there are some differences,
consistent with changes in tryptophan microenvironments, as would be
expected for the substitution of serine and the possible concomitant
burying of water in the folded domain. Conserved tryptophan residues
are also important components of each domain core (21, 40).
-sheet. This occurs for both wt and mutant protein, but the loss in the secondary structure signal is steeper in the mutant than the wt.
-crystallin (43). In the case of lens
- and
-crystallins there
is evidence that early unfolding intermediates characterized by
substantial amounts of secondary structure bind to
-crystallin (44).
Our experiments give no information on the structure of the aggregating
state, but are consistent with these views. In the high protein
concentrations of the lens, any protein that exposes surface
hydrophobic groups, even transiently, could find many protein partners
for irreversible aggregation. In this way, the unfolding of previously
well behaved proteins, perhaps due to accumulated insults such as
oxidation or through simple stochastic processes in the normal aging
lens, can lead to opacification once the endogenous
-crystallin is saturated. Opj illustrates this sort of process, with an
essentially normally folded protein that is near the edge of stability
at physiological temperatures. Histological examination reveals protein inclusion bodies in both Opj/+ and Opj/Opj which
would act as light-scattering centers, causing opacity.
-crystallins. In Opj/+
mice, which retain some normal
S protein, opacity occurs in lenses
that have essentially normal organization. However, in
Opj/Opj, cortical lens fiber cells, which happen to be the
locales of
S expression (34), are severely swollen, vacuolated, and
disordered. Cell junctions and the normal smooth layering of lens
fibers also appear to be highly aberrant. Interestingly, there is also
evidence of cell dysfunction in cell layers in contact with the
cortical fibers. This includes vacuole formation in epithelial cells
and what appears to be a failure of normal cell nuclei breakdown in
inner fiber cell layers (45, 46). This latter effect is reminiscent of other
-crystallin-related cataracts in mice which affect nuclear fibers (8, 11, 47). These effects outside the cells that express
S
may reflect "collateral damage," with loss of normal cell contacts
and transport processes in the outer cortical fibers causing disruption
of adjacent cell layers.
S itself.
S is an outlying
member of the
-crystallin family, and it has been suggested that it
might represent a hypothetical ancestor of the
-crystallins,
possibly predating their recruitment to the lens (35, 48, 49). This
family in turn belongs to the wider
-crystallin superfamily,
which has members in both prokaryotes and eukaryotes (14, 50, 51). The
members of this superfamily remain largely mysterious in function, but
observations from non-lens relatives of the crystallins have suggested
that one common functional theme of these proteins may be some
involvement in the control of cell morphology (18, 52).
S in maintenance of normal cell morphology and function in
cortical lens fiber cells. Indeed, in Opj/Opj, but not in
Opj/+ at most ages examined, electron microscopy reveals
unusual tangled structures within individual cells, resembling the
collapse of some components of internal structure within each cortical
fiber cell. This is evident even at 23 days in Opj/Opj. No
similar defects occur in +/+ or Opj/+ through a wide range
of ages. Not until 16 months were similar defects observed in one
Opj/+, and even then this lens was less severely affected
than younger Opj/Opj. This suggests that even normal
S
function may decline with age, eventually causing the heterozygous
phenotype to mimic that of the homozygote. If this is the case, some
effects of the loss of
S function might occur in normal individuals,
perhaps including humans, with extreme age.
S continue to differentiate throughout
life, this member of the
-crystallin family may play an especially
important part in maintaining transparency in the aging lens. Indeed,
recent results show that senile cataract in humans may be associated
with specific post-translational modifications to
S (53). To
separate the effects of the twin processes of protein aggregation and
loss of function in the Opj cataract, a homologous
recombination experiment is in progress to delete the Crygs gene.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Rachel Neal and Qiu-Fang Cheng for help with initial studies on aggregation behavior and Anne Groome for electron microscopy. We are grateful to Bonnie Wallace for expert advice on spectroscopy.
![]() |
FOOTNOTES |
---|
* 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 first three authors contributed equally to this work.
¶ Present address: Molecular Biology and Assay Development, MetaMorphix, Inc., Baltimore, MD 21227.
To whom correspondence should be addressed: Section on
Molecular Structure and Function, NEI, Bldg. 6, Rm. 331, National
Institutes of Health, Bethesda, MD 20892-2740. Tel.: 301-402-3452; Fax:
301-496-0078; E-mail: graeme@helix.nih.gov.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M010583200
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ABBREVIATIONS |
---|
The abbreviations used are:
S,
S-crystallin;
PCR, polymerase chain reaction;
wt, wild type;
MES, 2-(N-morpholino)ethanesulfonic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bron, A. J., Vrensen, G. F., Koretz, J., Maraini, G., and Harding, J. J. (2000) Ophthalmologica 214, 86-104[CrossRef][Medline] [Order article via Infotrieve] |
2. | Hejtmancik, J. F. (1998) Am. J. Hum. Genet. 62, 520-525[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Stephan, D. A.,
Gillanders, E.,
Vanderveen, D.,
Freas-Lutz, D.,
Wistow, G.,
Baxevanis, A. D.,
Robbins, C. M.,
VanAuken, A.,
Quesenberry, M. I.,
Bailey-Wilson, J.,
Juo, S. H.,
Trent, J. M.,
Smith, L.,
and Brownstein, M. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1008-1012 |
4. | Kannabiran, C., Rogan, P. K., Olmos, L., Basti, S., Rao, G. N., Kaiser-Kupfer, M., and Hejtmancik, J. F. (1998) Mol. Vis. 4, 21[Medline] [Order article via Infotrieve] |
5. |
Kmoch, S.,
Brynda, J.,
Asfaw, B.,
Bezouska, K.,
Novak, P.,
Rezacova, P.,
Ondrova, L.,
Filipec, M.,
Sedlacek, J.,
and Elleder, M.
(2000)
Hum. Mol. Genet.
9,
1779-1786 |
6. | Ren, Z., Li, A., Shastry, B. S., Padma, T., Ayyagari, R., Scott, M. H., Parks, M. M., Kaiser-Kupfer, M. I., and Hejtmancik, J. F. (2000) Hum. Genet. 106, 531-537[CrossRef][Medline] [Order article via Infotrieve] |
7. | Heon, E., Priston, M., Schorderet, D. F., Billingsley, G. D., Girard, P. O., Lubsen, N., and Munier, F. L. (1999) Am. J. Hum. Genet. 65, 1261-1267[CrossRef][Medline] [Order article via Infotrieve] |
8. | Cartier, M., Breitman, M. L., and Tsui, L. C. (1992) Nat. Genet. 2, 42-45[Medline] [Order article via Infotrieve] |
9. |
Chambers, C.,
and Russell, P.
(1991)
J. Biol. Chem.
266,
6742-6746 |
10. | Graw, J., Jung, M., Loster, J., Klopp, N., Soewarto, D., Fella, C., Fuchs, H., Reis, A., Wolf, E., Balling, R., and Hrabe de Angelis, M. (1999) Genomics 62, 67-73[CrossRef][Medline] [Order article via Infotrieve] |
11. | Klopp, N., Favor, J., Loster, J., Lutz, R. B., Neuhauser-Klaus, A., Prescott, A., Pretsch, W., Quinlan, R. A., Sandilands, A., Vrensen, G. F., and Graw, J. (1998) Genomics 52, 152-158[CrossRef][Medline] [Order article via Infotrieve] |
12. | Smith, R. S., Hawes, N. L., Chang, B., Roderick, T. H., Akeson, E. C., Heckenlively, J. R., Gong, X., Wang, X., and Davisson, M. T. (2000) Genomics 63, 314-320[CrossRef][Medline] [Order article via Infotrieve] |
13. | Chang, B., Hawes, N. L., Roderick, T. H., Smith, R. S., Heckenlively, J. R., Horwitz, J., and Davisson, M. T. (1999) Mol. Vis. 5, 21[Medline] [Order article via Infotrieve] |
14. | Wistow, G. (1995) Molecular Biology and Evolution of Crystallins: Gene Recruitment and Multifunctional Proteins in the Eye Lens. Molecular Biology Intelligence Series , R. G. Landes Company, Austin, TX |
15. | Wistow, G. (1993) Trends Biochem. Sci. 18, 301-306[Medline] [Order article via Infotrieve] |
16. | de Jong, W. W. (1981) in Molecular and Cellular Biology of the Eye Lens (Bloemendal, H., ed) , pp. 221-278, Wiley-Interscience, New York |
17. | Harding, J. J., and Crabbe, M. J. C. (1984) in The Eye (Davson, H., ed), Vol. 1B , pp. 207-492, Academic Press, New York |
18. |
Ray, M. E.,
Wistow, G.,
Su, Y. A.,
Meltzer, P. S.,
and Trent, J. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3229-3234 |
19. | Benedek, G. B. (1971) Appl. Optics 10, 459-473 |
20. | Delaye, M., and Tardieu, A. (1983) Nature 302, 415-417[Medline] [Order article via Infotrieve] |
21. | Slingsby, C., and Clout, N. J. (1999) Eye 13, 395-402[Medline] [Order article via Infotrieve] |
22. | Harding, J. J. (1998) Curr. Opin. Ophthalmol. 9, 10-13[Medline] [Order article via Infotrieve] |
23. | Bloemendal, H. (1991) Investig. Ophthalmol. Vis. Sci. 32, 445-455[Medline] [Order article via Infotrieve] |
24. | Vrensen, G. F. (1995) Comp. Biochem. Physiol. A. Physiol. 111, 519-532[CrossRef][Medline] [Order article via Infotrieve] |
25. | Eshagian, J. (1982) Trans. Ophthalmol. Soc. U. K. 102, 364-368[Medline] [Order article via Infotrieve] |
26. | Shiels, A., and Bassnett, S. (1996) Nat. Genet. 12, 212-215[Medline] [Order article via Infotrieve] |
27. | Berry, V., Francis, P., Kaushal, S., Moore, A., and Bhattacharya, S. (2000) Nat. Genet. 25, 15-17[CrossRef][Medline] [Order article via Infotrieve] |
28. | Jakobs, P. M., Hess, J. F., FitzGerald, P. G., Kramer, P., Weleber, R. G., and Litt, M. (2000) Am. J. Hum. Genet. 66, 1432-1436[CrossRef][Medline] [Order article via Infotrieve] |
29. | Conley, Y. P., Erturk, D., Keverline, A., Mah, T. S., Keravala, A., Barnes, L. R., Bruchis, A., Hess, J. F., FitzGerald, P. G., Weeks, D. E., Ferrell, R. E., and Gorin, M. B. (2000) Am. J. Hum. Genet. 66, 1426-1431[CrossRef][Medline] [Order article via Infotrieve] |
30. | Favor, J. (1983) Mutat. Res. 110, 367-382[Medline] [Order article via Infotrieve] |
31. | Favor, J. (1984) Genet. Res. 44, 183-197[Medline] [Order article via Infotrieve] |
32. | Everett, C. A., Glenister, P. H., Taylor, D. M., Lyon, M. F., Kratochvilova-Loester, J., and Favor, J. (1994) Genomics 20, 429-434[CrossRef][Medline] [Order article via Infotrieve] |
33. | Kerscher, S., Glenister, P. H., Favor, J., and Lyon, M. F. (1996) Genomics 36, 17-21[CrossRef][Medline] [Order article via Infotrieve] |
34. | Jaworski, C., and Wistow, G. (1996) Biochem. J. 320, 49-54[Medline] [Order article via Infotrieve] |
35. | Sinha, D., Esumi, N., Jaworski, C., Kozak, C. A., Pierce, E., and Wistow, G. (1998) Mol. Vis. 4, 8[Medline] [Order article via Infotrieve] |
36. | Wistow, G., Sardarian, L., Gan, W., and Wyatt, M. K. (2000) Mol. Vis. 6, 79-84[Medline] [Order article via Infotrieve] |
37. | Hogan, B., Costantini, F., and Lacy, E. (1986) Manipulating the Mouse Embryo: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
38. | Wallace, B. A., and Teeters, C. L. (1987) Biochemistry 26, 65-70[Medline] [Order article via Infotrieve] |
39. | Robison, W. G., Jr., Kador, P. F., Akagi, Y., Kinoshita, J. H., Gonzalez, R., and Dvornik, D. (1986) Diabetes 35, 295-299[Abstract] |
40. | Wistow, G., Turnell, B., Summers, L., Slingsby, C., Moss, D., Miller, L., Lindley, P., and Blundell, T. (1983) J. Mol. Biol. 170, 175-202[Medline] [Order article via Infotrieve] |
41. | Benedek, G. B. (1997) Investig. Ophthalmol. Vis. Sci. 38, 1911-1921[Medline] [Order article via Infotrieve] |
42. | Wetzel, R. (1996) Cell 86, 699-702[Medline] [Order article via Infotrieve] |
43. | Horwitz, J. (2000) Semin. Cell. Dev. Biol. 11, 53-60[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Das, K. P.,
Choo-Smith, L. P.,
Petrash, J. M.,
and Surewicz, W. K.
(1999)
J. Biol. Chem.
274,
33209-33212 |
45. | Gao, C. Y., Bassnett, S., and Zelenka, P. S. (1995) Dev. Biol. 169, 185-194[CrossRef][Medline] [Order article via Infotrieve] |
46. | Counis, M. F., Chaudun, E., Arruti, C., Oliver, L., Sanwal, M., Courtois, Y., and Torriglia, A. (1998) Cell. Death Differ. 5, 251-261[CrossRef][Medline] [Order article via Infotrieve] |
47. | Yoshiki, A., Hanazono, M., Oda, S., Wakasugi, N., Sakakura, T., and Kusakabe, M. (1991) Development 113, 1293-1304[Abstract] |
48. | Quax-Jeuken, Y., Driessen, H., Leunissen, J., Quax, W., de Jong, W., and Bloemendal, H. (1985) EMBO J. 4, 2597-2602[Abstract] |
49. | van Rens, G. L., Raats, J. M., Driessen, H. P., Oldenburg, M., Wijnen, J. T., Khan, P. M., de Jong, W. W., and Bloemendal, H. (1989) Gene (Amst.) 78, 225-233[Medline] [Order article via Infotrieve] |
50. | Wistow, G. (1990) J. Mol. Evol. 30, 140-145[Medline] [Order article via Infotrieve] |
51. | Clout, N. J., Slingsby, C., and Wistow, G. J. (1997) Nat. Struct. Biol. 4, 685[Medline] [Order article via Infotrieve] |
52. | Teichmann, U., Ray, M. E., Ellison, J., Graham, C., Wistow, G., Meltzer, P. S., Trent, J. M., and Pavan, W. J. (1998) Mamm. Genome 9, 715-720[CrossRef][Medline] [Order article via Infotrieve] |
53. | Takemoto, L., and Boyle, D. (2000) Mol. Vis. 6, 164-168[Medline] [Order article via Infotrieve] |