A Temperature-sensitive Mutation of Crygs in the Murine Opj Cataract*

Debasish SinhaDagger §, M. Keith WyattDagger §, Robert SarraDagger ||§, Cynthia JaworskiDagger , Christine Slingsby||, Caroline Thaung**Dagger Dagger , Lewis Pannell§§, W. Gerald RobisonDagger , Jack Favor¶¶, Mary Lyon**, and Graeme WistowDagger ||||

From the Dagger  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 Dagger Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for gamma 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 gamma S-crystallin which would be consistent with ideas that members of the beta gamma -crystallin superfamily may have roles associated with maintenance of cytoarchitecture.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -crystallins belong to the small heat-shock protein superfamily and can act as "junior chaperones" (14-17). The important beta - and gamma -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).

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). gamma S-crystallin (gamma S)1 is a major component of the adult mammalian lens, essentially replacing the expression of other gamma -crystallins (gamma 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). gamma S expression increases after birth and continues as the lens grows throughout life. Thus high levels of gamma S mark the cortical fibers of the mature lens, the same cells affected by the Opj cataract. Crygs, the gene for gamma S, was mapped to mouse chromosome 16, close to the locus of Opj (35).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma 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 gamma B-crystallin (PDB: 4GCR), essentially as described previously (3).

Recombinant Proteins-- Clones for the complete coding sequence of wild type (wt) and Opj mutant gamma 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.

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-beta -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).

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 gamma 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 gamma 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 gamma S was confirmed by Western blotting using antiserum GSp1 (35) and reagents from Vector Laboratories (Burlingame, CA).

                              
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Table I
Gradient profile in Wt and Opj gamma s elution column

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 -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.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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 gamma 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. beta gamma turn indicates a region of folded hairpin turn characteristic of members of the beta gamma -crystallin superfamily.

Molecular Modeling-- Mouse gamma S was modeled using the experimentally derived coordinates of bovine gamma B-crystallin (PDB: 4GCR). The sequences and structures of gamma -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 gamma S. The left panel shows a slice through the model for wt mouse gamma S, based on experimental coordinates for bovine gamma 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).

Recombinant Proteins-- To test the consequences of the Opj Phe right-arrow 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 gamma 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 gamma S (pETgamma S) after induction by isopropylthio-beta -D-galactoside is shown. Lanes gamma S and Opj show the purified soluble proteins for both wt and mutant.

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 gamma 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 gamma 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.

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 gamma -crystallin family, corresponding to a predominantly beta -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 gamma 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.

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.


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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.

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.


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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma S. This results in a non-conservative sequence change of Phe right-arrow 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 beta gamma -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 gamma 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 beta -sheet structure of the gamma -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).

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 beta -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.

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 alpha -crystallin (43). In the case of lens beta - and gamma -crystallins there is evidence that early unfolding intermediates characterized by substantial amounts of secondary structure bind to alpha -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 alpha -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.

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 beta gamma -crystallins. In Opj/+ mice, which retain some normal gamma 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 gamma 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 gamma -crystallin-related cataracts in mice which affect nuclear fibers (8, 11, 47). These effects outside the cells that express gamma 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.

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 gamma S itself.

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, gamma S is an outlying member of the gamma -crystallin family, and it has been suggested that it might represent a hypothetical ancestor of the gamma -crystallins, possibly predating their recruitment to the lens (35, 48, 49). This family in turn belongs to the wider beta gamma -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).

The severity of the Opj/Opj phenotype may thus reflect a role for gamma 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 gamma 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 gamma S function might occur in normal individuals, perhaps including humans, with extreme age.

Because new cells expressing gamma S continue to differentiate throughout life, this member of the gamma -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 gamma 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.

    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

    ABBREVIATIONS

The abbreviations used are: gamma S, gamma S-crystallin; PCR, polymerase chain reaction; wt, wild type; MES, 2-(N-morpholino)ethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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